Assessment of digestive enzyme activities ... - Wiley Online Library

Journal of Fish Biology (2003) 63, 48–58 doi:10.1046/j.1095-8649.2003.00120.x, available online at http://www.blackwell-synergy.com

Assessment of digestive enzyme activities during larval development of white bream J. B. C A R A *, F. J. M O Y A N O *†, S. C A´ R D E N A S ‡, C. F E R N A´ N D E Z - D I´ A Z § A N D M. Y U´ F E R A § *Dpto. Biologıá Aplicada. E.P.S. Univ. Almerıá, E-04120 Almerıá, Spain, ‡CICEM El Torunõ, Junta de Andalucıá, P. O. Box 16, E-11500 El Puerto de Santa Marıá, Ca´diz, Spain and §Instituto de Ciencias Marinas de Andalucıá (CSIC). Apartado Oficial, E-11510 Puerto Real, Ca´diz, Spain (Received 21 May 2002, Accepted 11 April 2003) Activity of some of the main enzymes involved in protein digestion and absorption (acid and alkaline proteases, leucine-aminopeptidase, acid and alkaline phosphatases) as well as those of amylase and lipase, was assessed during larval development of white sea bream Diplodus sargus. All enzyme activity was detected at the moment of mouth opening. The variations observed in the activity profiles of the digestive enzymes were correlated either to developmental events, such as the functional start of the stomach (22 days after hatching), or to changes in the nature of the diet. The early and noticeable development of digestive enzyme activities was linked to a # 2003 The Fisheries Society of the British Isles high survival after weaning. Key words: digestive enzymes; Diplodus sargus; larval development; SDS-PAGE.

INTRODUCTION At present, Mediterranean finfish aquaculture is mainly based on the intensive production of two species: sea bass Dicentrarchus labrax (L.) and gilthead sea bream Sparus aurata (L.). The production techniques for these species are well developed and their production has risen greatly in recent years. There is a need, however, for diversification of fish species used in the future for finfish mariculture in the Mediterranean. In this respect, some species of autochthonous sparids such as the sharpsnout sea bream Diplodus puntazzo (Cetti) or the white sea bream Diplodus sargus (L.) have been reported as highly suitable for aquaculture (Abellań et al., 1994). Although there are some studies on larval morphological and behavioural development (Divanach et al., 1982; Kentouri & Divanach, 1982) and osteological ontogeny (Koumoundouros et al., 2001), the published information on biology and physiology is still scarce. There are an increasing number of papers dealing with the onset and development of digestive enzyme activities during larval development of fishes used in aquaculture (Baglole

†Author to whom correspondence should be addressed. Tel.: þ34 950015294; fax: þ34 950015476; email: [email protected]

48 #

2003 The Fisheries Society of the British Isles

DIGESTIVE ENZYMES IN LARVAL DIPLODUS SARGUS

49

et al., 1988; Kolkowski et al., 1993; Oozeki & Bailey, 1995; Dı´ az et al., 1997; Kim et al., 2001). There are two areas of interest: (1) the assessment of the presence and level of activity of certain enzymes (e.g. trypsin) may be used as an indicator of larval development, as well as a predictor of their future survival (Hjelmeland et al., 1984); (2) the possibility of early weaning or the potential ability of the larvae to be fed exclusively on artificial diets, which can only be based on ontogenetic changes in digestive enzyme activity (Moyano & Sarasquete, 1993; Zambonino Infante & Cahu, 1994; Moyano et al., 1996; Martı´ nez et al., 1999). In this context, the aims of the present study were to assess the development and activity of the main digestive enzymes present in larvae of white sea bream. MATERIALS AND METHODS FI S H L A R V AE White sea bream eggs were obtained during 2001 from a captive broodstock held at temperatures ranging from 17 to 20 C at the experimental facilities of the CICEM El Torunõ, Ca´diz, Spain. After hatching, larvae were reared in 250 l tanks at 195 C with constant illumination and a salinity of 33. It was noticed that 24 h after mouth opening, almost 100% of larvae were actively feeding. From the opening of the mouth, 3 days after hatching (DAH) to 15 DAH, the larvae were fed rotifers, Brachionus rotundiformis and Brachionus plicatilis. From 12 to 30 DAH larvae were fed recently hatched Artemia nauplii and from day 31 onwards, commercial fish feed was supplied. Changes in the feeding regime were based on those routinely used for S. aurata, taking into account the size of larvae and the relative proportion in the population of a size class able to ingest such prey. At different times during their development, groups of 40–500 larvae were sampled, rinsed in distilled water and freeze-dried until analysed. Larvae obtained from two different batches were pooled to form a sample. Larval dry mass was determined in triplicates of 15–40 individuals for each sample point.

