An energetic and conceptual model of the

physiological role of dietary carbohydrates and

salinity on Litopenaeus vannamei juveniles

Carlos Rosas a,*, Gerard Cuzon b, Gabriela Gaxiola a,Cristina Pascual a, Gabriel Taboada a, Leticia Arena a,

Alain van Wormhoudt c

aGrupo de Biologıa Marina Experimental, Fac. de Ciencias UNAM, Apdo. Post. 69, Cd. del Carmen,

Campeche, MexicobCentre Oceanologique du Pacifique (COP), IFREMER, BP 700, BP 7004, Taravao, Tahiti, French Polynesia

cStation de Biologie Marine du Museum National d’Histoire Naturelle et du College de France, BP 225,

29900, Concarneau, France

Received 9 July 2001; received in revised form 2 October 2001; accepted 31 October 2001

Abstract

We are reporting results directed to explain the relation between carbohydrates (CHO), protein

metabolism, and the energetic balance of Litopenaeus vannamei juveniles. The interaction of

dietary CHO and salinity was measured to try to understand the relation between osmotic control

and metabolism, both from a biochemical and energetic point of view. Two experiments were

done. In the first experiment, shrimp were fed with 0%, 5%, 33%, and 61% CHO and maintained

at 15xand 40xsalinity. Glucose, lactate protein, hemocyanin, ammonia concentration, and

osmotic pressure were measured in blood. Digestive gland glycogen (DGG) was measured also. In

the second experiment, shrimp were fed with 0% and 38% dietary CHO and maintained at 15xand 40xsalinity. From that shrimp, absorbed energy (Abs) was calculated as: Abs = respiration

(R) + ammonia excretion (U) and production (P); assimilated energy (As) was calculated as the

product of R�P. Osmotic pressure, hemocyanin, protein, lactate, and blood ammonia increased

with the reduction in dietary CHO. In contrast, an increase in blood glucose was observed with an

increase in dietary CHO. Digestive gland glycogen (DGG) increased following a saturation curve

with a DGG maximum at 33% dietary CHO. Blood metabolites of fasting and feeding shrimp

showed the same behavior. Energy balance results showed that shrimp maintained in low salinity

and fed without CHO waste more energy in U production than for shrimp maintained in high

0022-0981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022-0981 (01 )00370 -7

* Corresponding author. Tel.: +52-938-28-730; fax: +52-938-28-730.

E-mail address: crv@hp.fciencias.unam.mx (C. Rosas).

www.elsevier.com/locate/jembe

Journal of Experimental Marine Biology and Ecology

268 (2002) 47–67

salinity and fed with high CHO levels. Notwithstanding, the production efficiency was higher in

shrimp fed without CHO than that observed in shrimp fed with high CHO independent of salinity. A

scheme trying to integrate the relation between CHO and protein metabolism and the way in which

both are modulated by salinity is presented. From published and present results, there are two factors

that apparently control the use of high dietary CHO levels; a-amylase enzyme-dietary CHO level

capacity and glycogen saturation in DG. Production of glucose is limited in shrimp because of

saturation of a-amylase when shrimp are fed with diets above 33% CHO. This is the first control

point of starch metabolism. The digestive gland is saturated with glycogen in shrimp fed with

dietary CHO levels > 33%. This is apparently the second control point of CHO metabolism that

limits growth rate in such conditions. The high metabolic cost related to high CHO diets could

explain why shrimp are well adapted to use protein as a source of energy. D 2002 Elsevier Science

B.V. All rights reserved.

Keywords: Carbohydrate metabolism; Salinity; Litopenaeus vannamei hemolymph proteins; Oxygen con-

sumption; Ammonia excretion; Internal ammonia

1. Introduction

In general, shrimp species are described as omnivorous, with constant feeding activity.

When stomach contents are examined, shrimp, polychaet worms, bivalves, ophiuroids,

nematodes, and plant debris are observed. According to Cousin (1995), carbohydrates

(CHO) from plants represent a small fraction ingested by shrimp, with glycogen the most

common source of CHO in wild populations. To feed cultured populations, diets have been

designed using starch as the principal source of CHO because monosaccharides produced

abnormal mortality (Abdel Rahman, 1996). For this reason, many researchers have

suggested that more complex carbohydrates be used to prepare shrimp feed, such as

starch, which undergoes enzymatic hydrolysis before assimilation, permitting glucose to

be absorbed in the gut at a slower rate than by using free glucose (Alava and Pascual,

1987; Pascual et al., 1983; Shiau and Peng, 1992; Shiau, 1998).

Once starches are ingested, a system located in digestive gland (DG) with a-amylase

and a-glucosidase gives hydrolysis to produce glucose, which is liberated slowly into the

blood (Cousin, 1995; Loret, 1993; Santos and Keller, 1993; Mc Donald et al., 1989).

Rosas et al. (2000) observed that the a-amylase activity of Litopenaeus stylirostris was

saturated with a dietary starch level of 21%, indicating that the digestibility of starch in this

species is limited by the a-amylase enzyme–substrate characteristics. Taking into account

that this enzyme controls the first step in starch degradation pathway, this limitation could

mean the relatively poor adaptation of shrimp to use CHO as a source of energy. Recently,

observed the maximum growth rate for L. vannamei juveniles occurred between 5% and

32% of dietary CHO. The dietary CHO level of 32% coincided with the point of inflexion

of a saturation curve measured between dietary CHO level and digestive gland glycogen,

indicating that digestive gland is rapidly saturated with diets above that CHO level. Rosas

et al. (2001a) observed that a very low CHO dietary level (around 1%) can induce the

phosphoenolpyruvate carboxykinase activity (PEPCK). Because this enzyme is a key in

the regulation of gluconeogenesis, and it is induced by dietary protein, it was proposed that

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–6748

shrimp such as other crustaceans are well adapted to synthesize CHO from protein

(Vinagre and Da Silva, 1992).

