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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: mggc@hp.fciencias.unam.mx (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.
References
Alava, V.R., Pascual, F.P., 1987. Carbohydrate requirements of P. monodon
(Fabricius) juveniles. Aquaculture 61, 211–217.
Al-Mohanna, S.Y., Nott, J.A., 1989. Functional cytology of the hepato-
pancreas of Penaeus semisulcatus (Crustacea: Decapoda) during moult
cycle. Mar. Biol. 101, 535–544.
G. Gaxiola et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 29–3938
Alvarado, F., Robinson, W.J.L., 1979. A kinetic study of the interaction
between amino acids and monosaccharides at the intestinal brush-
border membrane. J. Physiol. 29, 457–475.
Aquacop, Bourgeois, B., Cuzon, G., 1975. Determination des stades
d’intermue chez Macrobrachium rosenbergii (Caridae) and Penaeus
merguiensis (Penaeidae). Cnexo/COP, Internal Report, 40 pp.
Arena, L., Gaxiola, G., Cuzon, G., Rosas, C., Soyez, C., van Wormhoudt,
A., Aquacop, 2003. Growth and physiology of juveniles Litopenaeus
vannamei, wild or domesticated under laboratory conditions. J. Shell-
fish Res. 22, 1–11.
Bernfeld, B., 1955. Sur une methode de dosage des amylases. Methods
Enzymol. 1, 149–154.
Boulton, A.P., Higgins, A.K., 1970. Glycolytic activity in crustaceans.
Comp. Biochem. Physiol. 33, 491–498.
Cardenas, M.L., Cornish-Bowden, A., Ureta, T., 1998. Evolution and regu-
latory role of the hexokinases. Biochim. Biophys. Acta 1401, 242–264.
Chuang, N.-N., Yang, B.-C., Lin, K.-S., 1992. Purification and character-
ization of acidic Apha d-mannosidase from the hepatopancreas of the
shrimp Penaeus monodon (Crustacea: Decapoda). J. Exp. Zool. 261,
387–393.
Cousin, M., 1995. Etude de l’utilisation des glucides et du rapport proteins-
energie chez deux especes de crevettes peneides: Penaeus vannamei et
Penaeus stylirostris, 1995. THESE Docteur de L’Institut National
Agronomique Paris-Grignon, France, 199 pp.
Cuzon, G., Guillaume, J., 1997. Energy and protein: energy ratio. In:
D’Abramo, L.R., Conklin, D.E., Akiyama, D.M. (Eds.), Crustacean
Nutrition, Advances in World Aquaculture Society, vol. 6. World
Aquaculture Society, Baton Rouge, LA, pp. 51–70.
Cuzon, G., Cahu, C., Aldrin, J.F., Messager, J.L., Stephan, G., Mevel, M.,
1980. Starvation effect on metabolism Penaous japonicus. Proc.World
Maricult. Soc. 11, 410–423.
Cuzon, G., Rosas, C., Gaxiola, G., Taboada, G., Van Wormhoudt, A., 2001.
Effect of dietary carbohydrates absence on gluconeogenesis enhance in
pre-moult Litopenaeus stylirostris juveniles and pre-adults. Nat. Shell-
fish Assoc. 20, 931–937.
Douglas, S.E., Mandla, S., Gallant, J.W., 2000. Molecular analysis of the
amylase gene and its expression during development in the winter
flounder Pleuronectes americanus. Aquaculture 190, 247–260.
Drach, P., Tchernigovtzeff, C., 1967. Sur la methode de determination des
stades d’intermue et son application generale aux crustaces. Vie Milieu
18, 595–617.
Engel, D.W., Brouwer, M., Mercaldo-Allen, R., 2001. Effects of moulting
and environmental factors on trace metal body-burdens and hemocyanin
concentrations on the American lobster Homarus americanus. Mar.
Environ. Res. 52, 257–269.
Fernandez-Gimenez, A.V., Garcıa-Carreno, F.L., Navarrete del Toro, M.A.,
Fenucci, J.L., 2001. Digestive proteinases of red shrimp Penaeus
stylirostris (Decapada, Penaeoidea) partial characterization and relation-
ship with molting. Comp. Biochem. Physiol., B 130, 331–338.
