Factorial effects of salinity, dietary carbohydrate and moult cycle on digestive carbohydrases and hexokinases in Litopenaeus vannamei (Boone, 1931

www.elsevier.com/locate/cbpa

Comparative Biochemistry and Physio

Factorial effects of salinity, dietary carbohydrate and moult

cycle on digestive carbohydrases and hexokinases

in Litopenaeus vannamei (Boone, 1931)

Gabriela Gaxiolaa,*, Gerard Cuzonb, Tomas Garcıaa, Gabriel Taboadaa, Roberto Britoc,

Marıa Eugenia Chimala, Adriana Paredesa, Luis Sotod, Carlos Rosasa, Alain van Wormhoudte

aUnidad de Docencia e Investigacion de Sisal, Facultad de Ciencias, UNAM, Puerto de Abrigo, Sisal, Yucatan, cp 97000, MexicobCentre Oceanologique du Pacifique (COP) IFREMER/COP, Tahiti, France

cFacultad de Ciencias Pesqueras, Universidad Autonoma del Carmen, MexicodInstituto de Ciencias del Mar y Limnologıa, UNAM, Mexico

eStation de Biologie Marine du Museum National d’Histoire Naturelle et du College de France, France

Received 31 July 2003; received in revised form 24 October 2004; accepted 26 October 2004

Abstract

Litopenaeus vannamei were reared in close cycle over seven generations and tested for their capacity to digest starch and to metabolise

glucose at different stages of the moulting cycle. After acclimation with 42.3% of carbohydrates (HCBH) or 2.3% carbohydrates (LCBH)

diets and at high salinity (40 g kg�1) or low salinity (15 g kg�1), shrimp were sampled and hepatopancreas (HP) were stored. Total soluble

protein in HP was affected by the interaction between salinity and moult stages ( pb0.05). Specific activity of a-amylase ranged from 44 to

241 U mg protein�1 and a significant interaction between salinity and moult stages was observed ( pb0.05), resulting in highest values at

stage C for low salinity (mean value 196.4 U mg protein�1), and at D0 in high salinity (mean value 175.7 U mg protein�1). Specific activity

of a-glucosidase ranged between 0.09 and 0.63 U mg protein�1, an interaction between dietary CBH and salinity was observed for the a-

glucosidase ( pb0.05) and highest mean value was found in low salinity–LCBH diet treatment (0.329 U mg protein�1). Hexokinase specific

activity (range 9–113 mU mg protein�1) showed no significant differences when measured at 5 mM glucose ( pN0.05). Total hexokinase

specific activity (range 17–215 mU mg protein�1) showed a significant interaction between dietary CBH and salinity ( pb0.05) with highest

value (mean value 78.5 mU mg protein�1) found in HCBH–high salinity treatment, whereas in the other treatments the activity was not

significantly different (mean value 35.93 mU mg protein�1). A synergistic effect of dietary CBH, salinity and moult stages over hexokinase

IV-like specific activity was also observed ( pb0.05). As result of this interaction, the highest value (135.5F81 mU mg protein�1) was

observed in HCBH, high salinity at D0 moult stage. Digestive enzymes activity is enhanced in the presence of high starch diet (HCBH) and

hexokinase can be induced at certain moulting stages under the influence of blood glucose level. Perspectives are opened to add more

carbohydrates in a growing diet, exemplifying the potential approach for less-polluting feed.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Hexokinases; Penaeid shrimp; Glucosidase; Amylase; Carbohydrates metabolism

1. Introduction

After a review of shrimp nutrition, it seems that

carbohydrates (CBH) could be one of the most interesting

1095-6433/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpb.2004.10.018

* Corresponding author. Tel.: +52 9889120147; fax: +57 9889120020.

E-mail address: [email protected] (G. Gaxiola).

nutrients in shrimp diet. Shrimp can digest CBH mainly

disaccharides and starches (Pascual et al., 1983; Alava

and Pascual, 1987; Shiau and Peng, 1992; Shiau, 1998).

However, glucose is not well-tolerated by shrimp, it tends

to be absorbed very fast, peaks in the hemolymph and

then is metabolized through glycolysis or other pathways

(Santos and Keller, 1993). Crustacean CBH metabolism

varies according to stages of the moult cycle, through: (i)

logy, Part A 140 (2005) 29–39

G. Gaxiola et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 29–3930

glycogen synthesis and Embden–Meyerhof oxidation

pathway, dominant in the intermoult period (Wang and

Scheer, 1963); (ii) Pentose shunt increases during

premoult (McWhinnie and Corkill, 1964). A distinct

increase in pentose shunt oxidation in the hepatopancreas

represents an alternative pathway for the oxidation of

hexoses; the major functions of this pathway are

production of NADPH and supply of ribose-5-phosphate

(synthesis of nucleic acids); (iii) chitin synthesis through

the N-acetylglucosamine pathway occurs in late premoult

and early postmoult at the level of the hypodermis

(Meenaski and Sheer, 1961).

Growth and survival assays seem to indicate that starch

provided in the diet can reduce growth and decrease survival

rate. Poor growth could be explained by an influence of

glucose at the intestinal level on amino acid absorption

(Alvarado and Robinson, 1979; Pascual et al., 1983).