The regulation of internal osmotic pressure in diluted environments is related to the

use of dietary protein as a source of amino acids to be used as osmotic effectors and is

related to the protein level in food (Lignot et al., 1999; Pierce, 1982). Results obtained

in L. vannamei shrimp showed that low salinity levels modify the energetic substrate

favoring the protein metabolism (measured through O/N ratio), increasing the apparent

heat increment (AHI, measured through oxygen consumption), and can induce an

increase in glutamate dehydrogenase activity (mUI/mg protein GDH) as a consequence

of an increase in total internal ammonia levels and ammonia excretion (Rosas et al.,

2001a). Low salinity levels (between 5xto 15x) produced higher growth rate of L.

vannamei juveniles than shrimp maintained in high salinity (Bray et al., 1994) and was

optimum when shrimp were fed with low CHO levels and maintained at low salinity

(Rosas et al., 2001b).

The reason why low salinity is optimum for L. vannamei could be related to protein

metabolism. When shrimp are in a dilute environment, they are forced to use the free

amino acid pool (FAAP) to compensate for changes in cellular volume. That FAAP can

follow three pathways: (i) used as a source of energy and functioning as an energetic extra

source, improving the energy channeled to production through energy balance (Dall, 1975;

Pierce, 1982; Rosas et al., 1999); (ii) stored as hemocyanin to be used as a source of amino

acids for growth or as a source of energy (Chen and Cheng, 1995; Gellissen et al., 1991);

or (iii) transferred to the gastric fluids in the digestive gland to be eliminated in fecal

products (Dall, 1975). For hypothesis (i) and (ii), the use of FAAP for growth will depend

on regulatory mechanisms related to the energetic and molecular demand. Both hypotheses

can be considered parts of the same process. Because shrimp are well adapted to use

protein as an energetic substrate and for growth, we believe that hypotheses (i) and (ii) can

help to explain the adaptive mechanisms related to the tolerance of shrimp to low salinity

environments, where they naturally grow in the wild. Both hypotheses help explain why

shrimp fed with low CHO diets grow faster than those fed with high CHO. Energy

delivered from FAAP degradation or ammonia end product form glucosamine (precursor

of chitin) and is probably used for growth.

Production (P) is a measurement of anabolism and is determined by environmental

conditions. When P is integrated with respiration (R) and the energy lost through nitrogen

excretion (U), it is possible to calculate the energy assimilated (As =P +R) and the energy

absorbed (Abs =P +R +U). The energy values expressed as a ratio of absorbed or

assimilated energy can help us to understand how the energy is channeled to growth

and the form in which energy is used in a particular condition (Lucas, 1993). The net

growth efficiency (P/P +R =P/As), net respiratory efficiency (R/As), and the assimilation

efficiency (A/R +P +U =As/Abs) can be used to determine if salinity or dietary CHO can

modulate the allocation of the ingested energy in shrimp and if the extra bonus of energy

hypothesized in low-salinity-acclimated shrimp is evident.

Here we report results of new experiments trying to explain the relation between

CHO, protein metabolism, and energetic balance of L. vannamei juveniles. Information

obtained in other papers with the same shrimp and other crustacean species will be

merged, trying to make a conceptual model that explains the physiological adaptation of

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–67 49

shrimp with both dietary CHO and protein. This model will explain the adaptation of

shrimp to a dilute environment, trying to explain how salinity modulates the shrimp

efficiencies and growth through protein, CHO metabolism, and the shrimp energetic

balance.

2. Material and methods

Two experiments were done. The first was designed to evaluate the effect of CHO–

salinity combinations, hemolymph protein, glucose, lactate, hemocyanin, internal ammo-

nia digestive gland glycogen, and osmotic pressure of shrimp. The second was done to

evaluate the effect of dietary CHO level on energetic balance of shrimp, taking into

account the evaluation of routine (RRUT) and feeding respiration (RAHI), the energy lost in

routine (URUT) and feeding (UPPNE) ammonia excretion and the energy channeled to

production (P) measured through growth rate obtained in laboratory conditions. The O/N

ratio of L. vannamei were calculated also.

2.1. Preparation of diets

Juveniles of L. vannamei were fed with artificial diets, prepared with four levels of

CHO; 0%, 5%, 33%, and 61% in experiment 1 and with 0% and 38% of dietary CHO in

experiment 2 (Table 1). The experimental diets were prepared by thoroughly mixing the

dry ingredients with oil and then adding water until a stiff dough resulted. This was then

passed through a mincer with a 2-mm die, and the resulting spaghetti-like strings were air

dried at 60 �C. After drying, the material was broken up and sieved to a convenient pellet

size and stored at � 4 �C. Three tanks were randomly assigned to each CHO level.

2.2. Experiment 1

A group of 800 shrimp (1.5F 0.12 g wet weight) was used. L. vannamei were obtained

from Pecis Industries S.C., in Yucatan, Mexico. Shrimp were reared for 30 days in 90-l

tanks (10 shrimp/tank) and exposed to 15xor 40xsalinity. Every salinity–CHO

combination had three replicates. The photoperiod was 12:12 h, water temperature was

28F 1 �C, dissolved oxygen was >5.0 mg/l, and pH was >8.1. The shrimp were fed twice

a day (0800 and 2000). Uneaten food particles and feces were removed regularly.

After the 30-day acclimation period, metabolic measurements were made on living

animals of L. vannamei (n = 800; between 2.5 and 7 g wet weight). To do that, shrimp were

fasted for 12 h. After that, shrimp were sampled. After the fasting shrimp sampling, shrimp

in tanks were fed with their corresponding diet. After 30 min, remaining food was

removed, and 1.5 h after, shrimp were sampled. In both cases, (fasting and feeding

shrimp), shrimp were placed in chilled (18 �C) and aerated water for 5 min to reduce the

effect of manipulation before the hemolymph extraction. Only shrimp in intermoult stage

(C stage) were used. Hemolymph (approximately 200–300 ml/shrimp) was individually

sampled through a chilled syringe needle inserted at the base of the fifth pereiopod after

the shrimp had been dried with a paper towel.

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–6750

Osmotic pressures (OP) of hemolymph and seawater were measured in a micro-

osmometer with 20 ml of sample per titration (3 MO-PLUS; Advanced Instruments, USA).

For hemocyanin (HEM) measurements, 15 ml hemolymph was immediately diluted

with 985 ml distilled water in a 10-mm cuvette and the absorbance was measured at 335

nm. Using an extinction coefficient of E = 17.26 calculated on the basis of functional

subunit of 74000, the hemocyanin concentration was calculated (Chen and Cheng,

1993).

