Development of digestive enzymes in larvae of Mayancichlid Cichlasoma urophthalmus

G. Lopez-Ramırez • C. A. Cuenca-Soria • C. A. Alvarez-Gonzalez •

D. Tovar-Ramırez • J. L. Ortiz-Galindo • N. Perales-Garcıa • G. Marquez-Couturier •

L. Arias-Rodrıguez • J. R. Indy • W. M. Contreras-Sanchez • E. Gisbert •

F. J. Moyano

Received: 8 November 2009 / Accepted: 30 August 2010 / Published online: 14 September 2010

� Springer Science+Business Media B.V. 2010

Abstract The development of digestive enzymes

during the early ontogeny of the Mayan cichlid

(Cichlasoma urophthalmus) was studied using bio-

chemical and electrophoretic techniques. From yolk

absorption (6 days after hatching: dah), larvae were

fed Artemia nauplii until 15 dah, afterward they were

fed with commercial microparticulated trout food

(45% protein and 16% lipids) from 16 to 60 dah.

Several samples were collected including yolk-sac

larvae (considered as day 1 after hatching) and

specimens up to 60 dah. Most digestive enzymes

were present from yolk absorption (5–6 dah), except

for the specific acid proteases activity (pepsin-

like), which increase rapidly from 8 dah up to

20 dah. Three alkaline proteases isoforms (24.0,

24.8, 84.5 kDa) were detected at 8 dah using

SDS–PAGE zymogram, corresponding to trypsin,

chymotrypsin and probably leucine aminopeptidase

enzymes, and only one isoform was detected (relative

electromobility, Rf = 0.54) for acid proteases (pep-

sin-like) from 3 dah onwards using PAGE zymo-

gram. We concluded that C. urophthamus is a

precocious fish with a great capacity to digest all

kinds of food items, including artificial diets provided

from 13 dah.

Keywords a-Amylase � Cichlasoma urophthalmus �Lipase � Mayan cichlid � Phosphatase � Zymogram

Introduction

Commercial fish farming in Mexico has focused

mostly on exotic species such as tilapia (Oreochromis

nilotucus), rainbow trout (Oncorhynchus mykiss),

catfish (Ictalurus punctatus), and grass carp (Cten-

opharingodon idella) (SAGARPA 2003); however, in

G. Lopez-Ramırez � C. A. Cuenca-Soria �C. A. Alvarez-Gonzalez (&) � N. Perales-Garcıa �G. Marquez-Couturier � L. Arias-Rodrıguez �J. R. Indy � W. M. Contreras-Sanchez

Laboratorio de Acuicultura Tropical-DACBIOL,

Universidad Juarez Autonoma de Tabasco, Carretera

Villahermosa Cardenas km 0.5, 86139 Villahermosa,

Tabasco, Mexico

e-mail: alvarez_alfonso@hotmail.com

D. Tovar-Ramırez

Centro de Investigaciones Biologicas del Noroeste,

Mar Bermejo 145, Colonia Playa Palo de Santa Rita,

23090 La Paz, Baja California Sur, Mexico

C. A. Cuenca-Soria � J. L. Ortiz-Galindo

Centro Interdisciplinario de Ciencias Marinas

(CICIMAR-IPN), Av. IPN s/n, Col. Playa Palo de Santa

Rita, 23096 La Paz, Baja California Sur, Mexico

E. Gisbert

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

43540 Sant Carles de la Rapita, Tarragona, Spain

F. J. Moyano

Departamento de Biologıa Aplicada, Escuela Politecnica

Superior. La Canada de San Urbano, Universidad de

Almerıa, 04120 Almerıa, Spain

123

Fish Physiol Biochem (2011) 37:197–208

DOI 10.1007/s10695-010-9431-6

recent years, the technological development for the

culture of native species farming has been studied, in

the southeastern region of the country, which has a

great variety of native fish species. Among these, the

Mayan cichlid (Cichlasoma urophthalmus) is one of

the most important economically local fishery

resources, and it reaches high prices in the regional

market. This species is widely distributed from the

river Coatzacoalcos in Mexico, along the Atlantic

coast of Central America south to the river Prinza-

polka in Nicaragua, and it is considered as an

omnivorous species with a preferentially carnivorous

diet (Martınez-Palacios and Ross 1986, 1994; Espin-

osa et al. 1993). A great number of studies have been

carried out on this species with the purpose of

developing its farming technology. As a result of

these investigations, it was possible to control its

reproduction under laboratory conditions almost

throughout the year, the masculinisation of fry for

later out-growing (Real-Ehuan 2003), the larviculture

using Artemia nauplii during the first days and

artificial diets afterward (Domınguez-Palma 1990;

