1 23

Fish Physiology and Biochemistry ISSN 0920-1742Volume 38Number 2 Fish Physiol Biochem (2012) 38:441-454DOI 10.1007/s10695-011-9525-9

Digestive enzyme activities during earlyontogeny in Common snook (Centropomusundecimalis)

L. D. Jimenez-Martinez, C. A. Alvarez-González, D. Tovar-Ramírez, G. Gaxiola,A. Sanchez-Zamora, F. J. Moyano,F. J. Alarcón, G. Márquez-Couturier, etal.

1 23

Your article is protected by copyright and

all rights are held exclusively by Springer

Science+Business Media B.V.. This e-offprint

is for personal use only and shall not be self-

archived in electronic repositories. If you

wish to self-archive your work, please use the

accepted author’s version for posting to your

own website or your institution’s repository.

You may further deposit the accepted author’s

version on a funder’s repository at a funder’s

request, provided it is not made publicly

available until 12 months after publication.

Digestive enzyme activities during early ontogenyin Common snook (Centropomus undecimalis)

L. D. Jimenez-Martinez • C. A. Alvarez-Gonzalez • D. Tovar-Ramırez •

G. Gaxiola • A. Sanchez-Zamora • F. J. Moyano • F. J. Alarcon • G. Marquez-Couturier •

E. Gisbert • W. M. Contreras-Sanchez • N. Perales-Garcıa • L. Arias-Rodrıguez •

J. R. Indy • S. Paramo-Delgadillo • I. G. Palomino-Albarran

Received: 19 December 2009 / Accepted: 3 June 2011 / Published online: 14 June 2011

� Springer Science+Business Media B.V. 2011

Abstract Common snook (Centropomus undecimal-

is) is one of the most important marine species under

commercial exploitation in the Gulf of Mexico; for this

reason, interest in developing its culture is a priority.

However, larviculture remains as the main bottleneck

for massive production. In this sense, our objective was

to determine the changes of digestive enzymes activ-

ities using biochemical and electrophoretic techniques

during 36 days of Common snook larviculture fed with

live preys (microalgae, rotifers, and Artemia). During

larviculture, all digestive enzymatic activities were

detected with low values since yolk absorption, 2 days

after hatching (dah) onwards. However, the maximum

values for alkaline protease (6,500 U mg protein-1),

trypsin (0.053 mU 9 10-3 mg protein-1), and Leu-

cine aminopeptidase (1.4 9 10-3 mU mg protein-1)

were detected at 12 dah; for chymotrypsin at 25 dah

(3.8 9 10-3 mU mg protein-1), for carboxypeptidase

A (280 mU mg protein-1) and lipase at 36 dah

(480 U mg protein-1), for a-amylase at 7 dah

(1.5 U mg protein-1), for acid phosphatases at 34 dah

(5.5 U mg protein-1), and finally for alkaline phos-

phatase at 25 dah (70 U mg protein-1). The alkaline

protease zymogram showed two active bands, the first

(26.3 kDa) at 25 dah onwards, and the second

(51.6 kDa) at 36 dah. The acid protease zymogram

showed two bands (RF = 0.32 and 0.51, respectively)

at 34 dah. The digestive enzymatic ontogeny of

C. undecimalis is very similar to other strictly marine

carnivorous fish, and we suggest that weaning process

should be started at 34 dah.

Keywords a-Amylase � Centropomus undecimalis �Common snook � Lipase � PAGE � Phosphatase �Protease

L. D. Jimenez-Martinez � C. A. Alvarez-Gonzalez (&) �G. Marquez-Couturier � W. M. Contreras-Sanchez �N. Perales-Garcıa � L. Arias-Rodrıguez �J. R. Indy � S. Paramo-Delgadillo

DACBIOL Laboratorio de Acuacultura, Universidad

Juarez Autonoma de Tabasco, Carretera Villahermosa-

Cardenas km 0.5, 86139 Villahermosa, Tabasco, Mexico

e-mail: alvarez_alfonso@hotmail.com

G. Gaxiola � A. Sanchez-Zamora �I. G. Palomino-Albarran

Unidad Multidisciplinaria de Docencia e Investigacion,

Facultad de Ciencias, UNAM, Puerto de abrigo s/n, Sisal,

Yucatan, Mexico

F. J. Moyano � F. J. Alarcon

Departamento de Biologıa Aplicada, Escuela Politecnica

Superior, Universidad de Almerıa, 04120 La Canada de

San Urbano, Almerıa, Spain

D. Tovar-Ramırez

Centro de Investigaciones Biologicas del Noroeste

(CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa

Rita, 23090 La Paz, B.C.S., Mexico

E. Gisbert

IRTA – Sant Carles de la Rapita, Crta. Poble Nou km 5.5,

43540 Sant Carles de la Rapita, Tarragona, Spain

123

Fish Physiol Biochem (2012) 38:441–454

DOI 10.1007/s10695-011-9525-9

Author's personal copy

Introduction

Marine fish culture in Mexico is a recent activity

mainly based on some species with high economic

value and adequate biological characteristics for their

culture, such as spotted sand bass (Paralabrax

maculatofasciatus), leopard grouper (Mycteroperca

rosacea), bullseye puffer (Sphoeroides annulatus),

rose snapper (Lutjanus guttatus), and more recently

cobia (Rachycentrum canadum) (Aviles-Quevedo

et al. 1995; Gracia-Lopez et al. 2004; Komar et al.

2004; Ibarra-Castro and Duncan 2007; Holt et al.

2007). The fat snook (Centropomus parallelus) and

the common snook (Centropomus undecimalis) of the

family Centropomidae, a group with highly important

commercial value in Mexico and the United States,

have been widely studied to understand their basic

biology and aiming for developing their culture

(Chavez 1961; Stephen and Shafland 1982; Sherwood

et al. 1993; Ramirez and Cerqueira 1994; Grier and

Taylor 1998; Grier 2000; Cequeira and Brugger

2001; Alvarez-Lajonchere et al. 2002; Tarcisio et al.

2005; Gracia-Lopez et al. 2006; Wainwright et al.

2006; Yanes-Roca et al. 2009). However, for

C. undecimalis, the bottleneck is still the massive

fry production due to feeding problems when micro-

algae, rotifers, and Artemia nauplii are used. These

live feeds are considered not adequate for fish larval

culture (Versichelle et al. 1989; Garcıa-Ortega et al.

1998), resulting in low growth and survival. For this

reason, many studies have been conducted to under-

stand the digestive physiology during early ontogeny

with many species, such as the seabream, Sparus

aurata (Moyano et al. 1996), Siberian sturgeon,

Acipenser baeri (Gisbert et al. 1999), white bream,

Diplodus sargus (Cara et al. 2003), yellowtail

amberjack, Seriola lalandi (Chen et al. 2006),

common seabream, Pagrus pagrus (Darias et al.

2006), P. maculatofasciatus (Alvarez-Gonzalez et al.

2008, 2010), and the orange-spotted grouper Epi-

nephelus coioides (Shaozhen et al. 2008). These

studies allow understanding the right moment to

conduct early weaning using artificial diets (Brock

et al. 1992; Zambonino-Infante and Cahu 1994;

Ribeiro et al. 1999; Cara et al. 2003; Fabillo et al.

2004). In consequence, the objective of this work was

to assess the development of digestive enzymes using

biochemical and electrophoretical techniques during

early ontogeny of C. undecimalis.

Materials and methods

Rearing and sampling of larvae

Twelve adult common snook C. undecimalis (4–5 kg

per fish) were maintained under controlled conditions

in four 13-m3 circular plastic tanks at the Unidad

Multidisciplinaria de Docencia e Investigacion

(UMDI) from UNAM in Sisal, Merida, Mexico for

2 years. Spawning induction was done in adults using

cholesterol implants with 150 lg of sGnRHa. fish-1

(Ovaplant, Syndel, Western Chemical, Fendale, WA,

USA). A total of 1,10,000 embryos were obtained after

32 h after injection. After the embryos hatched, the

yolk-sac larvae (3,250) were collected by siphoning

and placed in a 400-l cylinder-conical tank with

constant water exchange, and continuous aeration until

the larvae absorbed the yolk (24 h later). Salinity

(35.2 ± 1.1 ppt), dissolved oxygen (6.0 ± 0.3

mg l-1), and temperature (29.9 ± 1.1�C) were mon-

itored daily.