EN Z Y M E E X T R A C T S Enzyme extracts were prepared by homogenization of pooled whole larvae (20 mg ml1 for biochemical assays; 35 mg ml1 for zymograms) in cold 100 mM Tris–HCl buffer þ 20 mM CaCl2, pH 80, followed by centrifugation (16 000g, 30 min, 4 C). Supernatants were stored at 20 C until analysis. Concentration of soluble protein in extracts was determined by the Bradford method (Bradford, 1976), using bovine serum albumin (1 mg ml1) as a standard. Larval extracts were assayed for the determination of acid and alkaline protease, amylase, lipase, leucine-aminopeptidase and acid and alkaline phosphatases.

EN Z Y M E A C T I V I T Y A S SA Y S Protein digestion in larvae was assessed through the quantification of total acid and alkaline protease activities. The scope was completed measuring activities of enzymes commonly used as indicators of either protein pynocitosis (acid phosphatase), complete protein hydrolysis at an intestinal level (leucine-aminopeptidase) or absorption of amino acids (alkaline phosphatase). Acid protease activity was measured using the method detailed by Anson (1938) using haemoglobin as substrate. One unit of activity was defined as 1 mg of tyrosine released min1. Alkaline protease activity was measured using azocasein as substrate (Sarath et al., 1989); one unit of activity was defined as the amount of enzyme able to produce an increase of one unit of absorbance min1. The activity of a-amylase was estimated using the Somogy–Nelson procedure described by Robyt & Whelan (1968) and one unit was defined as the amount of enzyme able to

#

2003 The Fisheries Society of the British Isles, Journal of Fish Biology 2003, 63, 48–58

50

J. B. CARA ET AL.

produce 1 mg of maltose min1. Acid and alkaline phosphatases were measured at pH 55 and 75, following the method described by Bergmeyer (1974) using 4-nitrophenol as substrate and considering one unit of activity as the amount of enzyme able to produce an increase of 001 units of absorbance. The activity of lipase was measured using the method of Mckellar & Cholette (1986) as modified by Versaw et al. (1989), one unit of activity was 1 mg of b-naphtol released min1. Leucine-aminopeptidase was measured by the modified method of Pfeiderer (1970), one unit of activity was the amount of enzyme able to produce an increase of one unit of absorbance min1. The activities of the different enzymes assayed were expressed in relation to soluble protein in the extracts (specific activity). All measurements were carried out in triplicate and results are given as mean S.D.

SUBSTRATE SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS SDS-PAGE of the proteins in the enzyme preparations was done according to Laemmli (1970), using 12% acrylamide and 8 10 0075 cm gels. Molecular mass marker (5 ml) was loaded on each plate. Preparation of samples and zymograms of proteinase activities of fractions separated by electrophoresis were done according to Garcı´ a-Carrenõ et al. (1993). Previously, samples were filtered throughout a Sephadex G-25S column (1/10, v/v). Electrophoresis was performed at a constant voltage of 100 V per gel for 60 min at 5 C. After electrophoresis, gels were washed and incubated in 05% casein Hammerstein, pH 9, for 30 min at 5 C, and were then transferred to the same solution at 37 C for 150 min without agitation. Thereafter, gels were washed and fixed in 12% TCA prior to staining with 01% Coomassie brillant blue (BBC R-250) in a methanol-acetic acid water solution (40 : 10 : 50). Destaining was carried out in a methanol-acetic acid water solution (40 : 10 : 50).

RESULTS The mass increase of D. sargus was high since larvae multiplied their body mass by a factor of 100 from mouth opening at 3 DAH to weaning at 30 DAH (Fig. 1). The pattern determined for alkaline protease showed a very early appearance of this activity, with peaks at 3 and 13 DAH, which were correlated to mouth opening and to the change from rotifer to Artemia, respectively [Fig. 2(a)]. Another peak was measured at 22 DAH, being followed

Dry mass (µg larva−1)

100 000 10 000

1000 100 10 0

10

20

30

40

50

Days after hatching FIG. 1. Growth of Diplodus sargus larvae along the experimental period. The curve fitted was by y ¼ 23998e01474x, r2 ¼ 09671.

#


51


250

(a)

mU mg−1

200 150 100 50 0 20

(b)

U mg–1

15 10 5

(c)

mU mg–1

40 35 30 25 20 15 10 5 0 80 70 60 50 40 30 20 10 0

(d)

mU mg–1

Specific activity

0

3000

(e)

U mg–1

2500 2000 1500 1000 500 0 250

(f )

U mg–1

200 150 100 50 0 0

5

10 Rotifer

Mouth opening

15

20

25

30

35

40

45

Artemia Weaning

Days after hatching FIG. 2. Specific activity of (a) alkaline protease, (b) acid protease, (c) leucine-aminopeptidase, (d) phosphatases (*, alkaline and *, acid), (e) amylase and (f) lipase assayed during larval development of Diplodus sargus.