Commercial kits were used for glucose (GH) (GOD-PAD, Merck-740393) and lactate

(Sigma-cat. 735). These determinations were adapted to a microplate using 20 ml of

plasma and 200 ml of enzyme chromogen reagent. Absorbance was recorded on a

Table 1

Ingredient composition of experimental diets

Ingredients Carbohydrate levels (g/kg)

0b 0a 5a 32.67a 38b 61a

Squid meal 150 399 500 254 150 108

High-quality fish protein concentrated * 313 50 199.6 30.1

Casein 400 200 50

Precooked corn starch 400

Native wheat starch 0 0 50 363.0 679

Cellulose 40

Robimix (vitamins) * * 57 100 36.3 108

Vitamins* * * 20 20

Soy bean lecithin 30 100 30 32.3

Sunflower oil 20 20

Cod oil 30 45.4 30

Fish oil 71.2

Cholesterol 6 45.4 6

Na2HPO4 71.2 10 10.8

KH2PO4 71.2 10 45.4 10.8

Ballast 224 17 10.8 224

Minerals 10

Vitamin C 20 20

Cellulose 40

Carboximethyl cellulose 50

Carbohydrate (g/kg) 0 0 5 32.67 38 61

Protein (g/kg) 477 567.1 565 357.4 146.5 102

Lipid (g/kg) 70 70 70 80 80 70

MJ/kg 14 10.4 16.9 17.4 13.6 17.2

* Soluble fish protein concentrate: 90% protein (Sopropeche, Boulogne s/mer, France).

** Robimix from Hoffman La Roche, Bale, Suisse. #1720: retenal palmitate (vitamin A): 8,000,000 UI;

cholacalcypherol (vitamin D3): 196,000 UI; a-tocopherol acetate (vitamin E): 10,000 mg/kg; vitamin K3: 800 mg/

kg; ascorbyl phosphate (vitamin C): 15,000 mg/kg; thiamin (vitamin B1): 700 mg/kg; riboflavin (vitamin B2):

2000 mg/kg; pyridoxine (vitamin B6) 1000 mg/kg; niacin (vitamin PP) 10,000 mg/kg; calcium pantothenate: 5000

mg/kg; cyanocobalamin (vitamin B12): 50 mg/kg; folic acid: 250 mg/kg; biotin: 30 mg/kg; inositol: 30,000 mg/kg

(Hofmann La Roche). Coefficient for energy concentration: 21/39/17 kJ for protein, lipid and carbohydrate

(Cousin, 1995).

*** Disodium phosphate and monopotassium phosphate in equal amount. a = Experiment 1, b =Experi-

ment 2.

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–67 51

microplate reader (BIO-RAD model 550) and concentrations were calculated from a

standard solution of substrate. Plasma was further diluted 1:500 for protein (HP)

determination by the Bradford (1976) technique adapted to a microplate method using

commercial chromogen reagent (Sigma, cat. 610) and bovine serum albumin as a

standard solution.

2.2.1. Glycogen concentration in digestive gland (DGG)

Glycogen was extracted in the presence of sulfuric acid and phenol (Dubois et al.,

1965). The digestive gland was first homogenized in trichloroacetic acid (TCA, 5%) for 2

min at 6000 rpm. After centrifugation (3000 rpm), the supernatant was quantified. This

procedure was done twice. One milliliter of TCAwas pipetted into a tube and mixed with

five volumes of 95% ethanol. The tubes were placed in an oven at 37–40 �C for 3 h. After

precipitation, the tubes were centrifuged at 3000 rpm for 15 min. The glycogen (pellet)

was dissolved by addition of 0.5 ml of boiling water and then 5 ml of concentrated sulfuric

acid and phenol (5%) were added and mixed. The content of the tubes were transferred to a

cuvette and read at 490 nm in a spectrophotometer.

2.2.2. Ammonia concentration in hemolymph (HAC)

A subsample of 10 ml of hemolymph without anticoagulant was obtained for HAC

measurements. The sample was diluted with distilled water to 100 ml. The concentration of

ammonia (total ammonia; NH4+ +NH3) was measured using flow injection-gas diffusion

(Hunter and Uglow, 1993). This technique consists of a carrier stream of NaOH (0.01 M)

separated from an indicator solution (Bromothymol blue 0.5 g l�1) by a gas permeable

membrane (PTFE). All ammonia in the sample is converted to gaseous NH3, which

diffuses across the membrane and reacts with the indicator to produce a pH-dependent

color change that is detected by a photometer. A calibration curve was made using

different concentrations of (NH4)2SO4.

2.3. Experiment 2

2.3.1. Energy balance

Energy balance was estimated using the equation (Lucas, 1993)

Ab ¼ Rþ U þ P

where R indicates respiration (R =RRUT +RAHI), U is the energy lost through ammonia

excretion (U =URUT +UPPNE), and P is the energy invested in production of biomass.

Assimilated energy (As) was estimated using the equation (Rosas et al., 1998)

As ¼ P þ R

Production (P) was obtained measuring the growth rate of the shrimp fed with two

different CHO levels (0% and 38%) and maintained at two salinities (15xand 40x). We

used 120 juvenile L. vannamei with an initial weight of 0.51F 0.007 g ww (meanF S.E.),

obtained from the Industrias Pecis hatchery. Shrimp were randomly placed in 12 flow-

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–6752

through 70-l tanks with 0.28-m2 surface and an average density of 36 shrimp m � 2 (10

shrimp tank � 1). Each CHO and salinity combination had three tanks.

Natural seawater, previously filtered with sand (20 mm) and with cartridge and diatom

filters, was used. During the experiment, water temperature was kept at 28F 5 �C,dissolved oxygen >5 mg l� 1 and pH at 8F 1. In low salinity tanks, sea water was diluted

with 24 h-dechlorinated tap water.