Larvae were fed four times per day (8:00, 12:00,

16:00, and 20:00 h), starting with green water culture,

using the microalgae Nannochloropsis sp.

(20 9 106 cells ml-1) and S-type rotifers Brachionus

rotundiormis (R, 2–10 preys ml-1) from mouth open-

ing (day 1 after hatching) until 10 dah. Rotifers were

mixed with newly hatched Artemia nauplii (AN, INVE

Aquaculture, Belgium, 2–10 preys ml-1) from day 10

until 25 dah and offered to the larvae. Finally, larvae

were fed exclusively with lipidic enriched (SELCO,

INVE Aquaculture, Belgium) Artemia meta-nauplii

(EAMN, 2–15 preys ml-1) from day 25 up to 36 dah

(end of the experiment). Nine samples of feed larvae

were taken from one culture tank using a 500-lm-

diameter mesh, in triplicate (the numbers in parenthe-

ses are the numbers of larvae sampled per replicate) on

day 0 (embryos, 80), 1 (600), 3 (600), 5 (600), 7 (600),

12 (400), 25 (100), 34 (50), and 36 (30) after hatching.

They were frozen with liquid nitrogen and stored at

-80�C until analysis was done. For growth analysis,

samples of 10 larvae were taken and fixed with

buffered borate formalin solution (4%) to measure

the total length for each larvae using a digital caliper

(Neiko-HKMUD473, Neiko-HKMUD473, CA, USA).

The individual wet weight (mg) of each larva was also

recorded with an analytic balance (OHAUS-Phoenix

GH-300, Pine Brook, NJ, USA; precision of 10-4 g)

after elimination of water excess with filter paper.

442 Fish Physiol Biochem (2012) 38:441–454

123

Author's personal copy

Biochemical analyses

Sampled larvae were dissected individually to

remove the head and tail, and the visceral bulks

were homogenized as pool (30 mg ml-1) in cold

50 mmol l-1 Tris–HCl 20 mmol l-1 CaCl2 buffer,

pH 7.5. The supernatant obtained after centrifugation

(16,000g for 15 min at 5�C) was stored at -20�C

until enzyme analysis. The concentration of soluble

protein was determined by the Bradford (1976)

method using bovine serum albumin as a standard.

Total alkaline protease activity was measured

using casein (0.5%) in 50 mmol l-1 Tris–HCl buffer,

pH 9.0, following Kunitz’s (1947) method, modified

by Walter (1984). Acid protease activity was evalu-

ated according to Anson (1938) using 0.5% hemo-

globin in 0.1 mmol l-1 glycine–HCl, pH 2.0. One

unit of enzyme activity was defined as 1 lg tyrosine

released per minute using a coefficient of molar

extinction of 0.008 at 280 nm. Trypsin activity was

assayed using BAPNA (N-a-benzoyl-DL-arginine

4-nitroanilide hydrochloride) as substrate according

to Erlanger et al. (1961). Chymotrypsin activity in

extracts was determined using SAAPNA (N-succinyl-

ala–ala-pro-phe p-nitroanilide) according to DelMar

et al. (1979). Leucine aminopeptidase was deter-

mined using leucine p-nitroanilide (0.1 mmol l-1 in

DMSO) as substrate, according to Maraux et al.

(1973). For trypsin, chymotrypsin, and leucine ami-

nopeptidase activities, one unit of enzyme activity

was defined as 1 lmol p-nitroaniline released per

minute using coefficients of molar extinction of 8.2 at

410 nm. Carboxypeptidase A activity was measured

following the protocol of Folk and Schirmer (1963)

using HPA (hippuryl-L-phenyl-alanine) as substrate

dissolved in 25 mmol l-1 Tris–HCl, 10 mmol l-1

CaCl2 buffer, pH 7.8. One unit of enzyme activity

was defined as 1 lmol of hydrolyzed hippuryl per

minute using a coefficient of molar extinction of 0.36

at 254 nm.

Determination of a-amylase activity was carried out

following the Somoyi-Nelson procedure described by

Robyt and Whelan (1968). One unit of activity was

defined as the amount of enzyme able to produce 1 lg

of maltose per minute at 600 nm. Lipase activity was

quantified using b-naphthyl caprylate as substrate

according to Versaw et al. (1989). One unit of activity

was defined as 1 lg of naphthol released per minute

using a molar extinction coefficient of 0.02 at 540 nm.

Acid and alkaline phosphatases were assayed using

4-nitrophenyl phosphate in acid citrate buffer (pH 5.5)

or glycine–NaOH buffer (pH 10.1) according to

Bergmeyer (1974). One unit was defined as 1 lg of

nitrophenyl released per minute using a molar extinc-

tion coefficient of 18.5 at 405 nm. All assays were

performed by triplicate at 37�C.

Digestive enzyme activities were expressed as

U mg protein-1 and U larva-1 using the total number

of larvae in each homogenized pooled sample.

Electrophoretic analysis

The analysis of the alkaline protease isoforms was

done using sodium dodecyl sulfate–polyacrylamide

gel electrophoresis (SDS–PAGE; 10% polyacryl-

amide) for each larval enzyme preparation in a Mini

Protean II chamber (Bio-Rad) according to Laemmli

(1970) using 8 9 10 9 0.075-cm gels. Zymograms of

alkaline protease activities were obtained as described

by Garcıa-Carreno et al. (1993). Electrophoresis was

carried out during 60 min at a constant voltage of

100 V per gel at 5�C. After electrophoresis, the gels

were washed and incubated for 30 min at 5�C in a 0.5%

casein Hammerstein (Research Organics) solution at

pH 9.0. The gels were then incubated for 90 min in the

same solution at 25�C without agitation. Finally, the

gels were washed and fixed in 12% trichloroacetic acid

(Sigma–Aldrich) prior to staining with 0.1% Coomas-

sie brilliant blue R-250 (Research Organics) in a

solution of methanol-acetic acid (Sigma–Aldrich)-

water (50:20:50). Distaining was carried out in a

solution of methanol-acetic acid–water (35:10:55).

Clear zones, which indicated activity of alkaline

proteases, were visible after 24 h.

The acid protease activities in larval extracts were

analyzed by neutral native polyacrylamide electro-

phoresis (Williams and Reisfeld 1964). All electro-

phoresis procedures were performed at a constant

voltage and amperage (100 V and 64 mA). Acid

protease isoforms were revealed according to the

procedure of Dıaz-Lopez et al. (1998). Same quantity

of protein (30 lg per well) was applied to carry out

each electrophoresis. The gels were removed from

the cell and soaked in 100 mmol l-1 HCl to reach pH

2.0 where the enzymes become active. After 15 min,

the gel was soaked for 30 min at 4�C in a solution

Fish Physiol Biochem (2012) 38:441–454 443

123

Author's personal copy

containing 0.25% hemoglobin in 100 mmol l-1 Gly-

cine–HCl, pH 2.0, and then for 90 min in a fresh

hemoglobin solution at 37�C. The gels were washed

in distilled water and fixed for 15 min in a 12%

trichloroacetic acid solution. When the clear areas of

enzyme activity appeared, the gels were stained using

Coomassie brilliant blue R-250 solution. Destaining

was carried out as mentioned earlier. Clear zones

revealed the activity of acid proteases within a few

minutes although well-defined zones were obtained

only after 2–4 h of staining. A low-range molecular

weight marker (5 ll per well) containing phosphor-

ylase b (97 kDa), bovine serum albumin (66 kDa),

egg albumin (45 kDa), carbonic anhydrase (29 kDa),

trypsinogen (24 kDa), and soybean trypsin inhibitor

(20 kDa) was applied to each SDS–PAGE. The

relative electromobility (Rf) was calculated for all

zymograms (Igbokwe and Downe 1978), and the

molecular weight (MW) of each band in the SDS-

zymograms (alkaline protease) was calculated by a

linearly adjusted model between the Rf and the

decimal logarithm of MW proteins using Quality One

V. 4.6.5 (Hercules, CA) software program.

Statistical analysis

Larval growth was determined with an exponential

model Y = aebX, with logarithm base 10 transformed

data, and the model parameters were calculated by

using the least-squares technique. A Kruskal–Wallis

test was used to compare enzyme activity between

ages for each activity. A nonparametric Nemenyi test

was used when significant differences were detected.