Martınez-Palacios and Ross 1994; Chavez-Sanchez

et al. 2000; Martınez-Palacios and Ross 2004), the

definition of the optimum farming temperature at

26–28�C (Guerrero-Zarate 2007) and the optimum

stocking density in closed recirculation systems at

10 larvae l-1 (Jimenez-Martınez et al. 2009). How-

ever, the production of fish fry usually needs live

food during larviculture, and this does not necessarily

cover the nutritional requirements of this period

(Versichelle et al. 1989). For this reason, many

studies have focused for several years on the

digestive physiology of the fish throughout the initial

ontogeny and have proved that they are able to

assimilate the nutrients in the diet even before the

first exogenous feeding (Zambonino-Infante and

Cahu 1994; Ribeiro et al. 1999; Green and

McCormick 2001; Cara et al. 2003; Fabillo et al.

2004). Some studies include the Siberian sturgeon,

Acipenser baeri (Gisbert et al. 1999), the yellowtail,

Seriola lalandi (Chen et al. 2006), the red sea bream,

Pagrus pagrus (Darias et al. 2006), the Mossambique

tilapia, Oreochromis mossambicus (Ming-Ji and

Chin-Feng 2006), the spotted sand bass, Paralabrax

maculatofasciatus (Alvarez-Gonzalez et al. 2008;

Alvarez-Gonzalez et al. 2010) and the grouper,

Epinephelus coioides (Shaozhen et al. 2008). These

studies emphasize the importance of knowing the

development of the digestive physiology to improve

the feeding protocols of larviculture and to offer a

more suitable food item. The purpose of this paper

was to investigate the activity of several digestive

enzymes during the initial ontogeny of C. urophthal-

mus using biochemical techniques and determine the

most probable time of an early weaning.

Materials and methods

Sampling and farming of larvae

Cichlasoma urophthalmus larvae were obtained from

a single spontaneous spawning of broodstock kept in

1.7 m3 tanks in an open system in the Laboratorio de

Acuicultura Tropical of the Division Academica de

Ciencias Biologicas (DACBIOL), Universidad Juarez

Autonoma de Tabasco (UJAT), Mexico. The larvae

were placed in a 200-l plastic tank in an open system,

at 29�C, with 5.43 mg l-1 of dissolved oxygen and a

pH of 7.4. The water parameters were measured daily

with an oxymeter (YSI 55, California, USA) and a

pH-meter (Denver Instrument UB-10, Denver, Colo-

rado, USA). Food was provided three times per day

(0800, 1200 and 1800 h) ad libitum, using Artemia

nauplii (INVE Aquaculture Nutrition) from the time

of yolk absorption (6 dah) to 15 dah; afterward, Silver

Cup trout artificial feed (45% protein and 16% lipids)

was provided after grinding, taking into consideration

the oral overture of the larvae up to 60 dah.

A variable number of individuals (approximately

from 400 embryos to 100 larvae) were collected with

a net of approximately 500-lm mesh size. Sampling

took place on days 1, 3, 6, 8, 11, 13, 15, 17, 19, 21,

24, 27, 30, 33, 36, 39, 42, 45, and 60 dah. The

specimens were anesthetized at a low temperature,

frozen in liquid nitrogen, and stored at -20�C for

later use. Additionally, samples of 10 larvae were

collected on the same days and preserved in 4%

formalin neutralized with borate to record growth as

individual wet weight on an analytical balance

(Sartorious AG, Gottingen, Germany; precision

1 9 10-4 g).

Biochemical analyses

Whole body homogenate was prepared from eggs

until 11 dah, cutting heads and tails (visceral bulks

198 Fish Physiol Biochem (2011) 37:197–208

123

dissection) to prepare multienzymatic extracts. Each

sample was homogenized in cold (4�C) Tris–HCl

50 mmol l-1, pH 7.5 (15 mg ml-1) and centrifuged

at 16,000g for 15 min at 4�C. The supernatant was

collected and stored at -20�C before the biochemical

analysis. Concentration of soluble protein in samples

was measured according to Bradford (1976), using

bovine serum albumin as a standard.

Alkaline protease activity was measured following

the method established by Walter (1984), using

casein (0.5%) as substrate in Tris–HCl 50 mmol l-1,

pH 9. Acid protease (pepsin activity) was measured

following the technique of Anson (1938) using

hemoglobin (0.5%) as substrate in Glycine–HCl

100 mmol l-1, pH 2. The mixtures were incubated

at 37�C, the reaction was stopped by adding 0.5 ml

20% TCA, and the absorbance of the reaction

products was measured at 280 nm. The unit of

enzymatic activity was defined as 1 lg of tyrosine

liberated per minute, based on the molar extinction

coefficient (MEC) of 0.005 at 280 nm. Trypsin

activity was measured following Erlanger et al.