Polysaccharides are the best dietary CBH fuel for shrimp

(Pascual et al., 1983; Alava and Pascual, 1987) In previous

studies, a possible effect of a-amylase specific activity on

glucose concentration in the hemolymph was shown in

function of dietary CBH (Rosas et al., 2000) and in shrimp

CBH metabolism is associated with changes in salinity and

dietary carbohydrates (Rosas et al., 2001b, 2002).

Once starch is ingested, two enzymes contribute to its

degradation. a-Amylase (a-1,4-alpha-d-glucan glucanohy-

drolase, EC 3.2.1.1) is responsible for the hydrolysis of a-

1,4 glycosidic bonds in starch and glycogen to form

oligosaccharides, branched a-dextrins, and maltose (van

Wormhoudt, 1980; van Wormhoudt and Favrel, 1988).

These final saccharides are efficiently hydrolyzed by the

complementary action of a-glucosidase (E.C. 3.2.1.20),

sucrase-isomaltase (E.C. 3.2.1.48), and a-dextrinase (E.C.

3.2.1.10); among these enzymes, a-glucosidase is directed

towards exo-hydrolysis of 1,4 a-glucosidic linkages (Le

Chevalier and van Wormhoudt, 1998; Douglas et al., 2000).

A regulatory role of this enzyme in glucose metabolism has

been proposed in crustaceans (Le Chevalier and van

Wormhoudt, 1998).

However, the first and obligatory step for glucose

utilization after sugar transport into the cell is its phosphor-

ylation. This reaction is catalysed by hexokinases (ATP:

hexose 6 phosphotransferases, E.C. 2.7.1.1), a family of

evolutionary and structurally related enzymes present in

eukaryotic cells from yeasts to mammals (Iynedjian, 1993).

Glucose entry into most cells is mediated by facilitated

diffusion, the transporters being part of membrane protein.

Certain hexokinases have been suggested to be involved in

glucose transport into mammalian cells (Cardenas et al.,

1998).

Hexokinase activity in crustacean hepatopancreas has

been shown for shore crab (Schatzkein et al., 1973), crab

(Loret and Devos, 1992), and shrimp (Rosas et al., 2001b).

A hexokinase with low affinity for glucose has been

reported in Homarus americanus (Stetten and Goldsmith,

1981).

The objective of this study is to evaluate possible

changes in the digestion system of dietary carbohydrates

to lead the bio-availability of glucose in the hepatopancreas

of L. vannamei juveniles, due three control factors of the

metabolism; dietary carbohydrates concentration; one hor-

monal: moult cycle; and one environmental: the salinity.

2. Materials and methods

2.1. Experimental design and diets

Effects of dietary carbohydrates and salinity were

assessed during the moult stages of L. vannamei juveniles.

A bi-factorial design of 2�2 (2 for dietary carbohydrates

and 2 for salinity factor) was used. Two hundred juveniles

(4.85 g wet mass) of each treatment were previously adapted

to the feeding regimen and salinity during 8 days, at

28F0.58C, according to Rosas et al. (2001b). Shrimp were

fed three times a day. After acclimation to the feeding

regimen and salinity conditions, 10 shrimp per treatment

were sampled daily during 20 days. The shrimp were

sampled 1 h after feeding to obtain postprandial values for

a-amylase, a-glucosidase, and hexokinases activities,

according to the peak of glucose in the hemolymph (Rosas

et al., 2000). Shrimp were placed in cold seawater prior to

quick dissection of the hepatopancreas. The samples were

immediately frozen in liquid nitrogen and stored at �708Cuntil determination of enzymatic activities. Then, the moult

stage was determined according to the method of Drach and

Tchernigovtzeff (1967) revised by Aquacop et al. (1975) for

penaeids. Intermoult was defined as stage C; premoult

stages were defined as stages D0, D1V, D1j and D2; and

postmoult stages were defined as stages A and B. The wet

mass (ww) of shrimp and hepatopancreas was also recorded

to calculate the hepatosomatic index (HI).

Two isocaloric diets were tested with two levels of CBH

(2.3% and 42.3%, Table 1). The experimental diets were

prepared by thoroughly mixing dry ingredients with oil and

then adding water until a stiff dough resulted. The dough

was then passed though a meat-mincer equipped with a 2

mm die, and the resulting spaghetti-like strands were air

dried at 608C. After drying, the material was broken up into

regular pieces sieved to a convenient pellet size and stored

at �48C.

2.2. Enzymatic analysis

2.2.1. Extract preparation

The hepatopancreas (mean wet mass 0.245 g) was

homogenised at 40C in 600 AL water containing benzami-

dine (6 mM), EDTA (10 mM), iodoacetamide (10 mM),

mercaptoethanol (10 mM) at pH 7.8 (Rossignyol, 2000;

Rosas et al., 2001b). The tissue to buffer ratio was 1:2.

Carbohydrases enzymes activities were measured in crude

tissue preparations.