All tests were carried out with Statistica v7.0

(StatSoft, Tulsa, OK, USA) software.

Results

Centropomus undecimalis larvae showed a typical

daily growth rate (0.140 mg day-1) expressed by

weight (Fig. 1a) and 0.078 mm day-1 expressed by

length (Fig. 1b).

For all specific and individual digestive enzyme

activities, statistical differences were detected

(P \ 0.05) when activities were compared between

ages (Fig. 2a–l). Specific alkaline protease was detected

from hatching (1 dah, 1,234.2 ± 0.5 U mg protein-1)

and increased until 3461.5 ± 0.5 U mg protein-1 at 3

dah, decreasing at 7 dah (393.4 ± 0.1 U mg protein-1)

reaching the maximum value at 12 dah (6729.0 ±

0.3 U mg protein-1), to finally decreased (6729.5 ±

0.3 U mg protein-1) up to 36 dah (Fig. 2a). Specific

acid protease activity was detected with low levels

from hatching; it increased slightly at 7 dah

(27.1 ± 0.8 U mg protein-1) remaining with statisti-

cally same values until 12 dah (19.2 ± 0.1 U mg pro-

tein-1), then increased rapidly at 34 dah (92.4 ±

6.0 U mg protein-1), reaching the maximum value at

36 dah (124.3 ± 0.8 U mg protein-1) (Fig. 2c). Spe-

cific trypsin activity showed low activity from hatching

(0.03 ± 0.03 mU 9 10-3 mg protein-1), reaching

two maximum peaks at 7 (0.05 ± 0.01 mU 9

10-3 mg protein-1) and 12 dah (0.05 ± 0.02 mU 9

10-3 mg protein-1), and then, it reduced rapidly at 34

and 36 dah (0.03 ± 0.04 and 0.03 ± 0.01 mU 9

10-3 mg protein-1, respectively) (Fig. 2e). Chymo-

trypsin-specific activity started with low values from

hatching (1.0 ± 0.1 mU 9 10-3 mg protein-1) until

day 5 after hatching (2.7 ± 0.3 mU 9 10-3 mg pro-

tein-1) and increased gradually, reaching the

Fig. 1 a Mean wet weight (lg larva-1 ± SD, n = 3 pooled

larvae), and b total length (mm larvae-1 ± SD, n = 3 pooled

larvae) of common snook larvae

444 Fish Physiol Biochem (2012) 38:441–454

123

Author's personal copy

Fig. 2 Digestive enzyme

activity during common

snook larviculture

(mean ± SD, n = 3 pooled

larvae). a Specific alkaline

protease and b individual

alkaline protease activities,

c specific acid protease and

d individual acid protease

activities, e specific trypsin

and f individual trypsin

activities, g specific

chymotrypsin and

h individual chymotrypsin

activities, i specific

carboxypeptidase A and

j individual

carboxypeptidase A

activities, k specific leucine

aminopeptidase and

l individual leucine

aminopeptidase activities.

R rotifers, AN Artemianauplii, EAMN Enriched

Artemia metanauplii

Fish Physiol Biochem (2012) 38:441–454 445

123

Author's personal copy

maximum activity at 25 dah (3.9 ± 0.2 mU 9

10-3 mg protein-1) and then decreasing rapidly from

this day onwards (2.0 ± 1.0 mU 9 10-3 mg pro-

tein-1 at 36 dah) (Fig. 2g). Carboxypeptidase A-spe-

cific activity was low during the first days of

larviculture around 25.1 ± 0.0 mU mg protein-1,

and increased rapidly at days 7 (128.3 ± 0.3

mU mg protein-1) and 12 days after hatching

(128.1 ± 0.2 mU mg protein-1) to reduce at 25 dah

(47.8 ± 0.2 mU mg protein-1), and increased again at

34 and 36 dah (158.4 ± 0.1 and 278.2 ± 0.2 mU

mg protein-1, respectively) (Fig. 2i). Specific Leucine

aminopeptidase was first detected from hatching

(1 dah, 0.23 ± 0.02 mU mg protein-1); it increased

gradually until it reached the maximum peak of activity

at 12 dah (1.4 ± 0.2 mU mg protein-1) and then

decreased from this day up to the end of the larviculture

at 36 dah (0.60 ± 0.03 mU mg protein-1) (Fig. 2k).

For individual digestive proteases activities, a general

pattern was observed with a gradual increase from the

beginning of the larviculture to reach the maximum

peak at 36 dah. For alkaline protease (Fig. 2b), the

activity started from 20 to 50 mU larva-1, increasing

to reach their maximum values at 34 and 36 dah

(210 ± 3 and 225 ±

1 mU larva-1, respectively), for acid proteases

(Fig. 2d) the lowest values varied from 0.1 to

0.25 mU larva-1 from hatching until 12 dah, reaching

the highest values at 34 and 36 dah (3.5 ± 0.2 and

5.2 ± 0.8 mU larva-1, respectively), trypsin activity

(Fig. 2f) had the lowest values from hatching until 12

dah (0.001–0.004 mU 9 10-3 larva-1), and reaching

the highest values at 34 and 36 dah (0.1 ± 0.0

and 0.01 ± 0.00 mU 9 10-3 larva-1, respectively),

finally, carboxypeptidase A (Fig. 2j) showed the

lowest values from hatching until 25 dah (from 0.3

until 0.4 mU 9 10-3 larva-1), increasing the activity

rapidly until the maximum values at 34 and 36 dah

(5.9 ± 0.1 and 11.0 ± 0.04 mU 9 10-3 larva-1,

respectively). Chymotrypsin individual activity

showed a similar gradual increment as the specific

activity from hatching until 25 dah (0.01–0.05 mU 9

10-3 larvae-1), and reaching the maximum peak

at 34 dah (0.1 ± 0.0 mU 9 10-3 larva-1) and then

decreased rapidly at 36 dah (0.1 ± 0.0 mU 9

10-3 larva-1) (Fig. 2h). Finally, the leucine amino-

peptidase individual activity was different from the

other enzyme patterns; it showed null activity from

hatching up to day 5 after hatching, with a rapid

increase at 7 dah (11.0 ± 0.1 mU 9 10-3 larva-1),

reaching the maximum peak at 12 dah (14.5 ±

0.2 mU 9 10-3 larva-1); and then decreased at 25

dah (10.3 ± 0.2 mU 9 10-3 larva-1), and increasing

again at 34 (13.2 ± 0.1 mU 9 10-3 larva-1) and 36

dah (12.5 ± 0.1 mU 9 10-3 larva-1) (Fig. 2i).

Specific lipase activity showed slight increments at

3 (130.5 ± 0.3 U mg protein-1) and 7 dah (190.2 ±

0.3 U mg protein-1); afterward, the activity

remained low (90–110 U mg protein-1) until 36

dah (480.4 ± 0.3 U mg protein-1) when it increased

rapidly, being this day the peak with the maximum

activity (Fig. 3a). The individual lipase activity

gradually increased its activity from hatching (10 ±

1 mU 9 10-3 larva-1) until 12 dah (1.8 ± 0.1

mU larva-1), increasing its value (3.8 ± 0.9 mU

larva-1) at 12 dah, reaching the maximum value at

34 dah (10.3 ± 0.12 mU larva-1), and finally

decreasing rapidly at 36 dah (1.3 ± 0.1 mU larva-1)