(1961), at 25�C using BAPNA (N-a-benzoyl-

DL-arginine p-nitroanilide) as substrate in Tris–HCl

50 mmol l-1, pH 8.2 and CaCl2 10 mmol l-1. Chy-

motrypsin activity was measured according to Del-

Mar et al. (1979) method, at 25�C using SAAPNA

(N-succinyl-ala-ala-pro-phe p-nitroanilide) as sub-

strate in DMSO 10 mmol l-1 and Tris–HCl 100

mmol l-1, pH 7.8 and CaCl2 10 mmol l-1. Carboxy-

peptidase A activity was measured using the method

described by Folk and Schirmer (1963) with Hippu-

ryl-L-phenyl-alanine as substrate in 25 mmol l-1

Tris–HCl and NaCl 500 mmol l-1 at pH 7.5. Leucine

aminopeptidase activity was measured with Leucine

p-Nitroanilide in DMSO 0.1 and 50 mmol l-1

sodium phosphate, pH 7.2 at 25�C (Maraux et al.

1973). The reactions in all the above-described

techniques were stopped with 30% acetic acid.

Enzymatic activity was defined as 1 lg of nitroani-

lide released per minute, using the molar extinction

coefficient of 8.8 for trypsin, chymotrypsin and

leucine aminopeptidase at 410 nm.

The a-amylase activity was measured using starch

(2%) as substrate in phosphate-citrate 100 mmol l-1,

NaCl 50 mmol l-1, pH 7.5, at 600 nm as described

by Robyt and Whelan (1968). Lipase activity was

quantified according to Versaw et al. (1989) using

b-naphthyl caprylate (200 mmol l-1) as substrate in

Tris–HCl 50 mmol l-1, pH 7.2 and sodium tauro-

cholate (100 mmol l-1). Incubation lasted 30 min

after which the reaction was stopped with TCA

(0.72 N); fast blue (100 mmol l-1) was added and

ethanol/ethyl acetate (1:1 v/v) was added to clarified.

The activity unit was defined as 1 lg of naphthol

released per minute at 540 nm, with a molar extinc-

tion coefficient of 0.02.

The acid and alkaline phosphatase activities were

estimated using 4-nitrophenyl phosphate as substrate,

in a citric-citrate 100 mmol l-1 at pH 5.5 for the acid

conditions and a NaOH-glycine 100 mmol l-1 buffer

at pH 10.1 for alkaline phosphatase, according to

Bergmeyer (1974). The activity unit was defined as

1 lg of nitrophenyl released per minute at 405 nm.

All the specific and individual activities of the

extracts were determined according to Alvarez-

Gonzalez et al. (2006). Assays were done in triplicate.

The classification of proteases was obtained by

SDS–PAGE electrophoresis with discontinuous gels

for alkaline proteases (Laemmli 1970; Garcıa-

Carreno et al. 1993) and by PAGE under native

conditions for acid proteases (Davis 1964). The

enzymatic extracts were mixed with the sample

buffer (Tris–HCl 500 mmol l-1, pH 6.8, glycerol,

SDS, bromophenol blue) at a v/v ratio of 1:1, and

20 ll of this mixture was applied on the gel wells

(8.3 cm 9 6.1 cm 9 0.75 cm). The discontinuous

zymograms consisted of a storage gel at 4% and a

separating gel at 10%. The gel was equilibrated at

80 V for 15 min, and the electrophoresis was done at

100 V and 120 mA for 100 min (Mini Protean III�

BIORAD� Laboratories, CA, USA). The gels were

submerged in a 2% casein solution in Tris–HCl

50 mmol l-1, pH 9 at 4�C for 60 min to allow the

gels to absorb the casein and then incubated at 37�C

during 18 h to allow the substrate hydrolysis. For

acid protease activities in larval extracts, neutral

native electrophoresis (10% of polyacrylamide) was

performed (Williams and Reisfeld 1964). All elec-

trophoresis procedures were performed at a constant

voltage and amperage (100 V and 64 mA) per gel.