Throughout the 40-day duration of the experiment, shrimp were fed ad libitum

rations two times a day (0800 and 2000). Feces, uningested food, and molts were

removed twice a day from each tank. Salinity, O2, temperature, and pH were measured

once a day. At the end of the experiment, the shrimp were weighed on a scale (F 0.05

g), sacrificed, and dried at 60 �C to constant weight. The mean value of 26.32F 0.66 J

g� 1 dw was used to transform the growth data into production units (P; J g� 1 dw

day� 1). This value was obtained from analyzing the energy content of the muscle of

five shrimp/treatment by means of a calorimeter (Parr) previously calibrated with ben-

zoic acid.

2.3.2. Oxygen consumption

Oxygen consumption was measured in 10 shrimp from each diet. Oxygen consumption

was determined individually by a continuous flow respirometer in a closed system (Rosas

et al., 1998). Oxygen consumption was calculated as VO2 =O2e�O2ex� Fr, where VO2 is

oxygen consumption (mg O2 h� 1 animal� 1), O2e indicates oxygen concentration at the

entrance to the chamber (mg l � 1), O2ex is oxygen concentration at the exit (mg l � 1), and

Fr is the flow rate (ml h� 1). Oxygen concentration was measured using a digital oximeter

(YSI 50B digital, USA) with a polarographic sensor (F 0.01 mg l � 1), previously

calibrated with oxygen-saturated seawater at 28 �C. The shrimp were afterwards fed food

pellet fragments of 0.06F 0.002 g each in the respirometric chambers. The same amount

of food was placed in a control chamber without organisms to estimate the oxygen lost by

food decomposition. Oxygen consumption of fed shrimp was measured every hour for a 4-

h period, between 0800 and 1300. Once the experiment was concluded, the shrimp were

weighed. Specific rate Rrout (mg g� 1 h� 1) was estimated from the VO2 of the unfed

shrimp. The specific rate of the apparent heat increase (RAHI; J g� 1 h� 1) was estimated

from the difference between VO2 of the unfed shrimp and the maximum value attained

after feeding. A 14.3 J mg � 1 conversion factor of oxygen consumption was used to

transform the unfed and fed VO2 to J g� 1 dry weight (dw) (Lucas, 1993).

At the same time, as the measurements of oxygen uptake were made, we also obtained

samples of water whose concentration of N–NH3 mg l � 1 was measured. The ammonia

excretion was determined from the differences between the ammonia concentration at

entrance and exit of each chamber and multiplying that by the rate of water flow. The

concentration of ammonia (total ammonia; NH4+ +NH3) was measured using a flow

injection-gas diffusion system (Hunter and Uglow, 1993). The ammonia excretion of

unfed and fed shrimp (postprandial nitrogen excretion; PPNE) was converted to energy

units using the value of 20.5 J mg � 1 N–NH3 excreted (Lucas, 1993) and defined as URUT

for the energy lost before feeding and UPPNE the energy lost after feeding.

The atomic ratio of the O/N was estimated for both fasting and feeding shrimp and used

values of oxygen consumption and ammonia excretion transformed to mg At g� 1 ww

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–67 53

h� 1. Feeding O/N was obtained using the maximum oxygen consumption and nitrogen

excretion obtained after feeding shrimp.

2.4. Statistical analysis

The effect of dietary carbohydrate–salinity combinations were analyzed using three-

way ANOVA (salinity–CHO levels–feeding condition) A two-way ANOVA was used to

determine the statistical interaction between salinity–dietary CHO in energy balance

results. A transformation arc sine was used to analyze data in percentage. Homogeneity of

variances was verified with Cochran’s test. Means obtained during the treatment were

compared by using Duncan’s multiple range test (Zar, 1974).

3. Results

3.1. Experiment 1

3.1.1. Osmotic pressure (OP)

A significantly lower OP was observed in shrimp exposed at 15xsalinity (between

710 to 748 mOsm/kg) in comparison to that obtained in shrimp maintained at 40xsalinity (between 800 to 830 mOsm/kg). The OP was not affected by feeding condition but

it was affected by CHO dietary levels (Fig. 1) (P < 0.05).

3.1.2. Hemocyanin (HEM)

An inverse relation was observed between dietary CHO levels and hemocyanin in

fasting and feeding shrimp in both salinities. Higher values were observed in low dietary

Fig. 1. Effect of salinity and dietary carbohydrate (CHO) levels on fasting and feeding osmotic pressure (mOsm/

kg) of L. vannamei juveniles. MeanF S.E. Different letters mean statistical differences between treatments.

Experiment 1.

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–6754

CHO levels and low values in shrimp fed with high CHO levels. A higher hemocyanin

was observed in almost shrimp maintained at 40xsalinity, both fasting and feeding

shrimp evidencing the statistical interaction between salinity and feeding condition

(Fig. 2).

3.1.3. Glucose in hemolymph (GH)

Both fasting and feeding shrimp GH increased with increasing dietary CHO levels

evidencing interaction with feeding condition (P < 0.05) (Fig. 3). In fasting shrimp, only

GH of shrimp fed with 61% dietary CHO was affected by salinity (P < 0.05) (Fig. 3). In

feeding shrimp, there were no differences between GH between salinities but a higher GH

was observed in shrimp fed with 61% dietary CHO (P > 0.05) (Fig. 3b).

Fig. 3. Effect of salinity and dietary carbohydrate (CHO) levels on fasting and feeding hemolymph glucose

concentration (mg/ml) of L. vannamei juveniles. MeanF S.E. Different letters mean statistical differences

between treatments. Experiment 1.

Fig. 2. Effect of salinity and dietary carbohydrate (CHO) levels on fasting and feeding oxyhemocyanin (mmol/l)

of L. vannamei juveniles. MeanF S.E. Different letters mean statistical differences between treatments.

Experiment 1.

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–67 55

3.1.4. Lactate in hemolymph (LH)

LH in both fasting and feeding shrimp maintained at 40xsalinity increased with the

reduction of dietary CHO levels (P < 0.05). In contrast, LH of shrimp maintained at 15xsalinity, both fasting and feeding, did not show a specific pattern with the dietary CHO

levels (Fig. 4). In shrimp maintained at 15xsalinity, a higher LH was observed in shrimp

fed than that observed in fasting shrimp (P < 0.05). A significant statistical interaction was

observed between salinity, dietary CHO, and feeding condition (P < 0.05).