(Fig. 3b). Specific a-amylase activity was present

from day 1 after hatching (0.2 ± 0.0 U mg pro-

tein-1); it increased from day 5 (0.9 ± 0.0

U mg protein-1) until day 12 after hatching (0.9 ±

0.0 U mg protein-1), reaching its maximum value at

7 dah (1.1 ± 0.0 U mg protein-1); the activity sud-

denly decreased from day 25 (0.1 ± 0.0 U mg pro-

tein- 1) until 34 dah (0.1 ± 0.0 U mg protein-1) and

to increase slightly 36 dah (0.5 ± 0.1 U mg pro-

tein-1) (Fig. 3c). For the individual activity of

a-amylase, two peaks were detected; the first one at 7

dah (8.0 ± 0.2 mU larva-1), with a gradual decrease

on the activity until day 34 after hatching (2.1 ±

0.4 mU larva-1) and a rapid increase at 36 dah

(18.2 ± 0.4 mU larva-1) being this the day of max-

imum activity (Fig. 3d). In the case of acid phospha-

tase-specific activity, low values were detected from

hatching until day 12 (0.5 ± 0.1 U mg protein-1);

however, from day 25 after hatching (3.8 ± 0.1

U mg protein-1) the activity increased, reaching the

maximum peak at 34 dah (5.1 ± 0.1 U mg pro-

tein-1) (Fig. 3e). The individual acid phosphatase

activity showed a similar tendency, being the max-

imum peaks obtained at 25 (50.5 ± 1.3 mU larva-1)

and 36 dah (Fig. 3f). Finally, the specific and

individual alkaline phosphatase activities (8–10

U mg protein-1 and 95–183 mU larva-1, respec-

tively) had the same pattern with low values from

446 Fish Physiol Biochem (2012) 38:441–454

123

Author's personal copy

hatching until day 12 after hatching, increas-

ing rapidly at 25 dah (78.1 ± 19.2 U mg protein-1

and 1,052.4 ± 11.8 mU larva-1, respectively) and

decreasing on 34 (27.2 ± 14.8 U mg protein-1 and

253.6 ± 9.5 mU larva-1, respectively) and 36 dah

(26.4 ± 9.5 U mg protein-1 and 322.6 ± 7.3 mU

larva-1, respectively) (Fig. 3g, h).

The zymogram for alkaline proteases showed only

one band from 25 up to 36 dah (26.4 kDa), and the

appearance of one additional active band of 51.6 kDa

at 36 dah (Fig. 4a). The zymogram for acid proteases

showed two active bands, the first band having a Rf

of 0.32, and the second with a Rf of 0.51, both bands

were observed at 34 and 36 dah (Fig. 4b).

Discussion

Growth

Larval culture is considered the bottleneck for

commercial production, especially in marine fish.

For C. undecimalis, the growth rate showed an

exponential value referred to wet weight and total

length, which was slow during the first days of life,

increasing rapidly after the day fifteen after hatching.

Same growth has been observed in several species,

such as Senegal sole (Solea senegalensis), California

halibut (Paralichthys californicus), Atlantic cod

(Gadus morhua), Atlantic halibut (Hippoglossus

Fig. 3 Digestive enzyme

activity during common

snook larviculture

(mean ± SD, n = 3 pooled

larvae). a Specific and

b individual lipase

activities, c specific and

d individual a-amylase

activities, e specific and

f individual acid

phosphatase activities,

g specific and h individual

alkaline phosphatase

activities. R Rotifers, ANArtemia nauplii, EAMNEnriched Artemia meta-

nauplii

Fish Physiol Biochem (2012) 38:441–454 447

123

Author's personal copy

hippoglossus), and spotted sand bass (Paralabrax

maculatofasciatus) (Ribeiro et al. 1999; Alvarez-

Gonzalez et al. 2006, 2008; Kvale et al. 2007), which

has been related with the live food quality and

quantity (mainly rotifers and Artemia nauplii), feed-

ing frequency, weaning period, and morphophysio-

logical changes that are related with digestion

(enzymatic activity) and absorption of nutrients by

enterocytes (Moyano et al. 1996).

Alkaline proteases

The trend observed for alkaline protease-, trypsin-,

and chymotrypsin-specific activities during the first

days of larviculture is similar to that of other

carnivorous fishes like S. lalandi (Chen et al. 2006),

P. californicus (Alvarez-Gonzalez et al. 2006), red

drum, Sciaenops ocellatus (Lazo et al. 2007), and

P. maculatofasciatus (Alvarez-Gonzalez et al. 2008a),

which suggest that these enzymes play an important

role in larval digestion, previous to the stomach

functionality (Moyano et al. 1996). These changes in

the alkaline enzymatic activity agree with Cahu and

Zambonino-Infante (1994), who observed that pan-

creatic enzymes are responsible for most protein

hydrolysis in the gut, which is initiated in the luminal

zone, and then is continued in the parietal cell

epithelium (microvilli), before the absorption of

nutrients by enterocytes.

These variations in the alkaline protease activities

during C. undecimalis larviculture could be associ-

ated to morphophysiological changes and co-feeding

process when rotifers were replaced by Artemia

nauplii. These changes may increase the secretion of

pancreatic zymogens, possibly influenced by the type

and quantity of protein of the feed; also, the first

feeding after yolk absorption may induce the activa-

tion of alkaline proteases such as chymotrypsin that

occurred at 3 dah, which has been detected as the first

alkaline exoprotease that acts in the digestive tract

(Hjelmeland et al. 1983). In contrast, the level of

trypsin in our species was detected early at low level

and increased rapidly at 12 dah. This enzyme has also

been early detected in Senegal sole (Solea senegal-

ensis) and P. olivaceus larvae, even before the first

feeding. Indeed, this enzyme is detected before the

mouth opening, and it is related to the activation of

other pancreatic zymogens and the protein hydrolysis

in the lumen of S. senegalensis and P. olivaceus

(Kurokawa and Suzuki 1996; Saenz et al. 2005). The

differences between trypsin and chymotrypsin activ-

ities observed in C. undecimalis larvae could be

considered as an indicator of nutritional status; for

example, under normal conditions, the proportion of

trypsin increases as a response to protein hydrolysis

demand; however, if the larvae is reared with an

inadequate diet or suffers a feeding restriction, this

proportion decreases because relatively less trypsin is

produced against a constant secretion of chymotryp-

sin (Moyano et al. 1996; Cara et al. 2003).

For carboxipeptidase A in C. undecimalis larvae,

low activity was detected from the embryos until 5

dah, increasing from 7 to 12 dah, decreasing at 25

dah, and reaching its maximum values at 34 and 36

dah. This enzyme has been primarily studied on

adults of common carp, Cyprinus carpio (Cohen et al.

1981), amur catfish, Parasilurus asotus (Yoshinaka

et al. 1985), S. maximus (Munilla-Moran and Stark

1990), and small-spotted catshark, Scyliorhinus

Fig. 4 Zymograms of acid (a) and alkaline digestive proteases

(b) during larval development of common snook. Numbers at

the top of the gel indicate the mean of days after hatching. The

first well indicates LWMM (kDa): 97 Phosphorylase; 66Bovine serum albumin; 45 Ovoalbumin; 29 Carbonic anhy-

drase; 24 Trypsinogen, 20 Trypsin soybean inhibitor

448 Fish Physiol Biochem (2012) 38:441–454

123

Author's personal copy

canicula (Hajjou et al. 1995), it is an important

digestive parietal enzyme that depends on Zn (me-

tallo-protease), and it is produced in the acinar

pancreatic cells to hydrolyze peptides from carboxyl

side (Vendrell et al. 2000). In P. olivaceus larvae has

only been reported procarboxypetidase A as the

major pancreatic enzyme, precursor for peptide

digestion in the intestinal lumen and is synthesized

at the first feeding (Srivastava et al. 2002).

Leucine aminopeptidase activity was detected with

low values in embryos, increasing between 2 and 12

dah, after that, decreasing was observed until the end

of the larviculture. This pattern coincides with turbot,

Scophthalmus maximus (Cousin et al. 1987) and

European sea bass, Dicentrarchus labrax (Cahu and

Zambonino-Infante 1997), where these parietal

enzymes were localized on microvilli of the entero-

cyte as proenzymes and then activated by alkaline

proteases (trypsin and chymotrypsin) to hydrolyze

peptides from the N-terminal amino acid.

Acid proteases (pepsin)

In C. undecimalis larvae, the maximum activity of

acidic protease was observed between 25 and 36 dah,

similarly to what is observed in other marine fish

larvae such as white bream (Diplodus sargus),

common dentex (Dentex dentex), and redbanded

seabream (Pagrus auriga) (Alarcon et al. 1998; Cara

et al. 2003; Moyano et al. 2005). It has been reported

that the appearance of pepsinogen like activity in the

bastard halibut Paralichthys olivaceus larvae at 45

dah (Kurokawa and Suzuki 1996) indicates the

starting of the juvenile stage and the settlement

period. Detection of high peaks of acid protease

activity (pepsin) indicates the presence of a func-

tional stomach (gastric cells and acid chloride

secretion) and could be taken, with other digestive

enzymes, as an indicator of maturation of the

digestive system (Ueberschaer 1993; Baglole et al.