Table 1

Diet composition (g kg�1)

Ingredients HCBH LCBH

Casein 190 550

Squid meal 200 250

Native wheat starch 440 30

Cod liver oil 80 80

Soybean lecithin 20 20

Cholesterol 2 2

Vitamin premixa 20 20

Rovimix Stay-Cb 20 20

Mineral premixa 10 10

Filling 18 18

Protein (N�6.25) % 30 66

Carbohydrates % 46.3 5.8

Lipids % 9 9

Digestible energy (kJ/g�1)c 18.6 19.0

HCBH, high dietary carbohydrates. LCBH, low dietary carbohydrates.a Vitamin and mineral premix provided by Agribrand de Mexico, S.A.

de CV.b Ascorbyl phosphate (Stay-C-35% Roche).c Digestible energy estimated using the following coefficients: 17.6 kJ

for carbohydrates, 39.5 kJ for lipids and 21.3 kJ for protein according to

Cuzon and Guillaume (1997).

G. Gaxiola et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 29–39 31

2.2.2. Protein measurement

Protein concentration of hepatopancreas was determined

by the method of Lowry et al. (1951) using bovine serum

albumin (BSA) as standard.

2.2.3. Enzyme determinations

a-Amylase activity was measured according to a

modified Bernfeld’s method (1955), using 1.5% glycogen

(Fluka, 50573) as substrate diluted in a 2.5 mM MnCl2,

10 mM NaCl, 10 mM phosphate buffer, at pH 7.

Enzymatic activity was expressed as milligrams of

maltose liberated per min at 378C, according to van

Wormhoudt (1980).

a-Glucosidase (E.C.3.2.1.20) was assayed spectrophoto-

metrically by using p-nitrophenyl-a-d-glucopyranoside

(Sigma-Aldrich, St. Louis, MO, USA) as substrate accord-

ing to Thirunavukkarasu and Pries (1983). The standard

reaction mixture—1 mM substrate in 50 mM sodium

phosphate buffer, pH 6, containing an aliquot of enzyme

solution—was incubated at 378C for 30 min (Le Chevalier

and van Wormhoudt, 1998). The reaction was stopped with

1 M Na2CO3 and absorbance was read at 405 nm. One unit

(U) of enzyme activity is defined as the amount of enzyme

capable of hydrolysing 1 Amol of substrate per min

(e=18,000 L/mol cm�1).

Hexokinase activity was measured in 50 mM HEPES

buffer, pH 7.8, containing 0.05 mM KCl, 10 mM MgCl2, 1

mg mL�1 bovine serum albumin, 10 mM amino caproic

acid, 3.2 mM DTT, and 0.6 mM NAD. After stabilisation of

the curve, 12.5 U of glucose-6-phosphate dehydrogenase

from Leuconostoc mesenteroides (G-6PDH, 2500 U mL�1

Roche) and 5 and 50 mM of d-glucose were added. The

reaction was started using 5 mM ATP and 50 AL of crude

extract. Activity is expressed as moles of NADH formed per

min at 258C per mg of protein (Grossbard and Schimke,

1966; Rossignyol, 2000; Rosas et al., 2001b). This protocol

was used to avoid the variable further reduction of

nucleotide caused by the action of 6-phosphogluconate

dehydrogenase (G-6PDH) when NADP is used with yeast

glucose 6-phosphate dehydrogenase, according to Stetten

and Goldsmith (1981). Glucokinase-like specific activity

was estimated from the difference between the measure-

ments of hexokinase specific activity at 50 mM (total

hexokinase activity) and at 5 mM glucose. Difference

between both glucose concentrations indicates hexokinase

IV-like activity.

2.3. Statistics

A tri-factorial ANOVA 2�2�8 (2 for dietary CBH, 2 for

salinity and 8 for moult stages) was used to determine

significant differences. When significant interaction

between two factors was found, data from the factors were

pooled (placed in the figures) and Duncan’s multiple range

test was used to identify differences between factors that

showed interaction. A one-way ANOVA and Duncan’s

multiple range test was conducted to determine more

precisely differences between the four treatments among

each moult stage. The level of confidence was set at 0.05

(Zar, 1996).

3. Results

Analysis of the results using tri-factorial ANOVA

showed the overall effect of the three assessed factors

(dietary carbohydrates, salinity, and moulting cycle).

3.1. Hepatosomatic index (HI)

In relation to HI, no significant differences ( pN0.05)

between the diet or salinity condition or moult stages were

found (mean value 4.4F0.02%).

3.2. Total soluble protein

The tri-factorial ANOVA showed no significant differ-

ences among treatments for the main factors (dietary CBH,

salinity, and moult stages) ( pN0.05). However, interaction

between salinity and moult stages was significant ( pb0.05)

(Table 2). The highest value of soluble protein was

obtained in moult stage C, with LCBH and high salinity

(Fig. 1).

3.3. a-Amylase

Tri-factorial analysis showed a significant interaction

between salinity and moult stages on a-amylase activity

( pb0.05) (Table 3). Shrimp in stage C with either HCBH

or LCBH diets at low salinity showed the highest value of

Table 2

Total soluble protein (mg mL�1) of the hepatopancreas and three-factorial ANOVA analysis in L. vannamei juveniles at different moulting stages and dietary

carbohydrate–salinity combinations

Carbohydrate and

salinity

Moulting stages

A B1 B2 C D0 D1V D1j D2

HCBH 15 g kg�1 41.9F19 (4) 81.5F21 (3) 126.0F45 (7) 72.0F43 (7) 44.0F8 (5) 65.0F9 (7) 119.6F43 (6) 83.0F26 (3)