After electrophoresis, gels were revealed for acid

protease isoforms according to the procedure of Dıaz-

Lopez et al. (1998); additionally, porcine pepsin

solution (1 mg ml-1, SIGMA, P-7012, 2500–3500 U

mg-1 of protein) was used as reference enzyme using

a concentration of 30 lg well-1. Gels were removed

from the cell and soaked in 0.1 mol l-1 HCl

Fish Physiol Biochem (2011) 37:197–208 199

123

(SIGMA–Aldrich, 320331) to lower the pH to 2.0 for

the enzymes to become active. After 15 min, the gel

was soaked for 30 min in a solution containing 0.25%

hemoglobin (SIGMA, H-2505) in 0.1 mol l-1 Gly-

HCl (SIGMA–Aldrich, 410225), pH 2.0 and 4�C,

then for 90 min in a fresh hemoglobin solution at

37�C. Gels were washed in distilled water and fixed

for 15 min in 12% TCA solution. After develop-

ment of enzyme activity, gels were stained by using

the same Coomassie brilliant blue R-250 solution.

Destaining was carried out as mentioned earlier.

Clear zones revealed activity of acid proteases within

a few min, although well-defined zones were

obtained only after 2–4 h staining.

Rf and molecular weight calculations

A low molecular weight marker (LRMWM) from

Pharmacia Biotech (Uppsala, Sweden) was applied to

each SDS–PAGE adding 5 ll well-1. The LRMWM

contained the following proteins: phosphorylase b

(97 kDa), serum bovine albumin (66 kDa), egg

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

trypsinogen (24 kDa), and soybean trypsin inhibitor

(20 kDa). The relative electromobility (Rf) for all

zymograms was calculated according to Igbokwe and

Downe (1978), and the molecular weight (MW) of

each band with alkaline protease activity was calcu-

lated as the linear fit between the Rf and the decimal

logarithm of the molecular weights of the proteins

used as markers, using the software Quality One V

4.6.5 (Hercules, CA, USA).

Statistical analyses

Larval growth was evaluated with the model of expo-

nential growth y = aebx, where y = individual fresh

weight, e = exponential base, a = initial weight,

b = growth rate, and x = larval age in days. The param-

eters of the model were evaluated through Mardquat

interactions. After review normality (Kolmogorov–

Smirnof test) and homoscedasticity (Levine test), an

ANOVA was used to compare specific and individ-

ual enzymatic activities between the ages of larvae,

and a post hoc Tukey test was used when signif-

icant differences were detected. All tests were car-

ried out using a 0.05 significance level with the

software StatisticaTM v. 8.0 (Analytical Software, AZ,

USA).

Results

Individual growth during larviculture showed an

exponential trend over the first 60 dah in which the

wet weight (lg larvae-1) varied in close co-linearity

with age (R = 0.93) in accordance with the equation

y = aebx, where a (initial wet weight) had a value of

5.1 and b (growth rate, lg larvae-1 day-1) had a

value of 0.038 (Fig. 1).

Statistical analysis showed significant differences

(P \ 0.05) for all specific and individual digestive

enzymes between ages of larvae as observed in

Figs. 2 and 3. The changes for the protease digestive

enzymatic activities are presented in Fig. 2a–l. The

specific activity of the alkaline protease was first

detected at 11 dah, with maximum values between 13

and 36 dah to suddenly decrease at days 39 and 42

after hatching, increasing at 45 dah to finally

decrease at 60 dah (Fig. 2a). The hydrolysis of the

specific acid protease began at 8 dah, presented

several peaks of high activity at 11, 21, 30, 36, and

45 dah, which had multiple variations along larvi-

culture to finally decrease at 60 dah (Fig. 2c).

Specific trypsin activity was to low from 1 to

11 dah, presented a slightly increase at 13 dah

decreasing again at 17 dah, and reaching the peak

of maximum activity at 24 dah; however, from this

age until 42 dah, the activity gradually decreases,

reaching a second statistically higher peak at 45 dah

and decreasing at 60 dah (Fig. 2e). A slight specific

chymotrypsin activity was observed in the yolk-sac

larvae on the day 3 after hatching, after which it

Fig. 1 Average wet weight (lg larva-1 ± SD, n = 3) of the

Mexican cichlid larvae

200 Fish Physiol Biochem (2011) 37:197–208

123

decreased at 6 dah and then increased gradually until

13 dah, decreasing rapidly at 19 dah until reaching

higher activity values from 21 to 27 dah, decreasing

again at 30 dah, before reaching the maximum

activity at 33 dah, for 36, 39, and 42 dah, lower

values of activity were detected, increasing rapidly at

45 dah and decreasing at 60 dah (Fig. 2g). Specific

carboxypeptidase A was detected early with two

strong statistically maximum peaks at 6 and 13 dah;

however, between 8 and 11 dah, lower activity values

were detected, after 13 dah the activity rapidly

decreases until 27 dah to reach two slightly high

peaks of activity at 30 and 42 dah, decreasing at

45 dah and increasing again at 60 dah (Fig. 2i). Spe-

cific leucine aminopeptidase activity was observed

early at 3 and 8 dah, increasing gradually until the

maximum peak of activity at 13 dah, to suddenly

decrease until 19 dah, then increasing at 21 dah; after

this age, low activity values were detected for 24, 27,

30, 36, and 36 dah, to reach a high peak of activity at

Fig. 2 Digestive enzymatic

activity of proteases during

initial ontogeny of the

Mayan cichlid

(average ± SD, n = 3).