3.1.5. Hemolymph protein (HP)

In fasting and feeding shrimp, the higher HP was observed in shrimp fed with 5% CHO

at both salinities (P < 0.05) (Fig. 5). In fasting and feeding shrimp, the lower HP value was

Fig. 5. Effect of salinity and dietary carbohydrate (CHO) levels on fasting and feeding hemolymph protein

concentration (mg/ml) of L. vannamei juveniles. MeanF S.E. Different letters mean statistical differences

between treatments. Experiment 1.

Fig. 4. Effect of salinity and dietary carbohydrate (CHO) levels on fasting and feeding hemolymph lactate

concentration (mg/ml) of L. vannamei juveniles. MeanF S.E. Different letters mean statistical differences

between treatments. Experiment 1.

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–6756

obtained in shrimp fed with 33% and 61% CHO and maintained at 40xsalinity and in

shrimp fed with 61% CHO and maintained at 15xsalinity (P < 0.05) (Fig. 5). In

consequence, a significant interaction was observed between salinity and dietary CHO and

dietary CHO and feeding condition (P < 0.05).

3.1.6. Digestive gland glycogen (DGG)

A saturation curve was observed in fasting and feeding shrimp maintained at both

salinities (Fig. 6). In fasting shrimp, DGG increased with the dietary CHO level, showing a

saturation curve with the inflexion point in shrimp fed with 33% CHO at both salinities. In

fasting shrimp, DGG concentration was higher in shrimp maintained at 40xthan at

15xsalinity (Fig. 6). A saturation curve of DGG level was observed with an increase in

dietary CHO level in feeding shrimp but with the inflexion point at the 5% dietary CHO

level in shrimp maintained at 40xsalinity and in 33% dietary CHO level in shrimp

maintained at 15xsalinity (Fig. 6). In both fasting and feeding shrimp, a significantly

lower DGG concentration was observed in shrimp maintained at 15xthan in shrimp

maintained at 40xsalinity (P < 0.05). The differences were supported by the statistical

interaction observed between salinity and dietary CHO and dietary CHO and feeding

condition (P < 0.05).

3.1.7. Ammonia concentration in hemolymph (HAC)

In fasting shrimp, the high HAC value was obtained in shrimp fed 0% CHO and

maintained at 15x salinity and the low value in shrimp fed with 38% CHO and

maintained at 15xsalinity(P < 0.05) (Fig. 7). In feeding shrimp, a higher HAC was

obtained in shrimp fed with 0% and 38% CHO and maintained at 15xsalinity than in

shrimp maintained at 40xsalinity and fed with both diets, evidencing the statistical

interaction between salinity and feeding condition (Fig. 7) (P < 0.05).

Fig. 6. Effect of salinity and dietary carbohydrate (CHO) levels on fasting and feeding digestive gland glycogen

(mg/g) of L. vannamei juveniles. MeanF S.E. Different letters mean statistical differences between treatments.

Experiment 1.

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–67 57

3.2. Experiment 2

3.2.1. Energetic balance

The amount of energy directed to production (P) varied according to salinity and

dietary CHO levels (Table 2). In both salinities, a higher P was obtained in shrimp fed with

0% CHO, than in shrimp fed with 38% CHO (P < 0.05). There was no interaction between

salinity and dietary CHO levels on P values (P > 0.05).

RAHI of shrimp maintained at 15xsalinity and fed with 0% CHO (28 J g� 1 dw

day � 1) was 1.8, 2.4, and 4.6 times higher than in shrimp maintained at 40xsalinity

and fed with 38% CHO, 40xsalinity and fed with 0% CHO, and 15xsalinity and

fed with 38% CHO (Table 2). The RRUT obtained in shrimp fed 0% CHO and maintained

at 40xsalinity (844 J g� 1 dw day � 1) was significantly higher than in shrimp fed 38%

CHO at 15xand 40xsalinity (mean value of 610 g� 1 dw day � 1) and in shrimp

fed with 0% CHB and maintained at 15x salinity (P < 0.05). In consequence, R

(RAHI +R RUT) was higher in shrimp fed with 0% CHO and maintained at 40xsalinity

than in all other treatments (P < 0.05; Table 2). The lowest of R (308.3 J g� 1 dw

day � 1) was observed in shrimp fed with 0% CHO and maintained at 15xsalinity. As

a result, a significant interaction between salinity and dietary CHO on R was observed

(P < 0.05).

Energy lost in ammonia excretion (U =UPPNE +URUT) was strongly affected by the

dietary CHO level and salinity (P < 0.05) (Table 2). The high URUT observed in shrimp fed

with 0% and maintained at 15xsalinity (906.6 J g� 1 dw day � 1) was 12.7 times higher

than the lower value obtained in shrimp fed with 38% CHO and maintained at 15xsalinity (71.4 J g� 1 dw day � 1) (P < 0.0001). Intermediate values were obtained in shrimp

Fig. 7. Effect of salinity and dietary carbohydrate (CHO) levels on fasting and feeding hemolymph ammonia

concentration of L. vannamei juveniles. MeanF S.E. Different letters mean statistical differences between

treatments. Experiment 2.

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–6758

maintained at 40xsalinity and fed with 0% (247.6 J g� 1 dw day � 1) and 38% CHO

(522.5 J g� 1 dw day � 1) (P < 0.05). The high and low UPPNE values were obtained in

shrimp maintained at 15xsalinity and fed with 0% and 38% CHO (Table 2). The daily U

obtained in shrimp fed 0% and maintained at 15xsalinity was 15, 4, and 2 times higher

than at 15xsalinity 38% CHO, 40xsalinity 0% CHO, and 40xsalinity 38% CHO

(P < 0.05).

3.2.2. O/N ratio

Both fasting and feeding O/N ratio of shrimp maintained in 15xsalinity and fed with

38% dietary CHO were significantly higher than that observed in remaining treatments

showing a significant interaction between salinity and dietary CHO (P < 0.05).

3.2.3. Absorption (Abs)

The Abs also varied in relation to the dietary CHO level and salinity (Table 2). The

higher and the lower Abs values were obtained in shrimp maintained at 15xsalinity and

fed with 0% CHO (1819.9 J g� 1 dw day � 1) and 38% CHO (857.8 J g� 1 dw day � 1).

Intermediate values were obtained in shrimp maintained at 40xsalinity and fed with both

diets (P < 0.05, Table 2).