LCBH15 g kg�1 81.3F13 (5) 89.8F48 (5) 82.0F25 (4) 59.0F23 (9) 51.0F8 (7) 56.0F7 (5) 65.8F12 (6) 135.0F33 (6)

HCBH 40 g kg�1 62.4F24 (6) 85.8F29 (9) 73.4F11 (5) 83.0F43 (6) 24.0F6 (7) 65.0F12 (6) 54.0F9 (5) 31.9F10 (3)

LCBH40 g kg�1 51.3F9 (5) 82.0F40 (5) 47.6F7 (6) 228.0F134 (4) 105.0F39 (7) 76.0F20 (5) 54.0F8 (6) 35.7F4 (5)

Effect df effect F P

CBH 1 0.50 0.470

Salinity 1 0.02 0.880

Moult Stage 7 1.48 0.170

CBH�Salinity 1 1.15 0.283

CBH�Moult Stage 7 1.40 0.208

Salinity�Moult Stage 7 3.70 0.001*

CBH�Salinity�Moult Stage 7 0.80 0.581

Mean valuesFstandard error, number of observations in parenthesis. HCBH, high dietary carbohydrates. LCBH, low dietary carbohydrates. Salinity in g kg�1.

G. Gaxiola et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 29–3932

a-amylase (mean specific activity 196 U mg protein�1,

Fig. 2). The lowest values were found at high salinity

condition, either with HCBH or LCBH, in moult stages

D1j and D2 (11.7 and 33.8 U mg protein�1, Fig. 2).

3.4. a-Glucosidase

Regarding the specific activity of a-glucosidase, a

significant interaction between carbohydrate content of the

diet and salinity conditions ( pb0.05) was observed (Table

4). The highest mean specific glucosidase activity was 0.33

U mg protein�1 with LCBH diet and low salinity ( pb0.05

(Fig. 3); the lowest mean specific activity was 0.19 U mg

protein�1 obtained in shrimp fed on HCBH diet at low

salinity (Fig. 3).

Fig. 1. Duncan’s multiple range test results after three-factor analysis of

soluble protein content (mg mL�1) in the hepatopancreas of L. vannamei

juveniles, showing the significant interaction between salinity and moult

stages. Different letters indicate significant differences ( pb0.05).

3.5. Hexokinase

The tri-factorial analysis of hexokinase specific activity

using 5 mM glucose as substrate showed no significant

differences ( pN0.05) neither or main factors or in their

interactions (Table 5). However, with total hexokinase

specific activity showed an interaction ( pb0.05) between

dietary carbohydrates and salinity conditions (Table 6). The

highest mean value was obtained with high salinity and

HCBH diet (78.5 mU mg protein�1) (Fig. 4).

3.6. Hexokinase IV-like specific activity

In relation to glucokinase-like specific activity, the tri-

factorial analysis showed a significant effect of dietary

carbohydrates as main factor ( pb0.05), reaching the highest

mean value with the HCBH diet (21.7 mU mg protein�1),

whereas the lowest activity was recorded with LCBH (mean

value 8.5 mU mg protein�1) (Fig. 5). An interaction

between dietary carbohydrates and salinity as well as

interaction between salinity and moult stages and all the

three factors were observed ( pb0.05) (Table 7).

4. Discussion

Our results demonstrate that dietary carbohydrates and

salinity changes affected the digestive carbohydrases and

hexokinase activities during the moult cycle of L. vannamei

juveniles. Results show the omnivorous habit of shrimp in

euryhaline condition and the different metabolic strategies

that juveniles can adopt in relation to environmental and

feeding changes.

In our study, the effect of dietary carbohydrates and

salinity exerted different consequences on the soluble

protein content of hepatopancreas and the activity of

Table 3

a-Amylase specific activity (U mg protein�1) of the hepatopancreas and three-factorial ANOVA analysis in L. vannamei juveniles at different moulting stages

and dietary carbohydrate–salinity combinations

Carbohydrate and salinity Moulting stages

A B1 B2 C D0 D1V D1j D2

HCBH 15 g kg�1 162F62 (4) 99F18 (2) 94F35 (7) 171F52 (7) 127F16 (5) 89F15 (7) 58F14 (6) 66F12 (3)

LCBH15 g kg�1 76F18 (5) 106F29 (5) 110F34 (4) 222F61 (9) 126F16 (7) 123F8 (5) 103F18 (6) 62F17 (6)

HCBH 40 g kg�1 172F64 (6) 188F62 (9) 98F15 (5) 114F36 (6) 241F31 (7) 94F11 (6) 123F28 (5) 169F48 (3)

LCBH40 g kg�1 91F8 (5) 114F37 (5) 124F20 (6) 44F18 (4) 111F32 (7) 94F22 (5) 100F13 (6) 136F34 (5)

Effect df effect F P

CBH 1 1.95 0.160

Salinity 1 0.92 0.340

Moult Stage 7 1.06 0.390

CBH�Salinity 1 3.84 0.050

CBH�Moult Stage 7 0.97 0.454

Salinity�Moult Stage 7 2.43 0.022*

CBH�Salinity�Moult Stage 7 0.47 0.852

Mean valuesFstandard error, number of observations in parenthesis. HCBH, high dietary carbohydrates. LCBH, low dietary carbohydrates. Salinity in g kg�1.