a Specific and b individual

activities of alkaline

protease, c specific and

d individual activities of

acid protease, e specific and

f individual activities of

trypsin, g specific and

h individual activities of

chymotrypsin, i specific and

j individual activities of

carboxypeptidase A,

k specific and l individual

activities of leucine

aminopeptidase

Fish Physiol Biochem (2011) 37:197–208 201

123

39 dah, decreasing gradually until the end of the

study (Fig. 2k). The individual activities of the

alkaline and acid proteases, trypsin, and chymotryp-

sin (Fig. 2b, d, f, h) presented similar patterns,

showing low activity values from yolk-sac larvae

until 11 dah, increasing gradually up to a maximum

value at 45 dah, and decreasing at 60 dah. Individual

carboxypeptidase A activity had lower values from

yolk-sac larvae until 27 dah, reaching two peaks at 30

and 42 dah, and reaching it maximum activity value

at 60 dah (Fig. 2j). In contrast with the other

activities, the individual leucine aminopeptidase had

low activity values from yolk-sac larvae until 8 dah,

increasing gradually to reach it maximum value at

39 dah and decrease markedly at days 42, 45, and 60

after hatching (Fig. 2l).

Specific and individual lipase activities had the

same pattern where measurable low activities were

detected from day 13 after hatching until 42 dah,

after which it rapidly increased at 45 dah until

reaching the maximum peak at 60 dah (Fig. 3a, b).

The specific a-amylase activity was low from yolk-

sac larvae (1 dah) until 17 dah, afterward three points

of high activity were found (21, 33 and 45 dah), and

no further change in the activity was detected up to

60 dah (Fig. 3c). The individual activity of the aamylase (Fig. 3d) presented two peaks of maximum

activity (24 and 36 dah), decreasing at 39 and 42 dah,

to increase its value at 45 dah and decrease again at

60 dah.

The specific activity of the acid phosphatase

(Fig. 3e) was detected at 3 dah, reaching the maxi-

mum activity at 17 dah, decreasing at 19 dah,

increasing again at 21 dah with a slight fall between

the days 24 and 27 after hatching, increasing again at

30 dah, to finally decrease until the end of the study.

Individual acid phosphatase activity (Fig. 3f) was

first detected, to at 11 dah, increasing gradually with

Fig. 3 Digestive enzymatic

activity during initial

ontogeny of Mayan cichlid

(average ± SD, n = 3).

a Specific and b individual

lipase activities, c specific

and d individual a-amylase

activities, e specific and

f individual alkaline

phosphatases activities,

g specific and h individual

acid phosphatases activities

202 Fish Physiol Biochem (2011) 37:197–208

123

a drop of the activity at 19 dah and increase again at

21 dah to reach its maximum activity between days

39 and 45 after hatching to decrease at 60 dah.

Specific alkaline phosphatase activity started at

3 dah, reaching the first high peak between 13 and

15 dah and the highest peak at 21 dah, decreasing

rapidly onwards (Fig. 3g). Individual alkaline phos-

phatase activity was first detected at 11 dah having a

gradual increase in the activity until reaching three

peaks of high activity at 21 (the peak with the highest

activity), 45, and 60 dah (Fig. 3h).

The SDS–PAGE zymogram for alkaline proteases

showed one band that appeared at 0 dah (24.8 kDa), a

second band that was detected at 3 dah (24.0 kDa),

and a third band that was observed at 8 dah

(84.5 kDa), three bands were present until the end of

the larviculture; however, the intensity of these bands

increased gradually until 21 dah. (Fig. 4a). The PAGE

technique under native conditions for the detection of

acid proteases allowed the observation of one band

with an rf of 0.52 from 3 dah onwards that remained

present throughout the development of the larvae, and

increasing its intensity until 21 dah (Fig. 4b).