Table 2

Energy balance of L. vannamei juveniles fed with different dietary CBH levels and salinities

Salinity (x)

15 40

Dietary CBH (%) 0 38 0 38

Respiration (R)

RRUT 280F 138a 597.7F 106b 844F 137c 623F 119a

RAHI 28.0F 2.4c 6.0F 1a 11.7F 1.8b 16F 2.4b

R(RUT + AHI) 308.3F 46.2a 603.7F 72.4b 855.4F 128.3c 638.9F 76.5b

Nitrogen excretion (U)

URUT 906.6F 178.6d 71.4F 8.9a 247.6F 40.6b 522.5F 40.1c

UPPNE 203.3F 37.7c 1.76F 0.29a 18.9F 2.50b 19.2F 1.40b

U(RUT + PPNE) 1109.9F 205d 73.1F12.0a 266.5F 42.3b 541.7F 39.5c

Production ( P) 402F 48.2b 181F 27.1a 451F 72.1b 154F 24.6a

Absorption (Abs =R+U +P) 1819F 127.4c 857.8F 111.5a 1572.9F 101b 1334.6F 213.5b

Assimilation (As =R +P) 710.3F 7.8a 784.7F 86.4a 1306.4F 118c 792.9F 95.1ab

Absorption efficiencies

R/Abs (%) 16.9 70.3 54.4 47.8

U/Abs (%) 60.9 8.5 16.9 40.6

P/Abs (%) 22.0 21.0 28.7 11.5

Assimilation efficiencies

As/Abs (%) 39 91 83 59

R/As (%) 43.0 76.9 65.5 80.6

P/As (%) 56.6 23.1 34.0 19.0

Values in J day� 1 g� 1 dry weight. MeanF S.E.

Different letters mean statistical differences between treatments, P < 0.05.

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–67 59

3.2.4. Assimilation (As)

The energy As was mainly affected by salinity rather than dietary CHO levels

(P < 0.05) (Table 2). The As obtained in shrimp fed with 0% CHO and maintained at

40xsalinity (1306.4 J g� 1 dw day � 1) was 1.8 times higher than in shrimp fed with all

remaining treatments (Table 2).

Between 17% and 70% of Abs was invested in R with the lower and higher values in

shrimp maintained at 15xsalinity and fed with 0% and 38% CHO (Table 2). The energy

lost in U was between 9% and 61% of Abs newly with high (U/Abs = 60.9%, 0% CHO)

and low (U/Abs = 9%, 38% CHO) levels in shrimp maintained at 15xsalinity and fed

with 0% and 38% CHO (Table 2). The energy invested in P was similar in shrimp

maintained at 15xsalinity (P/Abs =mean value of 21.5%, both diets) and 40xsalinity

and 0% CHO (P/Abs = 29%). These values were two times higher than in shrimp

maintained at 40xsalinity and fed with 38% CHO (P/Abs = 11.5%).

A part of the energy Abs is assimilated. That energy can be calculated as a proportion of

Abs and is called assimilation efficiency (AE%= [As/Abs]100) (Table 2). The higher

assimilation efficiency was obtained in shrimp maintained at 15xsalinity and fed with

38% CHO (91%) and in shrimp maintained at 40xsalinity and fed 0% CHO (83%). A low

AE% was obtained in shrimp fed with 38% CHO and maintained at 40xsalinity (59%)

and in shrimp fed with 0% CHO and maintained at 15xsalinity (39%) (Table 2).

Between 43% and 81% of As was invested in R with the low values in shrimp

maintained at 15xsalinity and fed 0% CHO (43%) and the high in shrimp maintained at

40xsalinity and fed with 38% CHO (81%) (Table 2). Intermediate and relatively high R/

As values were recorded in shrimp maintained in other treatments. This behavior affected

the proportion of the As energy that was channeled to the net production efficiency (P/

As). The high value was obtained in shrimp fed with 0% CHO and maintained at 15xsalinity (57%) and the low in shrimp maintained at 40xsalinity and fed with 38% CHO

(Table 2).

4. Discussion

The dietary CHO levels and salinity affected the CHO and protein metabolism of L.

vannamei juveniles. In Fig. 9 is a scheme trying to integrate the relation between CHO and

protein metabolism and the way in which both are modulated by salinity. That scheme was

elaborated with data from literature, present experiments, and papers published previously

(Fig. 9).

After feeding, ingested food is partially digested in stomach where the chyme is formed

(Al-Mohanna and Nott, 1987). Starch is absorbed in DG and hydrolyzed by a-amylase

(Al-Mohanna and Nott, 1987; Wigglesworth and Griffith, 1994). Recent results obtained

in L. sytlirostris demonstrated that the a-amylase activity is saturated when dietary CHO

level exceeded 33% (Rosas et al., 2000). From those results, we believe that in L.

vannamei, the hydrolyzation of dietary starch by a-amylase could be limited by dietary

CHO such we observed in L. stylirostris. With L. stylirostris, we reported that the a-glucosidase activity was directly related to the dietary CHO level, indicating that the

hydrolysis of oligosaccharides to produce glucose was not limited by diet CHO although it

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–6760

is controlled for the oligosaccharides delivered by the a-amylase (Rosas et al., 2000). In

Fig. 9, a similar figure was assumed for L. vannamei shrimp.

Once the glucose is produced in DG, it can be directed to glucose-6-P, which is

delivered to blood to be used as a source of energy in tissues. The pentose cycle leads to

synthesis of nucleotides and ribose units (Al-Mohanna and Nott, 1987; Santos and Keller,

1993). The results obtained in the present study showed that glucose increases with the

increase in dietary CHO. Such a trend has been reported by Abdel Rahman et al. (1979) in

P. japonicus, in L. vannamei (Cousin, 1995), in L. stylirostris (Cousin, 1995; Rosas et al.,

2000), and L. setiferus (Rosas et al., 2001b). According to Cousin (1995) and Abdel

Rahman et al. (1979), glucose is quickly released into the hemolymph, remaining in the

blood for several hours.