G. Gaxiola et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 29–39 33

carbohydrases and glucose phosphorylation during the

moulting cycle of Litopenaeus vannamei juveniles. Fernan-

dez-Gimenez et al. (2001) and Muhlia-Almazan and Garcıa-

Carreno (2002) already reported an enzymatic adaptation in

crustacean related to the physiological processes of moult-

ing and environmental parameters. Rosas et al. (2000,

2001b) demonstrated that digestive enzyme activities affect

also metabolic rates.

An interesting interaction was evidenced between sal-

inity and moult stages on soluble protein content of the

digestive gland of L. vannamei juveniles (Fig. 1). The lower

soluble protein content of the juveniles maintained in low

salinity conditions during intermoult (C) stage can be related

to the use of protein sources (free amino acid pool, FAA) for

Fig. 2. Duncan’s multiple range test results after three-factor analysis of a-

amylase specific activity in L. vannamei juveniles, showing the significant

interaction between salinity and moult stages. Different letters indicate

significant differences ( pb0.05).

osmotic regulation, whereas the juveniles maintained in

high salinity conditions did not need to use FAA to maintain

their osmotic condition. According to Rosas et al. (2001a), a

decrease in blood osmotic pressure in shrimp acclimated for

30 days to 15 g kg�1 of salinity indicates that extracellular

regulation is not powerful enough to ensure homeo-osmotic

control. The second point of interaction (Fig. 1) is between

D1V and D2, in which the soluble protein of juveniles

maintained at low salinity was higher than in those

acclimated to high salinity. Here the question is related to

the possibility of recycling protein derivates from the FAA

in premoult to maintain the homeosmoticity at the same

time that water uptake begins in the epidermis before

ecdysis (Ross-Stevenson, 1985).

Recently, it was demonstrated that the F cells of the

hepatopancreas are the site of hemocyanin production

(Lehnert and Johnson, 2002), which is the main component

of the hemolimph, accounting for up to 95% of the

hemolymph serum proteins (Sellos et al., 1997). Although

we did not measure hemocyanin concentration in the

hemolymph, we can address the increment of protein

content in the hepatopancreas to the increment of hemo-

cyanin in L. vannamei juveniles as reported by Rosas et al.

(2002) in premoult stages in low salinity conditions. A

synergistic effect of low salinity could be associated to

increased osmotic pressure and hemocyanin concentration

before moulting which would facilitate the uptake of water,

a component of the process of ecdysis, as reported for H.

americanus (Engel et al., 2001).

Regarding the specific activity of a-amylase, the feeding

regimen did not produce a significant difference in the

specific activity as Le Priol (1999) stated for juveniles of

this species. Specific activity of a-amylase was affected

only by the interaction between salinity and moult stages.

This interaction can be visualized between B2 and C moult

stages, during which the highest value of activity was

Table 4

a-Glucosidase specific acitity (U mg protein�1) of the hepatopancreas and three-factorial ANOVA analysis in L. vannamei juveniles at different moulting

stages and dietary carbohydrate–salinity combinations

Carbohydrate

and salinity

Moulting stages

A B1 B2 C D0 D1V D1j D2

HCBH

15 g kg�1

0.11F0.03 (4) 0.09F0.02 (3) 0.16F0.04 (7) 0.43F0.10 (7) 0.27F0.10 (5) 0.20F0.05 (7) 0.11F0.03 (6) 0.13F0.01 (2)

LCBH

15 g kg�1

0.21F0.08 (5) 0.42F0.10 (5) 0.18F0.04 (4) 0.35F0.05 (8) 0.36F0.10 (7) 0.40F0.08 (5) 0.48F0.10 (6) 0.22F0.08 (6)

HCBH

40 g kg�1

0.20F0.08 (6) 0.46F0.20 (9) 0.18F0.06 (5) 0.17F0.06 (6) 0.63F0.10 (7) 0.21F0.05 (6) 0.24F0.02 (5) 0.21F0.02 (3)

LCBH

40 g kg�1

0.28F0.06 (5) 0.32F0.10 (5) 0.30F0.05 (6) 0.12F0.05 (4) 0.24F0.10 (7) 0.21F0.03 (5) 0.39F0.08 (6) 0.35F0.04 (5)

Effect df effect F P

CBH 1 3.59 0.059

Salinity 1 0.44 0.507

Moult stage 7 1.73 0.107

CBH�Salinity 1 5.06 0.026*

CBH�Moult Stage 7 1.84 0.084

Salinity�Moult Stage 7 2.00 0.058

CBH�Salinity�Moult Stage 7 1.49 0.175

Mean valuesFstandard error, number of observations in parenthesis. HCBH, high dietary carbohydrates. LCBH, low dietary carbohydrates. Salinity in g kg�1.

G. Gaxiola et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 29–3934

obtained (196 U mg protein�1) in juveniles maintained in

low salinity conditions, whereas the highest value of

specific activity in high salinity conditions was observed

in moult stage D0 (131.6 U mg protein�1) (Fig. 2). This last

response has already been reported by van Wormhoudt

(1980) for P. serratus, depicting an increment in this activity

starting in D0 and reaching the highest value in D1V.Regarding the premoult period, the a-amylase specific

activity of juveniles in both salinity conditions showed the

same decreasing pattern (Fig. 2), which can be related to the

hormone control of moulting. van Wormhoudt (1980),

studying the effect of temperature and eyestalk ablation in

Fig. 3. Duncan’s multiple range test results after three-factor analysis of a-

glucosidase specific activity in L. vannamei juveniles, showing the

significant interaction between salinity and dietary carbohydrates. Different

letters indicate significant differences ( pb0.05).