Discussion

Trypsin and chymotrypsin of C. urophthalmus were

active since yolk-sac larvae (0 dah) onwards,

although levels were very low and became stronger

from 13 dah on, which has also been reported for

species such as California halibut Paralichthys cal-

ifornicus (Alvarez-Gonzalez et al. 2006), S. lalandi

(Chen et al. 2006) and red drum Sciaenops ocellatus

(Lazo et al. 2007). Both enzymes have similar amino

acid structure although trypsin favors basic residues

like lysine and arginine; meanwhile, chymotrypsin

favors aromatic residues like phenylalanine, tyrosine,

and tryptophan (De Haen et al. 1975). In this sense,

trypsin has been reported as the most responsible for

the alkaline proteolytic activity in P. maculatofasci-

atus larvae (Alvarez-Gonzalez et al. 2008), in con-

trast with our results where most of the alkaline

proteolysis was represented by chymotrypsin type.

This is explained because the chymotrypsin has been

reported as a part of omnivorous and herbivorous fish

rather than the trypsin type, whereas the opposite

occurs in carnivorous fish (Jonas et al. 1983), which

has been detected in anchovy (Engraulis japonicus)

and it is responsible for more protein hydrolysis than

trypsin (Heu et al. 1995), also has been detected as

the main intestinal proteinases of fish bentho- and

planktophages (Kuz’mina and Ushakova 2007), that

could be related to the different food items that

freshwater fish prefer. On the other hand, trypsin has

also been detected more frequently during the change

from larval to juvenile stages, while chymotrypsin

compensates the lack of a functional stomach during

Fig. 4 Zymograms of digestive proteases during larval devel-

opment of Mayan cichlid. a SDS–PAGE for alkaline proteases.

M: Marker of molecular weights. Serum bovine albumin

(66 kDa); egg albumin (43 kDa); carbonic anhydrase (29 kDa);

trypsinogen (24 kDa); soybean trypsic inhibitor (20 kDa).

b PAGE for acid proteases

Fish Physiol Biochem (2011) 37:197–208 203

123

the first days of life in fish larvae (Kolkovski 2001).

Thus, trypsin activity was early detected between 8

and 11 dah, which showed the functionality of the

exocrine pancreas and become more important at

13 dah, reaching the strongest value at 24 dah,

coinciding with the ending of the larval stage and

the beginning of the juvenile stage and the maturation

of the enterocite and the increment of the alkaline

phosphatase activity (Cahu and Zambonino-Infante,

1995). It has also been observed in the group

of prickleback fish that includes the species Cebid-

ichthys violaceus, Xiphister mucosus, Xiphister

atropurpureus, and Anoplarchus purpurescens, two

herbivores and two carnivores, respectively (Chan

et al. 2004).

The exopeptidases (leucine aminopeptidase and

carboxypeptidase A) were present in low levels in

yolk-sac larvae until total absorption of yolk

(5–6 dah). They are considered important in the

digestion of proteins where they liberate free amino

acids from either the carboxylic or amino terminal

side of the proteins during the initial development of

two pleuronectiforms, the yellowtail flounder Pleu-

ronectes ferruginea and the Winter flounder Pseu-

dopleuronectes americanus (Baglole et al. 1998). The

Nile tilapia (Oreochromis niloticus) also presents

L-aminopeptidase and bipeptidase IV at the moment

of hatching (Tengjaroenkul et al. 2002), and their

presence has been correlated with the maturation of

the enterocytes (Cahu and Zambonino-Infante, 1995;

Gawlicka et al. 1995). The general activity of the

alkaline protease was clearly detected from day 13

until 36 dah. The presence at this period, of most

pancreatic enzymes associated with nutrients hydro-

lysis, indicates that the stomach becomes functional

(Cahu and Zambonino-Infante 1994; Baglole et al.

1998; Kvale et al. 2007).

The active secretion of chlorhydric acid and

pepsinogen from glandular cells indicates the differ-

entiation of the stomach primordium, as occurs in the

tilapia O. mossambicus approximately at 20–30 dah

(Chan et al. 2004; Ming-Ji and Chin-Feng 2006).

Whereas the Japanese flounder Paralichthys olivaceus

pepsinogen-secreting stomach glands appears around

the time of metamorphosis, at 45 dah (Kurokawa and

Suzuki 1996). However, the presence of gastric glands

does not imply they are functional, as does pepsin

activity during the first days of life of the larvae

(Ueberschaer 1993). Douglas et al. (1999) suggested

that the stomach of winter flounder, Pleuronectes

americanus, is not functional until the onset of

metamorphosis through an examination of pepsin

activity, structural development of the stomach and

gene expression of pepsinogen and the gastric proton

pump. Thus, an important specific activity of the acid

protease in C. urophthalmus larvae was observed from

8 dah (2 days after yolk absorption) onwards, and the

highest activity level after 21 dah, similar to reports of

O. mossambicus (Ming-Ji and Chin-Feng 2006), could

be an indicator to establish of a complete gastric cells

functionality and the entire stomach development.