1998; Kvale et al. 2007). In this sense, the change

from an undifferentiated straight tube in larvae to a

differentiated gastrointestinal tract, including the

presence on gastric cells, allows to start the weaning

period (Lazo et al. 2007). However, it is adequate to

complement the information of enzymatic activity

(biochemistry techniques) with the presence of gas-

tric cells through histology, as was done with the

D. dentex (Gisbert et al. 2009). On the other hand,

low activity pepsin activity was detected during the

first days of larviculture (from yolk-sac larvae until 12

dah) and is not necessarily from stomach origin, but it

could be another type of hydrolases (cathepsines),

which work at intracellular level under acidic condi-

tions and could be detected when whole-body

extracts are prepared (Moyano et al. 1996).

Lipase

Lipase activity was detected at 3 dah, increasing

rapidly at 36 dah, in accordance to Green and

McCormick (2001) who suggested that the presence

of these digestive enzymes before hatching is

strongly related with the absorption of nutritional

components of the yolk sac. Specifically for lipase

activity, diverse studies have been conducted through

larval development of marine fish, where the activity

shows two high peeks in a recurrent manner: the first

one occurs at early days of life related to lipid

hydrolysis of the yolk, and the second one when the

digestive system maturation was reached (Oozeki and

Baley 1995). However, these peeks could present

fluctuations, which are strictly related to either

changes in feed supplies and feed enrichment with

lipid emulsifiers (Hoehne-Reitan et al. 2001), as it

was observed in S. senegalensis larvae (Martınez

et al. 1999), in which two peeks of maximum activity

were detected at 7 and 36 dah. It is admitted that lipid

catabolism is performed primarily by esterase action

hydrolyzing fatty acids as energy source in the first

days of life, while true lipase is dependant on colipase

and bile salts, acting over phospholipids and triacyl-

glycerols (van Tilbeurgh et al. 1992); this enzyme is

responsible for releasing highly polyunsaturated fatty

acids and other more complex compounds, generally

observed when maturation of the digestive system

arrives (Ribeiro et al. 1999; Zambonino-Infante and

Cahu 1999; Gawlicka et al. 2000; Sidell and Hazel

2002; Murray et al. 2003; Morais et al. 2005; Gisbert

et al. 2009)

Amylase

Regarding a-amylase activity during ontogeny of

C. undecimalis, maximum values were detected between

5 and 12 dah, and later decreased, in agreement with

to earlier observations for other marine fish species,

where the highest activity was reported before

Fish Physiol Biochem (2012) 38:441–454 449

123

Author's personal copy

hatching and during the first days of life when

the absorption of yolk-sac occurred and was followed

by a subsequent reduction in activity (Cahu and

Zambonino-Infante 1994; Zambonino-Infante and

Cahu 2001). a-amylase activity and expression have

been detected in fish fertilized eggs by biochemical or

molecular approaches (Naz 2008; Darias et al. 2006)

though its function has not been understood at early

stages of fish development (Moyano et al. 1996;

Peres et al. 1996; Martınez et al. 1999; Buchet et al.

2000; Cuvier-Peres and Kestemont 2002). In larvae,

it has been considered as a digestive system matu-

ration indicator as it occurs in mammals with lactase

(Zambonino-Infante and Cahu 1994). Fish larvae

tend to maintain a-amylase at low levels of activity to

utilize carbohydrates in the feed in carnivorous

species (Munilla-Moran and Saborido-Rey 1996;

Alvarez-Gonzalez et al. 2008). Fange and Grove

(1979), Ugolev et al. (1983), and Hidalgo et al.

(1999) have proved that a-amylase activity tends to

be higher with a progressive activity increasing in

herbivorous and omnivorous fish when compared to

carnivorous fish.

Phosphatases

On the other hand, parietal digestive enzymes, such

as the brush border enzyme phosphatases, are very

important because they are responsible for conclud-

ing digestion at intestinal epithelium level besides

helping on hydrolyzed nutrient transport into the

enterocytes (Harpaz and Uni 1999; Smith et al. 2000;

Zambonino-Infante and Cahu 2001). Alvarez-Gon-

zalez et al. (2008) reported that phosphatases have

two main functions: the first one is to hydrolyze

inorganic phosphate used for energy production, and

the second one is to transport nutrients through cell

membranes (absorption process). In this sense,

Gawlicka et al. (2000) reported that when enterocytes

reach their maximum hydrolysis and absorption

capacity, phosphatase activity increases, which is

related to a drop in leucine-alanine peptidase activity

and maturation of enterocytes, as it has been detected

in D. labrax larvae (Peres et al. 1997) and conse-

quently determines the most adequate moment to

carry on the substitution of live prey to artificial feeds

(Ribeiro et al. 2002; Zambonino-Infante and Cahu

2007). In this manner, phosphatases act in the

digestion process facilitating other enzyme actions,

besides initiating the migration of nutrient processes

from the cryptic region to the microvilli border to

promote cell absorption (Copeland 1996).

Zymogram analysis

Zymogram of acid protease activity in C. undecimalis

larvae allowed the detection of two isoforms (0.32

and 0.51 Rf’s) up to 34 dah, which is similar in

juveniles as reported by Concha-Frıas (2008); this

author found that the total acid protease activity was

86% inhibited using pepstatin A in the stomach of

C. undecimalis juveniles. In this sense, pepsin activity

detection through this technique has been reported for

other species such as silk snapper, Lutjanus vivanus

(26.1 kDa active fraction); grunt, Haemulon plumi-

erii (24 kDa active fraction); spotted goatfish,

Pseudupeneus maculatus (24.0 kDa active fraction);

P. maculatofasciatus (one active fraction on 12 dah,

0.65 RF) (Rivera 2003; Rodrıguez 2004; Souza et al.

2007; Alvarez-Gonzalez et al. 2010) in agreement

that these detected enzymes are similar to the swine

pepsin A (35 kDa). However, the detection of a

second acid protease isoform in C. undecimalis larvae

could correspond not only to pepsin A, but also to

pepsin C that was reported for Pacific bluefin tuna

Thunnus orientalis juveniles using a molecular

approach (Tanji et al. 2009). For alkaline protease,

zymogram of C. undecimalis larvae showed only two

isoforms, which apparently correspond to trypsin-like

(51.6 kDa) detected at 36 dah, and chymotrypsin-like

(26.4 kDa) detected at 34 and 36 dah. In this sense,

an early detection using electrophoretical technique

of these isoforms was not possible because of the low

activity in whole-body homogenates until 25 dah,

which was corroborated using biochemical technique.

The molecular masses of active fractions found for

C. undecimalis larvae were similar to those detected

in wild juveniles of the same species (11 cm of total

length) by Concha-Frıas (2008); however, this

researcher found a third high molecular mass isoform

([75 kDa), which could correspond to an aminopep-

tidase. Our results are similar to those reported for

other strictly carnivorous marine fish species such as

the coho salmon (Oncorhynchus kisutch) and chinook

salmon (Oncorhynchus tschawytscha) with two

22 kDa isoforms, Northern bluefin tuna (Thunnus

thynnus) with three isoforms from 16.8 to 26.8 kDa

and spotted sand bass (P. maculatofasciatus) with

450 Fish Physiol Biochem (2012) 38:441–454

123

Author's personal copy

two isoforms of 20.1–56.5 kDa, respectively. They

are different from omnivorous fishes such as S.

aurata (five active fractions ranging 24.5–90 kDa),

D. dentex (eight fractions ranging from 24.5 to

69.5 kDa), pirapita, Brycon orbignyanus (nine frac-

tions ranging from 7 to 70 kDa), blue disk, Symphys-

odon aequifasciata (eight fractions ranging from 19.2

to76.5 kDa), and rohu, Labeo rohita (five fractions

ranging from 20.9 to 69.4 kDa) (Dimes et al. 1994;

Alarcon et al. 1998; Garcıa-Carreno et al. 2002;

Chong et al. 2002; Essed et al. 2002; Chakrabarti

et al. 2006; Alvarez-Gonzalez et al. 2010).