P. serratus, concluded that hormonal control exists over

enzymes synthesis and on enzymatic regulation.

The a-glucosidase activity measured at pH 6 can be

addressed based on the results of Mehrani and Storey (1993)

in liver of rainbow trout, who reported that this enzyme

seems to be associated with the lysosomes and is believed to

function to hydrolyze any glycogen or oligosaccharides that

are trapped in lysosomes as a result of cellular autophagy.

Mehrani and Storey (1993) stated that glycogenolysis

can occur in animal tissues by both phosphorolytic

(producing glucose 1-phosphate) and glucosidic (producing

glucose) pathways. The former, involving glycogen phos-

phorylase, is well known and has been well studied,

particularly because of its role in cellular energy metabolism

and its sensitivity to hormonal regulation. Glycogen can

also be hydrolyzed by enzymes that cleave glucose units off

the polymer; these include a-amylase, amylo-1,6-glucosi-

dase, acid a-glucosidase (with an optimal pH 4–5).

Chuang et al. (1992) purified a-glucosidase from

Marsupenaeus japonicus but with an optimal pH of 5,

and 90% of this lysosomal enzyme exist in the hepatopan-

creas of shrimp as a non-membrane-bound monomer. Le

Chevalier and van Wormhoudt (1998) reported an optimal

pH 6, which is the pH used in this work.

The a-glucosidase determined by Le Chevalier and van

Wormhoudt (1998) and Chuang et al. (1992) is located in B

cells (blister-like cells) as an acidic glucosidase. Besides,

glucose transport in the hepatopancreas has been hypothe-

sized to be Na+-dependent and only expressed in certain cell

types (F cells) during certain stages of the moult cycle (Verri

et al., 2001) and B cells are the main site of absorption and

digestion of nutrients (Al-Mohanna and Nott, 1989). In this

study, we observed an interaction between salinity and

Table 5

Hexokinase specific activity (with 5 mM glucose) (mU mg protein�1) of the hepatopancreas and three-factorial ANOVA analysis in L. vannamei juveniles at

different moulting stages and dietary carbohydrate–salinity combinations

Carbohydrate and salinity Moulting stages

A B1 B2 C D0 D1V D1j D2

HCBH 15 g kg�1 44F13 (4) 14F2 (2) 10F3 (5) 26F10 (6) 34F14 (4) 18.5F8 (5) 21F14 (5) 23F14 (3)

LCBH15 g kg�1 12F31 (4) 32F14 (5) 12F9 (2) 72F35 (8) 36F9 (6) 25F7 (4) 7F4 (6) 19F6 (5)

HCBH 40 g kg�1 51F31 (6) 65F36 (7) 19F2 (3) 30F4 (4) 113F43 (4) 38F16 (4) 12F5 (4) 60F19 (3)

LCBH40 g kg�1 31F9 (4) 42F14 (4) 48F12 (6) 17F12 (3) 28F17 (5) 9F6 (3) 36F12 (5) 49F9 (4)

Effect df effect F P

CBH 1 0.69 0.4069

Salinity 1 3.65 0.0585

Moult stage 7 1.05 0.3986

CBH�Salinity 1 1.45 0.2306

CBH�Moult Stage 7 0.83 0.5577

Salinity�Moult Stage 7 0.890 0.5169

CBH�Salinity�Moult Stage 7 1.037 0.4091

Mean valuesFstandard error, number of observations in parenthesis. HCBH, high dietary carbohydrates. LCBH, low dietary carbohydrates. Salinity in g kg�1.

G. Gaxiola et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 29–39 35

dietary carbohydrates on glucosidic activity (Fig. 3), with

induction of specific activity in the LCBH–low salinity

treatment and the lowest activity found in HCBH–low

salinity, whereas no changes were observed with either

HCBH or LCBH in high salinity. This could indicate that in

HCBH–low salinity there was a saturation of the specific

activity of a-glucosidase in response to the excess substrate

produced by the amylase activity. Since in low salinity

nitrogen metabolism is preferentially operating to maintain

the osmotic pressure (Hochachka and Somero, 1973);

probably in low salinity–LCBH conditions, no enough

products were available from starch hydrolysis.