Fields, J.H.A., 1985. A note on anaerobic metabolism in Callinectes
sapidus during intermolt cycle. J. Crust. Biol. 5, 242–248.
Gallou, S., 1977. Quantifications enzymatiques et caracterisation d’un
fragment de cDNA de la PEPCK chez Penaeus stylirostris. Rapport
IUT Quimper. 27 pp.
Grossbard, L., Schimke, R.T., 1966. Multiple hexokinases of rat tissues.
J. Biol. Chem. 241, 3546–3560.
Hochachka, PW., Somero, G.N., 1973. Water and solute problems.
Strategies of Biochemical Adaptation. Saunders, UK, pp. 97–143.
Iynedjian, P.B., 1993. Mammalian glukokinase and its gene. Biochem.
J. 293, 1–13.
Le Chevalier, P., Van Wormhoudt, A., 1998. Alpha glucosidase from the
hepatopancreas of the shrimp, Penaeus vannamei (Crustacea–Decap-
oda). J. Exp. Zool. 280, 384–394.
Le Priol, Y., 1999. Influence des regimens riches en glucides sur le
metabolisme et l’activite des enzymes digestives chez la crevette
Penaeus vannamei . These de Diploma. Universite de Bretagne
Occidentale IUT de Quimper. France, 23 pp.
Lehnert, S.A., Johnson, S.E., 2002. Expression of hemocyanin and
digestive enzyme messengere RNAs in the hepatopancreas of the black
tiger shrimp Penaeus monodon. Comp. Biochem. Physiol., B 133,
163–171.
Loret, S.M., Devos, P.E., 1992. Hydrolysis of G-6P by a microsomal
specific phosphatase and glucose phosphorylation by a low Km
hexokinase in the digestive gland of the crab Carcinus maenas.
Variations during the moult cycle. J. Comp. Physiol., B 162, 651–657.
Lowry, O., Rosebrough, N., Farr, A., Randall, R., 1951. Protein measure-
ment with the folin phenol reagent. J. Biol. Chem. 193, 256–275.
McWhinnie, M.A., Corkill, A.J., 1964. The HMP pathway and its
variations in the intermoult cycle in crayfish. Comp. Biochem. Physiol.
12, 81–93.
Meenaski, V.R., Sheer, B.T., 1961. Metabolism of glucose in the crab
cancer magister and Hemigrapsus nudus. Comp. Biochem. Physiol. 3,
30–41.
Mehrani, H., Storey, K.B., 1993. Characterization of a-glucosidases from
rainbow trout liver. Arch. Biochem. Biophys. 306, 188–194.
Muhlia-Almazan, A., Garcıa-Carreno, F.L., 2002. Influence of molting and
starvation on the synthesis of proteolytic enzymes in the midgut gland
of the white shrimp Penaeus vannamei. Comp. Biochem. Physiol., B
133, 383–394.
Needham, A.E., 1957. Factors affecting nitrogen excretion in Carcinus
maenas (Pennant). Physiol. Comp. Oecol. 4, 209–239.
Pascual, P.F., Coloso, R.M., Tamse, C.T., 1983. Survival and some
histological changes in Penaeus monodon Fabricius juveniles fed
various carbohydrates. Aquaculture 31, 169–180.
Rosas, C., Cuzon, G., Gaxiola, G., Arena, A., Lemaire, P., Soyes, C., van
Wormhoudt, A., 2000. Influence of dietary carbohydrate on the
metabolism of juvenile Litopenaeus stylirostris. J. Exp. Mar. Biol.
Ecol. 249, 181–198.
Rosas, C., Cuzon, G., Taboada, G., Pascual, C., Gaxiola, G., van
Wormhoudt, A., 2001a. Effect of dietary protein and energy levels (P/
E) on growth, oxygen consumption, hemolymph and digestive gland
carbohydrates, nitrogen excretion and osmotic pressure of Litopenaeus
vannamei and L. setiferus juveniles (Crustacea Decapoda, Penaeidae).
Aquac. Res. 32, 1–20.
Rosas, C., Cuzon, G., Gaxiola, G., Le Priol, Y., Pascual, C., Rossignyol, J.,
Contreras, F., Sanchez, A., van Wormhoudt, A., 2001b. Metabolism and
growth of juveniles of Litopenaeus vannamei: effect of salinity and
dietary carbohydrates level. J. Exp. Mar. Biol. Ecol. 259, 1–22.