We can conclude that the low activities of alkaline

protease, lipase, a-amylase, and phosphatase activity

detected in C. undecimalis larvae at hatching and

their increment throughout larviculture also the

presence of only two isoforms of alkaline protease

may be indicators of (1) genetic processes, specially

while observing a slight specific activity in starving

larvae during yolk absorption (for example, phos-

pholipids hydrolysis); (2) a response to ingestion

(feeding behavior), at the moment of adding live

feeds (rotifers, Artemia and enriched Artemia meta-

nauplii); and (3) progressive disappearance of the

metabolic function in which a specific enzyme is

involved (lactase in the case of mammals), or a

relative increase in the soluble protein pool in the

organism. Additionally, acidic protease activity,

which starts at 25 dah reaching its maximum activity

at 36 dah, allows us to consider C. undecimalis as a

juvenile focusing on a digestive physiology point of

view from 34 dah onwards, and let us situate this

species as a strictly carnivorous fish larvae, also the

weaning period for this species should be started after

this age.

Acknowledgments This work was made possible thanks to

the Project ‘‘Estudio sobre la fisiologıa digestiva del robalo

blanco Centropomus undecimalis’’ SEP-CONACyT (CB-2006-

1-58931). We thank Claudia Durruty Lagunes and Jaime

Suarez Bautista for their technical assistance. The Consejo

Nacional de Ciencia y Tecnologıa (CONACYT) of Mexico

provided a fellowship grant to the first author Luis Daniel

Jimenez-Martınez.

References

Alarcon FJ, Dıaz M, Moyano FJ, Abellan E (1998) Charac-

terization and functional properties of digestive proteases

in two sparids; gilthead seabream (Sparus aurata) and

common dentex (Dentex dentex). Fish Physiol Biochem

19:257–267

Alvarez-Gonzalez CA, Cervantes-Trujano M, Tovar-Ramırez

D, Conklin DE, Nolasco H, Gisbert E, Piedrahita R (2006)

Development of digestive enzymes in California halibut

Paralichthys californicus larvae. Fish Physiol Biochem

31:83–93

Alvarez-Gonzalez CA, Moyano-Lopez FJ, Civera-Cercedo R,

Carrasco-Chavez V, Ortiz-Galindo J, Dumas S (2008)

Development of digestive enzyme activity in larvae of

spotted sand bass (Palabrax maculatofasciatus). I: bio-

chemical analysis. Fish Physiol Biochem 34:373–384.

doi:10.1007/s10695-007-9197-7

Alvarez-Gonzalez CA, Moyano-Lopez FJ, Civera-Cercedo R,

Carrasco-Chavez V, Ortiz-Galindo J, Nolasco-Soria H,

Tovar-Ramırez D, Dumas S (2010) Development of diges-

tive enzyme activity in larvae of spotted sand bass (Palabraxmaculatofasciatus) II: electrophoretic analysis. Fish Physiol

Biochem 36:29–37. doi:10.1007/s10695-008-92976-4

Alvarez-Lajonchere L, Cequeira RV, Dos Reis M (2002) De-

sarrollo embrionario y primeros estadios larvales del robalo

chucumite, Centropomus parallelus Poey (Pices: Centrop-

omidae) con interes para su cultivo. Hidrobiologica 12(2):

89–100

Anson ML (1938) The estimation of pepsin, trypsin, papain

and cathepsin with hemoglobin. J Gen Physiol 22:79–89

Aviles-Quevedo A, McGregor-Pardo U, Rodrıguez-Ramos R,

Morales-Castro O, Huerta-Bello M, AIizawa M (1995)

Biologıa y cultivo de la cabrilla arenera Paralabrax ma-culatofasciatus (Steindachner, 1868). Secretarıa de Pesca.

Instituto Nacional de la Pesca. JICA. Mexico, p 85

Baglole CJ, Goff GP, Wright GM (1998) Distribution and

ontogeny of digestive enzymes in larval yellowtail and

winter flounder. J Fish Biol 53:767–784

Bergmeyer HV (1974) Phosphatases methods of enzymatic

analysis, vol 2. Academic Press, New York

Bradford MM (1976) A rapid and sensitive method for the

quantization of microgram quantities of protein utilizing the

principle of protein dye binding. Anal Biochem 72:248–254

Brock D, Robinette HR, Heinen J (1992) Culture system for

evaluating live and formulated diets for larval fish. Progr

Fish Cult 54:270–273

Buchet V, Zambonino-Infante JL, Cahu C (2000) Effect of lipid

level in a compound diet on the development of red drum

(Sciaenops ocellatus) larvae. Aquaculture 184:339–347

Cahu CL, Zambonino-Infante JL (1994) Early weaning of sea

bass (Dicentrarchus labrax) larvae with a compound diet:

effect on digestive enzymes. Comp Biochem Physiol 109A:

213–222

Cahu CL, Zambonino-Infante JL (1997) Is the digestive

capacity of marine fish larvae sufficient for compound diet

feeding? Aquacult Int 5:151–160

Cara JB, Moyano FJ, Cardenas S, Fernandez-Dıaz C, Yufera M

(2003) Assessment of digestive enzyme activities during

larval development of white bream. J Fish Biol 63:48–58

Cequeira RV, Brugger AM (2001) Effect of light intensity on

initial survival of fat snook (Centropomus parallelus, Pis-

ces: Centropomidae) larvae. Brazilian Arch Biol Technol

Int J 44(4):343–349

Chakrabarti R, Rathore RM, Kumar S (2006) Study of diges-

tive enzyme activities and partial characterization of

Fish Physiol Biochem (2012) 38:441–454 451

123

Author's personal copy

digestive proteases in a freshwater teleost, Labeo rohita,

during early ontogeny. Aquacult Nutr 12:35–43

Chavez H (1961) Estudio de una nueva especie de robalo del

Golfo de Mexico y redescripcion de Centropomus unde-cimalis (Bloch). Ciencia 2(2):75–86

Chen BN, Jian GQ, Martin SK, Wayne GH, Steven MC (2006)

Ontogenetic development of digestive enzymes in yel-

lowtail kingfish Seriola lalandi larvae. Aquaculture

256:489–501

Chong AS, Hashim R, Chow-Yang L, Ali AB (2002) Partial

characterization and activities of proteases from the

digestive tract of discus fish (Symphysodon aequifasciata).

Aquaculture 203:321–333

Cohen T, Gertler A, Birk Y (1981) Pancreatic proteolytic

enzymes from carp (Cyprinus carpio): 1. Purification and

physical properties of trypsin, chymotrypsin, elastase and

carboxypeptidase B. Comp Biochem Physiol Comp Bio-

chem 69B(3):639–646

Concha-Frıas B (2008) Evaluacion de la capacidad digestiva de

juveniles de Centropomus undecimalis (bloch, 1792) so-

bre diferentes ingredientes proteınicos. Master’s thesis,

Universidad Catolica del Norte de Chile, p 109

Copeland RA (ed) (1996) Structural components of enzymes.

In: Enzymes, a practical introduction to structure, mech-

anism and data analysis. Wiley, New York, pp 35–65

Cousin JCB, Baudin-Laurencin F, Gabaudan J (1987) Ontog-

eny of enzymatic activities in fed y fasting turbot,

Scophthalmus maximus L. J Fish Biol 30:15–33

Cuvier-Peres A, Kestemont P (2002) Development of some

digestive enzymes in Eurasian perch larvae Perca fluvia-tilis. Fish Physiol Biochem 24:279–285

Darias MJ, Murray HM, Gallant JW, Astola A, Douglas SE,

Yufera M, Martınez-Rodrıguez G (2006) Characterization

of a partial a-amylase clone from red porgy (Pagruspagrus): expression during larval development. Comp

Biochem Physiol 143:209–218

DelMar EG, Largman C, Broderick JW, Geokas MC (1979) A

sensitive new substrate for chymotrypsin. Anal Biochem

99:316–320

Dıaz-Lopez M, Moyano-lopez FJ, Alarcon-Lopez FJ, Garcıa-

Carreno FL, Navarrete del Toro MA (1998) Character-

ization of fish acid proteases by substrate-gel electro-

phoresis. Comp Biochem Physiol 121B:369–377

Dimes LE, Garcıa-Carreno FL, Haard NF (1994) Estimation of

protein digestibility. III. Studies on digestive enzyme from

the pyloric caeca of rainbow trout and salmon. Comp

Biochem Physiol 109:349–360

Erlanger B, Kokowsky N, Cohen W (1961) The preparation

and properties of two new chromogenic substrates of

trypsin. Arch Biochem Biophys 95:271–278

Essed Z, Fernandez I, Alarcon FJ, Moyano FJ (2002) Cara-

cterizacion de la actividad proteasa digestiva de atun rojo

Thunnus thynnus (Linnaeus, 1758). Bol Inst Esp Oceanogr

18(1–4):99–107

Fabillo MD, Herrera AA, Abucay JS (2004) Effects of delayed

first feeding on the development of the digestive tract y

skeletal muscles of Nile Tilapia, Oreochromis niloticus L.