Results obtained in O/N ratio in the present paper showed that the metabolic energy

obtained by shrimp in both diets and salinities is the result of breakdown of protein more

than CHO, indicating that HG has another more important role in metabolism of shrimp

than as a source of metabolic energy. However, an interaction between salinity and dietary

CHO level was observed. Shrimp maintained with 38% CHO and 15xsalinity showed the

high O/N values, indicating that in such conditions, shrimp used more CHO as a source of

energy than that observed in all remaining treatments. If in low salinity and high dietary

CHO (38%) the protein was limited (14.7%) (Table 1), we can expect that the FAAP to

regulate osmotic pressure were limited also. Although dietary CHO was directed to satisfy

the energetic demands of shrimp in low salinity (reflected in a high O/N ratio), it was not

enough to substitute for the proteins missing in the diet, because in such conditions, shrimp

Fig. 8. Effect of salinity and dietary carbohydrate (CHO) levels on fasting and feeding O/N ratio of L. vannamei

juveniles. MeanF S.E. Different letters mean statistical differences between treatments. Experiment 2.

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–67 61

production was lowest in comparison with all other treatments (Table 2). When shrimp were

fed with 0% CHO (and high protein: 48%) or with high CBH and maintained in 40xsalinity, the energetic substrate was protein (Fig. 8), indicating that when shrimp have

enough protein to regulate their osmotic pressure (48% protein) or when shrimp are in high

salinity (40x), the metabolism is directed mainly to use protein rather than CHO.

According to Santos and Keller (1993) and Stevenson (1985), hemolymph glucose-6-P can

be reabsorbed into muscle and digestive gland to synthesize glycogen or used as a source of

macromolecules with a specific physiological role, such as N-acetylglucosamine (GlcNAc),

a major molecule used to form chitin in crustaceans (Carey, 1965; Stevenson, 1985).

Glycogen in DG is saturatable (Fig. 6). The saturation curve of DGG concentration in

relation to the dietary CHO was noted by Rosas et al. (2000) in L. stylirostris. A similar

curve was noted in the present study and in another paper reported for juveniles of L.

vannamei indicating that the DG capacity to store glycogen is limited. When the digestive

gland is saturated, glycogen could act as a physical barrier preventing the amino acids and

peptides to flow into the B cells of the digestive gland. In that situation, a high dietary

CHO could indirectly affect growth because the amino acids and peptides could be lost

through fecal excretion. Recently, we observed that the growth rate of L. vannamei

juveniles was reduced just in shrimp fed with dietary CHO that produced a DGG

concentration above the inflexion point between DGG and dietary CHO curve. Similarly,

in the present paper, we observed that production (P) was higher in shrimp fed with low

dietary CHO levels than in shrimp fed with high CHO levels (Table 2).

Fig. 9. Schematic representation of the biochemical and physiological events that occur in shrimp fed with high

CHO (HCHO) dietary level or low CHO (LCHO) dietary levels. Dark words indicate the biochemical or

physiological events that has been measuring mainly in L. vannamei or in L. stylirostris. When shrimp are fed

with HCHO dietary levels, starch is processed by a-amylase to produce oligosaccharides and glucose. There are

two factors that control the use of high dietary CHO levels; a-amylase enzyme-dietary CHO capacity and

glycogen saturation in DG. Glucose 6-P produced is used as a fuel and glycogen synthesis or as a source of

glucosamine or glutamine in DG. Glucose can be used as a source of glucosamine in epidermis also. When

shrimp are fed with LCHO diets, shrimp are obligated to use proteins to form glycogen and glucose besides to

synthesize peptides and proteins. In low salinity, the free amino acid pool (FAAP) mobilization could help to

support the amino acids (AA) needs to produce protein, and hemocyanin could work as a store of protein to be

used for growth or metabolic energy. In that situation, FAAP could be used as a source of metabolic energy,

saving energy to be used to produce glycogen or for protein synthesis and growth. Glutamate dehydrogenase

enzyme (GDH) is enhanced in low salinity and in shrimp fed with LCHO diets, which helps to understand how

the energy from proteins is modulated by salinity. In high salinity, mobilization of FAAP are reduced, reducing at

the same time its catabolism and in consequence the GDH activity, hemolymph ammonia concentration (HAC),

and ammonia excretion. Notwithstanding FAAP could help to produce proteins in DG (e.g. hemocyanin or

peptides) when shrimp are fed with LCHO diets. HAC and the ammonia excretion (U ) are low, indicating that

metabolism of proteins is diminished in high salinity conditions (by the absence of the FAAP catalysis) and the

use of glucosamine as a carrier of ammonia to be excreted. Lactate was high in this condition, showing that

shrimp could use the energy from anaerobic metabolism. GLC= glutarate cycle; PCPK= phosphoenolpyruvate

carboxykinase, GDH= glutamate dehydrogenase, AA= amino acids, FAAP= free amino acid pool, TCA= three

carboxylic acid cycle. (1) Le Chevalier and Van Wormhoudt (1998); (2) Lallier and Walsh (1991); (3) Loret

(1993); (4) Rosas et al. (2000); (5) Stevenson (1985); (6) Santos and Keller (1993); (7) Rosas et al. (2001a,b); (8)

Vinagre and Da Silva (1992); (9) King et al. (1985); (10) Gibson and Barker (1979); (11) Dall (1975); (12) Pierce

(1982); (13) Hewitt (1992); (14) Claybrook (1983); (15) Chen and Cheng (1995); (16) Regnault (1993); (17); (18)

Williams and Lutz (1975); (19) Lynch and Webb (1973); (20) Morris and Taylor (1988); (21) Carey (1965); (22)

Dendinger and Schaltzlein (1973); (23) Wigglesworth and Griffith (1994).

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–6762

Shrimp fed diets with low CHO levels grow faster than shrimp fed with high CHO

levels. When a low CHO diet is combined with a low salinity, the growth rate usually is

greater than in shrimp fed with low CHO levels and high salinity (Rosas et al., 2001a).

When shrimp are fed with low CHO level and are maintained in low salinity, they are

forced to use protein as a substitute for CHO and simultaneously to compensate for the

changes in osmotic pressure (Fig. 1).