The specific highest activities of the trypsin and acid

protease in C. urophthalmus (24 and 21 dah respec-

tively), agrees with reports in which a high trypsin

activity (at 22 dah) is used as an indicator of stomach

functionality and of an increase in the capacity to

assimilate nutrients in the intestine (Lazo 1999; Lazo

et al. 2007). However, this may also be understood

considering the joined action of pepsin in the stomach

and trypsin (accompanied by chymotrypsin) in the

gut, which improves the process of fine hydrolysis of

nutrients and the absorption in the intestine (Ming-Ji

and Chin-Feng 2006). This is relevant as several

authors have indicated that the functionality of the

stomach is a first sign for inert feeding to start

(Lazo et al. 2007).

The zymograms obtained from C. urophthalmus

homogenates indicated the presence of three alkaline

proteolytic active bands. One of these has a high

molecular weight of approximately 84 kDa and

probably corresponds to the aminopeptidase type

digestive enzyme. It has been also detected in the

gilthead seabream (Sparus aurata), in the common

dentex (Dentex dentex), and in P. maculatofasciatus

(Alarcon et al. 1998; Alvarez-Gonzalez et al. 2010).

The other two isoforms of medium molecular weight

(24.0 and 24.8 kDa respectively) could correspond

to chymotrypsin and trypsin-like enzymes. These

enzymes have been also detected in the blue discus

(Symphysodon aequifasciata) with a range of molec-

ular weights of 19.2–21.8 kDa for chymotrypsin

(Chong et al. 2002), and in the S. aurata larvae with

values of 23.5–95.0 kDa for trypsin (Moyano et al.

1996). This agrees with that of Romano et al. (2004)

who described the cathepsin D as an intracellular

aspartic protease present in fish that plays the most

important role in the yolk proteins digestion and in

the eggs development. The band detected at 3 dah is

204 Fish Physiol Biochem (2011) 37:197–208

123

probably associated with cathepsin intracellular pro-

tease, which works properly at acid pH, because a

whole larvae homogenate was used. In this sense,

cathepsins D, E, and L have been detected in fish

eggs, and during the development of sea bass

(Dicentrarchus labrax) embryos (Carnevali et al.

2001). On the other band, the increase in intensity in

C. urophthalmus acid band from 8 dah onwards

corresponds to the functional activity of the pepsin as

was observed in other species, which are similar to

porcine pepsin (35 kDa). This enzyme has also

been detected in species such as C. idella with a

molecular mass of 28.5 kDa (Liu et al. 2008). When

compared C. urophthalmus with other similar fish

species such as O. niloticus, an early activity is noted

for alkaline protease in the intestine, which increases

notably at 11 dah and for acid proteases in the

stomach at 8 dah.

Regarding the lipase and a-amylase activities that

were detected on day 13 after hatching, it is

considered that the fish must be able to acquire and

assimilate lipids and carbohydrates after the yolk

sack is absorbed (5–6 dah) (Green and McCormick

2001). Martinez et al. (1999) observed a maximum

lipolytic activity at 10 dah in the sole Solea senegal-

ensis larvae and was correlated to the pancreas

development; whereas Cousin et al. (1987) observed

lipase activity at 15 dah in the turbot Scophthalmus

maximus. In the case of O. niloticus, lipase activity

was detected at 3 dah in the intestine (Tengjaroenkul

et al. 2002) In C. urophthalmus, lipolytic activity was

detected at 13 dah with a marked increase at 45 dah,

which corresponds to an opportunistic carnivorous

species and not an omnivorous fish species such as

O. nilotucus.

Amylase specific activity has been shown to be

high during larval stages and generally decreases

during the development when juvenile stage is

reached (Cahu et al. 2004), which has been used as

an indicator of pancreas maturation. Contrary to this

pattern observed in several carnivorous marine fish,

an increase in specific a-amylase activity of

C. urophthalmus was observed from 21 dah until

reaching the maximum value at 45 dah. Our results

did not indicate a delay of pancreas maturation but

probably associated with the feeding habits of the

species, which present a low capacity to digest the

carbohydrates provided in the diet. As we know, as

omnivorous fish such as O. niloticus show better

digestion rates of starch than opportunistic carnivo-

rous fish (Wilson 1994) such as C. urophthalmus.