In: Proceedings 6th international symposium on Tilapia in

aquaculture Philippine International Convention Center,

Roxas Boulevard, Manila, Philippines, pp 301–315

Fange R, Grove D (1979) Digestion. In: Hoar WS, Randall DJ,

Brett JR (eds) Fish physiology, vol 8. Academic Press,

NY, pp 161–260

Folk JE, Schirmer EW (1963) The porcine pancreatic car-

boxypeptidase a system. J Biol Chem 238:3884–3894

Garcıa-Carreno FL, Dimes LE, Haard NF (1993) Substrate-gel

electrophoresis for composition and molecular weight of

proteinases or proteinaceous proteinase inhibitors. Anal

Biochem 214:65–69

Garcıa-Carreno FL, Albuquerque-Cavalcanti C, Navarrete del

Toro MA, Zaniboni-Filho E (2002) Digestive proteinases

of Brycon orbignyanus (Characidae, Teleostei): charac-

teristics and effects of protein quality. Comp Biochem

Physiol 132B:343–352

Garcıa-Ortega A, Verreth JAJ, Coutteau P, Segner H, Huisman

EA, Sorgeloos P (1998) Biochemical and enzymatic

characterization of decapsulated cysts and nauplii of the

brine shrimp Artemia at different developmental stages.

Aquaculture 161:501–514

Gawlicka A, Parent B, Horn MH, Ross N, Opstad I, Torrinsen

OJ (2000) Activity of digestive enzymes in yolk-sac lar-

vae of Atlantic halibut (Hippoglossus hippoglossus):

indication of readiness for first feeding. Aquaculture

184:303–314

Gisbert E, Sarasquete MC, Willot P, Castello-Orvay F (1999)

Histochemistry of the development of the digestive sys-

tem of Siberian sturgeon during early ontogeny. J Fish

Biol 55:596–616

Gisbert E, Gimenez G, Fernandez I, Kotzamanis Y, Estevez A

(2009) Development of digestive enzymes in common

dentex Dentex dentex during early ontogeny. Aquaculture

287(3):381–387

Gracia-Lopez V, Kiewek-Martınez M, Maldonado-Garcıa M

(2004) Effects of temperature and salinity on artificially

reproduced eggs and larvae of the leopard grouper My-cteroperca rosacea. Aquaculture 237:485–498

Gracia-Lopez V, Rosas-Vazquez C, Brito-Perez R (2006)

Effects of salinity on physiological conditions in juvenile

common snook Centropomus undecimalis. Comp Bio-

chem Physiol 145A(3):340–345

Green BS, McCormick MI (2001) Ontogeny of the digestive

and feeding systems in the anemone fish Amphiprionmelanopus. Environ Biol Fishes 61:73–83

Grier H (2000) Ovarian germinal epithelium and folliculo-

genesis in the common snook, Centropomus undecimalis(Teleostei: centropomidae). J Morphol 243(3):265–281

Grier HJ, Taylor RG (1998) Testicular maturation and regression

in the common snook. J Fish Biol 53(3):521–542

Hajjou M, Smine A, Guerard F, Le Gal Y (1995) Purification

and some properties of a carboxypeptidase B from dogfish

Scyliorhinus canicula. Comp Biochem Physiol 110B(4):

791–798

Harpaz S, Uni Z (1999) Activity of intestinal mucosal brush

border membrane enzymes in relation to the feeding

habits of three aquaculture fish species. Comp Biochem

Physiol 124A:155–160

Hidalgo MC, Urea E, Sanz A (1999) Comparative study of

digestive enzymes in fish with different nutricional habits.

Proteolytic and amylase activities. Aquaculture 170:

267–283

452 Fish Physiol Biochem (2012) 38:441–454

123

Author's personal copy

Hjelmeland K, Huse I, Jorgensen T, Molvik G, Raa J (1983)

Trypsin and trypsinogen as indices of growth and survival

potential of cod (Gadus morhua L.) larvae. Flodevigen

Rapp 3:1–17

Hoehne-Reitan K, Kjorsvik E, Reitan KI (2001) Bile saltde-

pendent lipase in larval turbot, as influenced by density

and lipid content of fed prey. J Fish Biol 58:746–754

Holt JG, Faulk CK, Schwarz MH (2007) A review of the lar-

viculture of cobia Rachycentron canadum, a warm water

marine fish. Aquaculture 268:181–187

Ibarra-Castro L, Duncan NJ (2007) GnRHa-induced spawning

of wild-caught spotted rose snapper Lutjanus guttatus.

Aquaculture 272:737–746

Igbokwe EC, Downe AER (1978) Electrophoretic and histo-

chemical comparison of three strains of Aedes aegypti.Comp Biochem Physiol 60B:131–136

Komar C, Turnbull JF, Roque A, Fajer E, Duncan NJ (2004)

Effect of water treatment and aeration on the percentage

hatch of demersal, adhesive eggs of the bullseye puffer

(Sphoeroides annulatus). Aquaculture 229:147–158

Kunitz M (1947) Crystalline soybean trypsin inhibitor II.

General properties. J Gen Physiol 30:291–310

Kurokawa T, Suzuki T (1996) Formation of the diffuse pan-

creas and the development of digestive enzyme synthesis

in larvae of the Japanese flounder Paralichthys olivaceus.

Aquaculture 141:267–276

Kvale A, Mangor-Jensen A, Moren M, Espe M, Hamre K

(2007) Development and characterization of some intes-

tinal enzymes in Atlantic cod (Gadus morhua L.) and

Atlantic halibut (Hippoglossus hippoglossus L.) larvae.

Aquaculture 264:457–468

Laemmli UK (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature

227:680–685

Lazo JP, Mendoza R, Holt GJ, Aguilera C, Arnold CR (2007)

Characterization of digestive enzymes during larval

development of red drum (Sciaenops ocellatus). Aqua-

culture 265:194–205

Maraux S, Louvard D, Baratti J (1973) The aminopeptidase

from hog-intestinal brush border. Biochim Biophys Acta

321:282–295

Martınez MI, Moyano FJ, Fernandez-Dıaz C, Yufera M (1999)

Digestive enzyme activity during larval development of

the Senegal sole (Solea senegalensis). Fish Physiol Bio-

chem 21:317–323

Morais S, Rojas-Garcıa CR, Conceicao LEC, Rønnestad I

(2005) Digestion and absorption of a pure triacylglycerol

and a free fatty acid by Clupea harengus L. larvae. J Fish

Biol 67:223–238

Moyano FJ, Diaz M, Alarcon FJ, Sarasquete MC (1996)

Characterization of digestive enzyme activity during lar-

val development of gilthead sea bream (Sparus aurata).

Fish Physiol Biochem 15:121–130

Moyano FJ, Barros AM, Prieto A, Canavate JF, Cardenas S

(2005) Evaluacion de la ontogenia de enzimas digestivas

en larvas de hurta, Pagrus auriga (Pisces: Sparidae).