Hexokinase has been measured in several species

(Astacus fluviatilis, Cancer pagurus, Carcinus maenas,

Crangon crangon, and Homarus vulgaris) in a range of 7–

Table 6

Total hexokinase specific activity (with 50 mM glucose) (mU mg protein�1) of

juveniles at different moulting stages and dietary carbohydrate–salinity combinati

Carbohydrate and salinity Moulting stages

A B1 B2 C

HCBH 15 g kg�1 73F29 (4) 15F3 (2) 18F9 (5) 4

LCBH15 g kg�1 26F6 (4) 59F19 (5) 21F5 (2) 9

HCBH 40 g kg�1 79F42 (6) 109F60 (7) 32F5 (3) 4

LCBH40 g kg�1 29F20 (4) 35F9 (4) 54F17 (6) 2

Effect df effect

CBH 1

Salinity 1

Moult stage 7

CBH�Salinity 1

CBH�Moult Stage 7

Salinity�Moult Stage 7

CBH�Salinity�Moult Stage 7

Mean valuesFstandard error, number of observations in parenthesis. HCBH, high

19 AM pyridine nucleotides min�1 mg protein�1 (Boulton

and Higgins, 1970). In L. stylirostris, Gallou (1977) found

12 and 13 AMmg protein�1 with 50 and 5 mM of glucose as

substrate, respectively, concluding that no hexokinase IV-

like specific activity was present. For H. americanus, two

isoforms of hexokinase were evidenced (Stetten and Gold-

smith, 1981), one isoform II with low Km (0.008 mM), low

affinity for glucose and isoform I resembling the hexokinase

IV of vertebrates (Km=6 mM) leading to the assumption of

glucokinase activity. Shrimp possess many similarities with

other omnivorous crustaceans from a metabolic point of

view. Hence, the activity that was measured at high

substrate concentration (50 mM glucose) evidenced a

hexokinase IV-like activity of L. vannamei as in fish.

Shrimp in early premoult stages show high enzymatic

the hepatopancreas and three-factorial ANOVA analysis in L. vannamei

ons

D0 D1V D1j D2

2F11 (6) 20F9 (4) 25F5 (5) 43F17 (5) 20F6 (3)

4F44 (8) 41F11 (6) 30F5 (4) 30F7 (6) 18F6 (5)

2F22 (4) 215F63 (4) 55F23 (4) 17F6 (4) 79F25 (3)

3F14 (3) 29F17 (5) 17F6 (3) 54F14 (5) 50F9 (4)

F P

1.73 0.130

5.41 0.057

1.30 0.483

4.12 0.028*

1.28 0.272

1.35 0.156

1.33 0.086

dietary carbohydrates. LCBH, low dietary carbohydrates. Salinity in g kg�1.

Fig. 4. Duncan’s multiple range test results after three-factor analysis of

hexokinase (50 mM) specific activity in L. vannamei juveniles, showing the

significant interaction between salinity and dietary carbohydrates. Different

letters indicate significant differences ( pb0.05).

Fig. 5. Duncan’s multiple range test results after three-factor analysis of

glucokinase specific activity in L. vannamei juveniles, showing the

significant interaction between dietary CBH, salinity condition and moult

stages Different letters indicate significant differences ( pb0.05).

G. Gaxiola et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 29–3936

activity that could be related with the glycolytic pathway to

derive energy from glucose. Arena et al. (2003) reported that

L. vannamei juveniles fed HCBH (40% starch) depicted a

high glycaemia (7.7 mM glucose) compared to 1.5 mM

glucose with LCBH. Enhanced glycaemia indicates a

phosphorylation of dietary glucose that enters blood

circulation, and there is a combination of an elevated

hexokinase IV-like activity and a positive response of

crustacean hyperglycaemic hormone (CHH) to carbohydrate

intake. In early premoult stages, shrimp have a higher need

for glycogen and energy storage in preparation for large

physiological changes that occurs at each moult (Cuzon et

al., 2001).

From the point of view of the intermediary metabolism,

three major enzymes could be influenced by dietary

carbohydrates: hexokinase, glucokinase, and phosphoenol-

pyruvate kinase (PEPCK).

Similarly, glucose phosphorylation and hexokinase

(HK) measured at 5 mM glucose did not change in L.

vannamei when dietary starch level is increased, whereas

PEPCK activity significantly increased at low dietary

starch level compared to high starch level (4 vs. 2 DO

mg protein�1 min�1, respectively) (Rosas et al., 2001b). In

line with this result, L. stylirostris fed with HCBH or

LCBH and sampled exclusively at D0–D1j stages (Cuzon

et al., 2001) drove their oxidative pathway in the reverse

direction leading to glucose formation (hepatosomatic

index increased significantly with LCBH diet and glycogen

remained unchanged). Hexokinase activity decreased or

remained unchanged in L. vannamei considering premoult

stages (Table 5). There is a complex regulation along the

glycolytic route; furthermore, rate of transfer for 14C

glucose is low in trout, fast in rat, and probably low in

shrimp (although no indication of glucose turnover has

been found) and a role played by futile cycles to increase

metabolic flux has been hypothesized.

The present results give evidence of a synchronism

between a-amylase, a-glucosidase, and hexokinase, rather

dependent on the moult stages than on a variation of a factor

(biotic or abiotic). Diet alteration is particularly revealed at

intermoult stage (B2–C) when animals start feeding which

implies two functions, storage of glycogen and production

of energy through glycolysis.

Shrimp possess enough flexibility at the metabolic level

to adapt to a diet alteration and to cope with a drastic change

in salinity. Priority for growth makes enzymes react to diet

composition. LCBH, independently from the moulting

stages, induces a lower total hexokinase activity because

the neoglucogenic pathway is activated.

4.1. Enzymes and moult cycle: importance of intermoult C

and D0 stages

There is an interesting similarity in the expression of

digestive enzymes (amylase, glucosidase and total hexoki-

nase activity) along the intermoult period that can be

explained by the fact that enzymes would be produced and

expelled in a batch, as a whole (including proteases,

although amylases showed a remarkable resistance to it).