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–67 63

Crustaceans are well equipped to make CHO from proteins. Vinagre and Da Silva

(1992) demonstrated that 20-day starved crabs (Chasmagnathus granulata), previously

fed with a high protein diet for 2 weeks, were able to maintain a higher glucose

concentration and DGG than in starved crabs previously fed with diets CHO-rich. The

ability of crabs fed on a protein-rich diet to maintain a high HG concentration and DGG

level during fasting period probably resulted from an effective gluconeogenesis. In a

similar form, Rosas et al. (2001a) demonstrated that PECPK of L. vannamei juveniles was

enhanced in low salinity and in shrimp fed with low dietary CHO.

According to Gibson and Barker (1979), glucosamine (GlcNAc) could be synthesized

in DG and transported to the epidermis to the chitin synthesis. At the same time, glutamine

can be synthesized in DG and have a role as a precursor for glutamic acid or as

detoxificating carrier of ammonia (King et al., 1985). In our present work, we observed

that shrimp fed with low CHO diets and maintained in low salinity had a higher HAC and

a U production than in shrimp fed with high CHO diets. Part of the blood ammonia could

be excreted as a final product of dietary protein metabolism and of metabolism of free

amino acid pool (FAAP) that are mobilized to compensate for the change in osmotic

pressure (Fig. 9). A high GDH activity in gills has been reported in shrimp fed with low

CHO diet and maintained in diluted sea water (15xsalinity) showing that shrimp are

well adapted to respond at an increase of blood FAAP increasing FAAP catabolism in gills

(Rosas et al., 2001a). At the same time, ammonia could be used to explain the chitin

synthesis and the higher growth rate observed previously in low salinity in L. vannamei

(Bray et al., 1994; Rosas et al., 2001a,b).

In our work, a high Ab was obtained in shrimp fed with low CHO diets in both

salinities, indicating that shrimp are well adapted to derive energy from protein despite the

energy lost in U (Table 2). But how could shrimp produce more biomass (P) if they lost a

high quantity of energy as U? The energy channeled to P depends of the quantity of

assimilated energy and the efficiency in which this energy is channeled to make biomass

or is used as physiological energy (R) (Lucas, 1993). The present results demonstrated that

shrimp fed with low dietary CHO and maintained at low salinity have a net production

efficiency (P/As = 56.6%) higher than in other treatments (P/As = 19–23%). That high

efficiency was the consequence of the reduction in energy used as R in shrimp fed with

low CHO diet and maintained at low salinity (Fig. 9). Although no explanation exists, we

can hypothesize that shrimp maintained at low salinity could use the FAAP metabolism as

a source of energy, which could mean an energetic surplus that improves the energy

channeled to production through energy balance, improving the efficiency of growth

(hypothesis (i)).

LH increases with dietary CHO decrease from 0.04 to 0.09 mg/ml in shrimp maintained

at high salinity (Fig. 4). According to Vinagre and Da Silva (1992), muscle lactate might

be an important source of carbon chains for gluconeogenesis or to produce metabolic

energy when FAAP catabolism is not favored, such as we observed in high salinity

acclimated shrimp (Vinagre and Da Silva, 1992). LH has been used as an index of stress in

L. vannamei when values exceed 0.5 mg/ml (Racotta and Palacios, 1998). Taking into

account that in the present work, we observed values 10 times less than in stressed shrimp,

we can expect that observed LH has a metabolic role that helps to improve the

gluconeogenic pathway. That pathway could be less efficient than that observed when

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–6764

gluconeogenesis comes from FAAP in shrimp acclimated at low salinity affecting the

glucosamine production and at the end the growth rate of shrimp.

As we expected, in low salinity and low CHO diet, shrimp had a high U value (61%),

indicating the energy lost during protein process. After a salinity change, the loss of FAAP

from tissues resulted in its excretion into the blood. This process must impose an

additional osmotic load on the blood, which would increase water inflow from a diluted

external medium. Transfer of FAAP to the gastric fluids into the digestive gland provides a

mean of minimizing this additional load (Dall, 1975). The digestive gland is thought to be

an important organ for hemocyanin synthesis, and products of this synthesis might cause

an increase in hemolymph protein content when the FAAP are transferred from blood

(Gellissen et al., 1991). Present results demonstrated that shrimp fed with low CHO diets

had more hemocyanin and protein than that observed in shrimp fed with high CHO diets

(Figs. 2 and 5), confirming that the shrimp must have the capacity to use FAAP jointly

with dietary proteins as a source of amino acids to store proteins in hemolymph. The

results explain hypothesis (ii), in which FAAP metabolism is proposed as a form to store

proteins as hemocyanin to be used as a source of amino acids for growth or as a source of

metabolic energy.

The results obtained in the present paper and by many other researchers had

demonstrated that CHO metabolism is a complex process in shrimp and even in

crustaceans. It depends on the diet and salinity. The results obtained demonstrated that

shrimp are well adapted to live without dietary CHO channeling energy to growth

although protein metabolism produced a big loss of energy through ammonia excretion.

The proposed hypotheses were partially explained and proved. Notwithstanding, many

questions remain without an answer and we propose some to be addressed as im-

portant:

1. How are the lipids working jointly with protein–CHO metabolism?

2. How are the FAAP and hemocyanin metabolism controlled?

3. Is it possible to store N acetyl glucosamine in muscle and DG? How is this

metabolism working?

4. What is the relation between dietary energy, protein origin (animal or vegetal), and

CHO–protein metabolism?

5. What is the relation between CHO–protein metabolism and salinity when shrimp

are maintained in fresh water?

6. Is it possible to improve enzyme activity to increase starch metabolism?

Acknowledgements

Thanks are due to Dr. Ellis Glazier for editing this English-language text. We thank

the ECOS Mexico–France program for its support to researcher exchanges during this

study. Special thanks are given to Janick Le Priol, Wendy Caamal, Juan Jose Alpuche,

Gemma Martinez, Adriana Paredes, and Gabriela Palomino for their help during the

experiments. The present study was partially financed by CONACYT-FOSISIERRA.

C. Rosas et al. / J. Exp. Mar. Biol. Ecol. 268 (2002) 47–67 65

Special thanks are given to Industrias Pecis and Ocean. Ramon Mendez Lanz, Secretary

of Fisheries of Campeche State, and Dr. Eliseo Elcantara from Malta Clayton SA de CV

for its support. [SS]

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