AquaNIC 22:39–47

Munilla-Moran R, Saborido-Rey F (1996) Digestive enzymes

in marine species. I. Proteinase activities in gut from red

fish (Sebastes mentella), seabream (Sparus aurata) and

turbot (Scophthalmus maximus). Comp Biochem Physiol

113B:395–402

Munilla-Moran R, Stark JR (1990) Metabolism in marine

flatfish—VI. Effect of nutritional state on digestion in

turbor, Scophthalmus maximus (L.). Comp Biochem

Physiol 95B(3):625–653

Murray HM, Gallant JW, Perez-Casanova JC, Johnson SC,

Douglas SE (2003) Ontogeny of lipase expression in

winter flounder. J Fish Biol 62:816–833

Naz M (2008) The changes in the biochemical compositions

and enzymatic activities of rotifer (Brachionus plicatilis,

Muller) and Artemia during the enrichment and starvation

periods. Fish Physiol Biochem 34:391–404

Oozeki Y, Baley M (1995) Ontogenetic development of

digestive enzyme activities in larval walleye pollock,

Theragra chalcogramma. Mar Biol 122:177–186

Peres A, Cahu CL, Zambonino-Infante JL, Legall MM,

Quazuguel P (1996) Amylase and trypsin responses to

intake of dietary carbohydrate and protein depend on the

developmental stage in sea bass (Dicentrarchus labrax)

larvae. Fish Physiol Biochem 15:237–242

Peres A, Cahu CL, Zambonino-Infante JL (1997) Dietary

spermine supplementation induces intestinal maturation in

sea bass (Dicentrarchus labrax) larvae. Fish Physiol

Biochem 16:479–485

Ramirez AB, Cerqueira VR (1994) Feeding behavior of young

robalo (Centropomus undecimalis Bloch, 1792). I. The

effect of chemical attractants. Aquaculture 124(1):289–290

Ribeiro L, Zambonino-Infante JL, Cahu C, Dinis MT (1999)

Development of digestive enzymes in larvae of Soleasenegalensis, Kaup 1858. Aquaculture 170:465–473

Ribeiro L, Zambonino-Infante JL, Cahu C, Dinis MT (2002)

Digestive enzymes profile of Solea senegalensis post

larvae fed Artemia and a compound diet. Fish Physiol

Biochem 27:61–69

Rivera SM (2003) Purification and characterization of trypsin

from intestinal and pyloric caecal tissues of the silk

snapper, Lutjanus vivanus (Cuvier 1828). Master’s thesis,

p 40

Robyt JF, Whelan WJ (1968) The a-amylase. In: Radley JA

(ed) Starch and its derivates. Chapman and Hall, London,

pp 430–497

Rodrıguez MAR (2004) Purification and kinetic characteriza-

tion of trypsin from the intestine and pyloric caeca of the

white grunt, Haemulon plumierii, (Lacepede, 1801).

Master’s thesis, p 29

Saenz RM, Alarcon FJ, Martınez MI, Ruiz F, Dıaz M, Moyano

FJ (2005) Caracterizacion de las proteasas digestivas del

lenguado senegales Solea senegalensis Kaup, 1858. Bol

Inst Esp Oceanogr 21(1–4):95–104

Shaozhen F, Wensheng L, Haoran L (2008) Characterization

and expression of the pepsinogen C gene and determina-

tion of pepsin-like enzyme activity from orange-spotted

grouper (Epinephelus coioides). Comp Biochem Physiol

149B:275–284

Sherwood NM, Grier HJ, Warby C, Peute J, Taylor RG (1993)

Gonadotropin-releasing hormones, including a novel

form, in snook Centropomus undecimalis, in comparison

with forms in black sea bass Centropristis striata. Regul

Pept 46(3):523–534

Fish Physiol Biochem (2012) 38:441–454 453

123

Author's personal copy

Sidell BD, Hazel JR (2002) Triacylglycerol lipase activities in

tissues of Antarctic fishes. Polar Biol 25:517–522

Smith TK, Tapia-Salazar M, Cruz-Suarez LE, Ricque-Marie D

(2000) Feed-borne biogenic amines: natural toxicants or

growth promoters? In: Cruz-Suarez LE, Ricque-Marie D,

Tapia-Salazar M, Olvera-Novoa MA, Civera-Cerecedo R

(eds) Avances en Nutricion Acuıcola V. Memorias del V

Simposium Internacional de Nutricion Acuıcola. Merida,

Yucatan, Mexico, pp 24–32, 19–22 Nov

Souza AAG, Amaral IPG, Alberico RES, Carvalho LB,

Bezerra RS (2007) Trypsin-like enzyme from intestine

and pyloric caeca of spotted goatfish (Pseudupeneusmaculatus). Food Chem 100:1429–1434

Srivastava AS, Kurokawa T, Suzuki T (2002) mRNA expres-

sion of pancreatic enzyme precursors and estimation of

protein digestibility in first feeding larvae of the Japanese

flounder, Paralichthys olivaceus. Comp Biochem Physiol

l32A:629–635

Stephen R, Shafland P (1982) Larval development of snook,

Centropomus undecimalis (Pisces: Centropomidae).

Copeia 3:618–627

Tanji M, Yakabe E, Kubota K, Kageyama T, Ichinose M, Miki

K, Ito H, Takahashi K (2009) Structural and phylogenetic

comparison of three pepsinogens from Pacific bluefin

tuna: molecular evolution of fish pepsinogens. Comp

Biochem Physiol 152B:9–19

Tarcisio T, Vinicius R, Brown J (2005) Early weaning of fat

snook (Centropomus parallelus Poey 1864) larvae.

Aquaculture 253:334–342

Ueberschaer B (1993) Measurement of proteolytic enzyme

activity: significance and application in larval fish

research. In: Walther BT, Fyhn HJ (eds) Physiological and

biochemical aspects of fish development, part III. Univ. of

Bergen, Norway, pp 233–239

Ugolev AM, Yegorova VV, Kuz’mina VV, Gruzdkov AA

(1983) Comparative molecular characterization of mem-

brane digestion on fish and mammals. Comp Biochem

Physiol 76B:627–635

van Tilbeurgh H, Sarda L, Verger R, Cambillau C (1992)

Structure of the pancreatic lipase-procolipase complex.

Nature 359:159–162

Vendrell J, Querol E, Aviles FX (2000) Metallocarboxypep-

tidases and their protein inhibitors. Structure, function and

biomedical properties. Biochim Biophys Acta 1477:

284–298

Versaw W, Cuppett SL, Winters DD, Williams LE (1989) An

improved colorimetric assay for bacterial lipase in nonfat

dry milk. J Food Sci 54:232–254

Versichelle D, Leger P, Lavens P, Sorgeloos P (1989) L’util-

isation d’artemia. In: Barnabe G (ed) Aquaculture. Tech-

nique et Documentation, Lavoisier, Paris, pp 241–259

Wainwright PC, Huskey SH, Turingan RG, Carroll AM (2006)

Ontogeny of suction feeding capacity in snook, Centrop-omus undecimalis. J Exp Zool 305(3):246–252

Walter HE (1984) Proteinases: methods with hemoglobin,

casein and azocoll as substrates. In: Bergmeyern HJ (ed)

Methods of enzymatic analysis, vol V. Verlag Chemie,

Weinham, pp 270–277

Williams DE, Reisfeld RA (1964) Disc electrophoresis in

polyacrylamide gels: extension to new conditions of pH

and buffers. Ann N Y Acad Sci 121:373–381

Yanes-Roca C, Rhody N, Nystrom M, Main KL (2009) Effects

of fatty acid composition and spawning season patterns on

egg quality and larval survival in common snook. Aqua-

culture 287(3):335–340

Yoshinaka R, Sato M, Tanaka H, Ikeda S (1985) Some enzy-

matic properties and digestive function of a pancreatic

metalloproteinase in the catfish (Parasilurus asotus).

Comp Biochem Physiol 80B(2):223–226

Zambonino-Infante JL, Cahu C (1994) Development and

response to a diet change of some digestive enzymes in

sea bass (Dicentrarchus labrax) larvae. Fish Physiol

Biochem 12(5):399–408

Zambonino-Infante JL, Cahu CL (1999) High dietary lipid levels

enhance digestive tract maturation and improve Dicen-trarchus labrax larval development. J Nutr 129:1195–1200

Zambonino-Infante JL, Cahu CL (2001) Ontogeny of the

gastrointestinal tract of marine fish larvae. Comp Biochem

Physiol 130C:477–487

Zambonino-Infante JL, Cahu CL (2007) Dietary modulation of

some digestive enzymes and metabolic processes in

developing marine fish: applications to diet formulation.

Aquaculture 268:98–105

454 Fish Physiol Biochem (2012) 38:441–454

123

Author's personal copy