Some secretion granules can be visualized, explaining that

regulation is under poor control. Regulation of enzyme

activity is under hormonal control and an increase in

premoult stages (D0–D2) would come from an increase in

ecdysteroids, as occurs in M. japonicus (Cuzon et al.,

1980).

Variations of hexokinase activity were evidenced in the

hepatopancreas of C. maenas during the moult cycle (Loret

and Devos, 1992). Similar pattern of variations was found in

Table 7

Hexokinase IV like specific activity (mU mg proteın�1) of the hepatopancreas and three-factorial ANOVA analysis in L. vannamei juveniles at different

moulting stages and dietary carbohydrate–salinity combinations

Carbohydrate

and salinity

Moulting stages

A B1 B2 C D0 D1V D1j D2

HCBH15 g kg�1 38.5F20 (3) 0.8F0.8 (2) 8.0F8 (5) 16.0F10 (6) �13.5F17 (4) 6.0F6 (5) 21.5F116 (5) �2.8F8 (3)

LCBH15 g kg�1 14.0F10 (4) 27.0F9 (5) 9.0F4 (2) 22.0F9 (8) 5.0F4 (6) 5.0F2 (4) 23.0F10 (6) �0.4F0.6 (5)

HCBH40 g kg�1 28.0F13 (6) 44.0F26 (7) 13.0F8 (3) 12.5F13 (4) 135.4F81 (3) 17.0F11 (4) 5.0F2 (4) 19.0F7 (3)

LCBH40 g kg�1 �2.0F13 (4) �7.6F12 (4) 6.0F8 (6) 6.0F3 (3) 1.3F1.8 (5) 9.0F1 (3) 19.0F12 (5) 0.9F9 (4)

Effect df effect F P

CBH 1 5.08 0.026*

Salinity 1 1.83 0.178

Moult Stage 7 1.05 0.404

CBH�Salinity 1 8.42 0.005*

CBH�Moult Stage 7 1.69 0.118

Salinity�Moult Stage 7 2.99 0.007*

CBH�Salinity�Moult Stage 7 2.88 0.008*

Mean valuesFstandard error, number of observations in parenthesis. HCBH, high dietary carbohydrates. LCBH, low dietary carbohydrates. Salinity in g kg�1.

G. Gaxiola et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 29–39 37

the present study. The moult cycle exerts a major influence

on enzyme variation during different stages, and the

incidence of dietary glucose and starch exists through a

specific hormonal regulation by food (Samain et al., 1985).

It might be one of the factors determining enzyme activities.

a-Amylase have been measured in Palaemon serratus (van

Wormhoudt, 1980) with a Km of 2–10 mg starch mL�1

when fed a high starch diet vs. 0.5–2 mg starch mL�1 when

fed a low starch diet, indicating a response to a trophic

condition. The significant increase in blood glucose

concentration when L. vannamei juveniles are fed HCBH

diet (1.5 vs. 0.4 g L�1 with LCBH, Arena et al., 2003)

underlines the ability of phosphorylation shown by this

species. Besides, tissue growth is similar in wild L.

vannamei juveniles fed on HCBH or LCBH diet, whereas

decreased in domesticated animals fed on HCBH diet, due

to a loss of allelic frequency on the amylase gene that turns

around 95% (Arena et al., 2003).

If this loss of alleles does not affect digestibility, it could

be hypothesized that enzymes at intermediary metabolism

are depressed in such way as to reduce glucose utilization,

and consequently growth performances when animals are

fed on HCBH diet. As Santos y Keller (1993) pointed out,

the regulation of blood glucose during the postprandial stage

and CBH tend to maintain a high blood level, probably in

relation with a demand for glucose linked with formation of

glucosamine, nucleic acids, and unsaturated fatty acids, as

well as energy demand and glycogen storage.

5. Conclusion

L. vannamei can utilise glucose due to digestive enzymes

activity enhanced in the presence of high starch diet

(HCBH); hexokinase can be induced at certain moulting

stages under the influence of blood glucose level.

From a practical point of view, perspectives are opened

to add more carbohydrates in a growing diet, Cousin (1995)

experienced satisfactory performances in diets with starch

content up to 50%, showing over again that CBH can act as

a significant energy source, since it is known to have a

protein-sparing effect in C. maenas (Needham, 1957),

exemplifying the potential approach for low-pollution feed.

Variations measured in this study were difficult to

dissociate: moult stage examination provides the largest

range of variation. Soluble proteins did not decrease while

salinity decreases. Digestive a-amylase responded to

salinity change from 40 to 15 g kg�1 and to moult stages,

whereas glucosidase activity changed with diet and salinity

alterations.

At the intermediary metabolism level, it was important to

demonstrate (i) induction of hexokinases, and (ii) presence

of hexokinase IV-like activity, an enzyme slightly inducible

by glucose that has been detected previously in Callinectes

sapidus (Fields, 1985) and H. americanus (Stetten and

Goldsmith, 1981).

Acknowledgements

We thank the financial support for projects UNAM IN-

234596 and IN-220502-3, SEP-CONACyT 38193 and

41513-A1. We thank also to Industrias Pecis, S.A. de C.V

by shrimp supplied and Ingrid Mascher for editorial

assistance with the manuscript.

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