Our results suggest that the coincidence of both

increases in lipase and a-amylase activity at 45 dah is

a normal pattern or indicator of final pancreas

maturity in C. urophthalmus. In larval stages, the

lipid catabolism is mediated by esterases and later by

true lipases once the digestive tract is fully devel-

oped. For example, the presence of non-specific

esterases was detected in O. niloticus at hatching

(Tengjaroenkul et al. 2002). Additionally, the pres-

ence of the lipase detected in C. uropthalmus

eleuteroembryo is a common pattern in fish with an

indirect ontogeny, and contributes to the development

up to the juvenile stage (Ozkizilcik et al. 1996;

Ribeiro et al. 2002). The low a-amylase specific

activity and the high digestion rate of lipids could be

an indicator of the carnivorous habits of species, such

as S. lalandi, and indicate that they are capable to

digest lipids and carbohydrates at an early age (Chen

et al. 2006). Thus, a clearly opportunistic carnivorous

feeding habit may be seen in C. urophthalmus, as

both a-amylase and lipase are present with low

activity values from 13 dah onwards.

Phosphatases are hydrolases with a variety of

functions including the hydrolysis of inorganic phos-

phates used to produce energy; the nutrients trans-

portation through membranes into the cells; the

facilitation of enzymatic actions that modify lateral

amino acid chains and enhancing Ca2? absorption,

and transportation from the lumen to the enterocytes.

This type of enzymes has been detected in the

siberian sturgeon (Acipenser baeri) using histochem-

ical techniques previous to hatching (Gisbert et al.

1999). Baglole et al. (1998) reported the presence of

alkaline phosphatase activity in the microvilli of the

enterocytes in P. ferruginea and P. americanus at

3 dah. Alkaline and acid phosphatases have been

detected in common seabream (Pagrus pagrus) from

the time of the opening of the mouth (Cara et al.

2003), whereas in C. urophthalmus, this activity was

recorded after 3 dah. In O. niloticus, a higher activity

of alkaline phosphatases has been found in the four

anterior regions and to a lesser degree in the terminal

region of the intestine (Tengjaroenkul et al. 2000),

indicating that this enzymatic activity increases when

the enterocytes reach their higher capacity of hydro-

lysis and absorption, which is related to a decrease in

the activity of the exopeptidases (such as leucine

Fish Physiol Biochem (2011) 37:197–208 205

123

alanine aminopeptidase) (Copeland 1996). Thus, the

identification of an abrupt increase in phosphatase

activity during the larval stage is the result of the

intestine becoming mature (Ribeiro et al. 2002;

Zambonino-Infante and Cahu 2007). This was

corroborated in this study when both leucine amino-

peptidase and carboxypeptidase A (exopeptidases)

decreased after 13 dah onwards, and the alkaline and

acid phosphatase activities increased synchronically

after this day. In agreement with the aforementioned

and with the activities recorded with the colorimetric

techniques, intestinal absorption in C. urophthalmus

is complete from 17 dah onwards.

The general digestive enzymatic activity on the

third day of sampling, when most of the embryos

hatched, was characterized by the presence of lipase,

a-amylase, and phosphatases (alkaline and acid), as

well as acid protease, trypsin, chymotrypsin and

leucine aminopeptidase, all of which were measur-

able after yolk absorption (5–6 dah). On the sixth day

after hatching, when exogenous feeding with Artemia

nauplii started, carboxypeptidase A type enzymes

appeared with the second highest peak of activity

(13 dah). The change of live food to an artificial diet

coincided with the increase of this digestive enzyme,

and marked an increase in the activity of alkaline

protease, chymotrypsin, leucine aminopeptidase,

a-amylase, and alkaline and acid phosphatases. This

is explained because S. ocellatus, is capable of digest

proteins, lipids and carbohydrates before starting

exogenous feeding as was observed for other species

(Lazo et al. 2007). According to our results, it is

concluded that the best moment to substitute live

food (Artemia) for an artificial diet should be started

at 13 dah, when alkaline and acid proteases, and

lipase activities have increased, although they are

present when yolk absorption is conclude at 3 days

after hatching. For this reason, starting to provide

inert food at an early stage, while verifying both the

level of energetic elements (mainly proteins and

lipids), the size and quality of the ingredients used in

the preparation should guarantee the adequate diges-

tion and assimilation of nutrients at this critical stage

in the farming of C. urophthalmus and a better

growth and survival.

Acknowledgments First author thanks to the Programa

Estatal de Nuevos Talentos, of the Consejo de Ciencia y

Tecnologıa, of the state of Tabasco for the fellowship grant.

This study was financed by FOMIX CONACYT-Government

of the State of Tabasco, through the research project TAB-

2005-C06-16260 and partial funding for this research was

provided by the Aquaculture Collaborative Research Support.

Program accession number # 1373. The Aquaculture CRSP is

funded in part by United States Agency for International

Development (USAID) Grant No. LAG-G-00-96-90015-00 and

by participating institutions. The opinions expressed herein are

those of the author(s) and do not necessarily reflect the views

of the US Agency of International Development.

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