Rosas, C., Cuzon, G., Gaxiola, G., Pascual, C., Taboada, G., Arena, L., van
Wormhoudt, A., 2002. An energetic and conceptual model of the
physiological role of dietary carbohydrates and salinity on Litopenaeus
vannamei juveniles. J. Exp. Mar. Biol. Ecol. 268, 47–67.
Ross-Stevenson, J., 1985. Dynamics of the integument. In: Bliss, D.E. (Ed.),
The Biology of Crustacea, vol. 9. Academic Press, London, pp. 1–42.
Rossignyol, J., 2000. Hexokinase et phosphoenolpyruvate carboxykinase
chez une espece de crevette tropicale: Litopenaeus vannamei .
Memoire de fin d’etudes, Diplome D’etudes approfondies. Ecole
Nationale Superieure Agronomique de Rennes, Universite Rennes I.
France, 22 pp.
Samain, J.F., Hernandorena, A., Moal, J., Daniel, J., LeCoz, J.R., 1985.
Amylase and trypsin activities during artemia development on artificial
axenic media: effect of starvation and specific deletions. J. Exp. Mar.
Biol. Ecol. 86, 255–270.
Santos, E.A., Keller, R., 1993. Crustacean hyperglycemic hormona (CHH)
and the regulation of carbohydrate metabolism: current perspectives.
Comp. Biochem. Physiol., A 106, 405–411.
Schatzkein, F.C., Carpenter, H.M., Rogers, M.R., Sutko, J.L., 1973.
Carbohydrate metabolism in the striped shore crab, Pachygrapsus
crassipes: I. The glycolytic enzymes of gill, hepatopancreas, heart and
leg muscles. Comp. Biochem. Physiol., B 45, 393–405.
Sellos, D., Lemoine, S., Van Wormhoudt, A., 1997. Molecular cloning
of hemocyanin cDNA from Penaeus vannamei (Crustacea, Decap-
oda): structure, evolution and physiological aspects. FEBS Lett. 407,
153–158.
G. Gaxiola et al. / Comparative Biochemistry and Physiology, Part A 140 (2005) 29–39 39
Shiau, S.-Y., 1998. Nutrient requirements of penaeid shrimp. Aquaculture
164, 77–93.
Shiau, S.-Y., Peng, S.-Y., 1992. Utilization of different carbohydrates at
different dietary protein levels in grass prawn, reared in seawater.
Aquaculture 101, 241–250.
Stetten, M.J., Goldsmith, P.K., 1981. Two hexokinases of Homarus
americanus (lobster), one having great affinity for mannose and fructose
and low affinity for glucose. Biochim. Biophys. Acta 687, 468–481.
Thirunavukkarasu, M., Pries, F.G., 1983. Synthesis of a-amylase and a-
glucosidase by membrane bound ribosomes from Bacillus lichen-
iformis. Biochem. Biophys. Res. Commun. 114, 677–682.
vanWormhoudt, A., 1980. Regulation d’activite de l’ a amylase a differentes
temperatures d’adaptation et en fonction de l’ablation des pedoncules
oculaires et du stade de mue chez Palaemon serratus. Biochem. Syst.
Ecol. 8, 193–203.
van Wormhoudt, A., Favrel, P., 1988. Electrophoretic characterization of
Palemon elegans (crustacean: Decapoda) amylase system: study of
amylase polymorphism during intermoult cycle. Comp. Biochem.
Physiol. 89 B, 201–207.
Verri, T., Mandal, A., Zilli, L., Bosa, D., Mandal, P.K., Ingrosso, L., Zonno,
V., Vilella, S., Ahearn, G.A., Storelli, C., 2001. d-Glucose transport in
decapod crustacean hepatopancreas. Comp. Biochem. Physiol., A 130,
585–606.
Wang, D.H., Scheer, B.T., 1963. UDPG–glycogen transglucosylase and a
natural inhibitor in crustacean tissues. Comp. Biochem. Physiol. 9,
263–274.
Zar, J.H., 1996. Biostatistical Analysis. Prentice Hall, Englewood Cliff.
718 pp.