#


52

J. B. CARA ET AL.

by a decrease to very low levels, which were maintained from that day onwards. That peak was also measured in most of the other activities but was not related to a change in the feeding pattern. Acid protease activity also showed peaks at 3 and 22 DAH [Fig. 2(b)] but in contrast to what was observed for alkaline activity, it continued to increase from that day onwards. The evolution of leucine-aminopeptidase specific activity showed a fairly similar profile to that observed both for alkaline protease and alkaline phosphatase [Fig. 2(c)]. Acid phosphatase showed a high activity at early stages of larval development, followed by a sharp decrease and peaks at 13, 22 and 30 DAH [Fig. 2(d)]. Zymograms confirmed, to a great extent, results obtained with biochemical assays of alkaline protease activity. Different bands were already detected in extracts obtained from 3 day old larvae with molecular masses of 104, 60, 53, 40, 376, 31, 28 and 22 kDa (Fig. 3, lane 1). The decrease in specific activity detected at 8 DAH was evidenced by a lower intensity in bands 1, 2 and 4, while the peak of activity measured in 13 DAH larvae was correlated to an increased intensity in band 4, as well as to the appearance of another band of low molecular mass. The low activity measured from 15 to 20 DAH was correlated again to a reduction in the intensity of bands in the zymogram, while the peak of activity reached at day 22 was visualized through an increase in the intensity of such bands. Decrease in protease activity measured in weaned and postweaned larvae (30 and 40 DAH) is shown by the lower intensity in all bands. Both amylase and lipase activities showed similar patterns during the first 3 days of larval development. The sharp increases in activity were related to mouth opening [Fig. 2(e), (f)]. Nevertheless, while amylase activity progressively decreased with age, lipase activity remained notably high, showing only a decrease clearly related to weaning at 30 DAH. The ratio between lipase and alkaline protease activities is shown in Fig. 4. As a general rule, the maximum activity of lipase was related to minimum protease activity, and the ratios reached 4 : 1 to 5 : 1 during different periods of larval development. They decreased to 1 : 1 when changes in the composition of live 3

MWM

8

13

20

22

30

40 DAH

1> 66

2> 3>

45 36 29

20

4 5 6 7

> > > >

8>

14 FIG. 3. Zymogram of the alkaline proteases obtained from Diplodus sargus larvae sampled at different ages after hatching. The different bands appearing during development are indicated (>). MWM Sigma Marker LR (6500-66000) Ref M3913.

#


53

400

6

350

5

300 4

250

3

200 150

2

Lipase : protease

Lipase (U) and protease (mU) activities


100 1

50 0

0 1

2

3

4

6

8

9

13

20

22

25

30

35

40

44

DAH FIG. 4. Lipase ( ), acid protease (&), alkaline protease (&) and the ratio of lipase : protease (*) activities during larval development of Diplodus sargus larvae.

food took place. The only exception was found at 20 DAH, when both activities were high.

DISCUSSION Results obtained in the present study indicated that the rearing conditions were suitable for the development of D. sargus larvae, promoting growth equivalent or better than that obtained in larvae of other sparids. At weaning, larvae had multiplied their initial mass by a factor >100, but under the same conditions, larvae of other cultured species, e.g. sea bream and Dentex dentex (L.) increased their mass by a factor of 50 or 75 (Moyano et al., 1996; Yu´fera et al., 2000; F.J. Moyano, F.J. Alarcon, M. Dı´ az, E. Abellań, M. Yu´fera & C. Fernańdez, unpubl. data). The presence of all main digestive enzymes in fish larvae at the moment of mouth opening has been widely demonstrated not only in freshwater or estuarine species such as Oreochromis niloticus L. (Tengjaroenkul et al., 2001) or Acipenser sturio L. (Gawlicka et al., 1995), but also in marine species such as S. aurata (Moyano et al., 1996) or Solea senegalensis Kaup (Martı´ nez et al., 1999). Further development of such activities seems to be affected mainly by the progressive appearance of the digestive organs and by the response to changes in the amount and composition of available food, either genetically programmed or induced by substrate. This typically results in patterns showing a succession of increases and decreases with time (Moyano et al., 1996; Martı´ nez et al., 1999). Nevertheless, several authors have described a pattern characterized by a gradual decrease of specific activities of the main digestive enzyme, a result that may be explained by the progressive increase in soluble proteins taking place in growing larvae as the body tissues fraction becomes steadily #


54

J. B. CARA ET AL.

more important. This was not the case for enzymes in D. sargus larvae, which showed important fluctuations with time. The organogenesis of the digestive tract in D. sargus revealed an early appearance of gastric glands by 20 DAH and an almost fully functional stomach by 30 DAH (Ortiz-Delgado, in press). In the present study, a sharp increase in the activity of most of the enzymes was observed at c. 22 DAH, which may reflect the response to the onset of acid digestion and provides an increased supply of available substrates for other digestive enzymes. Such development in gastric digestion, as well as the development of muscles and mouth observed during the first month suggests that at this age this species would be able to catch and digest food of a greater range than just planktonic microcrustaceans. The other peaks in the pattern of protease activity could be better explained as a response to changes in diet, since the synthesis of the main digestive enzymes in larval and juvenile fishes is greatly dependent on the total amount and quality of the food. It has been demonstrated that in fish larvae enzyme response to changes in diet takes only 24 h for trypsin and