Metabolism and growth of juveniles of Litopenaeus vannamei: effect of salinity and dietary...

Journal of Experimental Marine Biology and EcologyŽ .259 2001 1–22

www.elsevier.nlrlocaterjembe

Metabolism and growth of juveniles ofLitopenaeusÕannamei: effect of salinity and dietary

carbohydrate levels

Carlos Rosasa,), Gerard Cuzonb, Gabriela Gaxiolaa,Yannick Le Priolc, Cristina Pascuala, Jordi Rossignyolc,

Fabian Contrerasa, Adolfo Sancheza, Alain Van Wormhoudtca Grupo de Biologıa Marina Experimental, Laboratorio de Ecofisiologıa, Facultad de Ciencias, UNAM,´ ´

Apdo. Post. 69, Cd. del Carmen, Campeche, Mexicob ( )Centre Oceanologique du Pacifique COP , IFREMER, BP 700, BP 7004, TaraÕao, Tahiti, French Polynesia

c Station de Biologie Marine du Museum National d’Histoire Naturelle et du College de France, BP 225,`29900, Concarneau, France

Received 7 September 2000; received in revised form 13 January 2001; accepted 8 February 2001

Abstract

Ž .The present study was designed to understand how carbohydrate CBH and protein metabolismare related in the penaeid shrimpLitopenaeus Õannamei. With this information, we obtained acomprehensive schedule of the protein–carbohydrate metabolism including enzymatic, energetic,and functional aspects. We used salinity to determine its role as a modulator of the protein–carbo-hydrate metabolism in shrimp. Two experiments were designed. The first experiment evaluatedthe effect of CBH–salinity combinations in growth and survival, and hemolymph glucose, protein,and ammonia levels, digestive gland glycogen, osmotic pressure, and glutamate dehydrogenaseŽ .GDH of L. Õannamei juveniles acclimated during 18 days at a salinity of 15‰ and 40‰. Thesecond experiment was done to evaluate the effect of dietary CBH level on pre- and postprandial

Ž .oxygen consumption, ammonia excretion, and the oxygen–nitrogen ratio OrN of juvenile L.Õannamei in shrimps acclimated at 40‰ salinity. We also evaluated the ability of shrimp to

Ž .carbohydrate adaptation. We made phosphoenolpyruvate carboxykinase PECPK and hexokinaseactivity measurements after a change in dietary carbohydrate levels at different times during 10days. The growth rate depended on the combination salinity–dietary CBH–protein level. Themaximum growth rate was obtained in shrimps maintained at 15‰ salinity and with a diet

) Corresponding author. Fax:q52-938-28730.Ž .E-mail address: [email protected] C. Rosas .

0022-0981r01r$ - see front matterq2001 Published by Elsevier Science B.V.Ž .PII: S0022-0981 01 00222-2

( )C. Rosas et al.rJ. Exp. Mar. Biol. Ecol. 259 2001 1–222

containing low CBH and high protein. The protein in hemolymph is related to the dietary proteinlevels; high dietary protein levels produced a high protein concentration in hemolymph. Thissuggests hemolymph is able to store proteins after a salinity acclimation. Depending on thesalinity, the hemolymph proteins could be used as a source of osmotic effectors or as metabolicenergy. The OrN values obtained show that shrimp used proteins as a source of energy, mainly

Ž .when shrimps were fed with low CBH. The role played by postprandial nitrogen excretion PPNEŽ . Ž .in apparent heat increase AHI PPNErAHI ratio is lower in shrimps fed diets containing high

CBH in comparison with shrimps fed diets containing low CBH levels. These results confirm thatthe metabolism ofL. Õannamei juveniles is controlled by dietary protein levels, affecting theprocesses involved in the mechanical and biochemical transformations of ingested food. A growthdepression effect was observed in shrimps fed with low-CBH protein diets and maintained in 40‰

Ž .salinity. In these shrimps, the hemolymph ammonia concentration HAC was significantly higherthan that observed in shrimps fed with low CBH and maintained in 15‰ salinity. That high HAClevel coincided with lower growth rate, which suggests that this level might be toxic for juvenilesof L. Õannamei. Results obtained for GDH activity showed this enzyme regulated both HAC andhemolymph protein levels, with high values in shrimps fed with low CBH levels and maintainedin 40‰ salinity, and lower in shrimps fed with high CBH and maintained in 15‰ salinity. Thesedifferences mean that shrimp with a high-gill GDH activity might waste more energy in oxidationof the excess proteins and amino acids, reducing the energy for growth. It was evident thatL.Õannamei can convert protein to glycogen by a gluconeogenic pathway, which permitted shrimpto maintain a minimum circulating glucose of 0.34 mgrml in hemolymph. A high PECPK activitywas observed in shrimps fed a diet containing low CBH level indicating that the gluconeogenicpathway is activated, as in vertebrates by low dietary CBH levels. After a change in diet, weobserved a change in PEPCK; however, it was lower and seems to depend on the way ofadaptation, because it occurred after 6 days when adapting to a high-CBH diet and with littlechange for the low-CBH diet.q2001 Published by Elsevier Science B.V.

Keywords: Carbohydrate metabolism; Penaeid shrimp; Oxygen consumption; Ammonia excretion; Salinity;Enzymatic activity;Litopenaeus Õannamei juveniles

1. Introduction

Gluconeogenesis in crustaceans is a biosynthetic pathway for de novo synthesis ofglucose from the precursors lactate or alanine. Phosphoenolpyruvate carboxykinaseŽ .PEPCK is a key regulatory enzyme in gluconeogenesis because it catalyzes the

Ž .conversion of oxaloacetate to phosphoenolpyruvate Seitz et al., 1980 . It is induced atthe level of transcription in some vertebrates, depending on the species and environmen-

Ž .tal factors Moon, 1988 . In crustaceans, the hepatopancreas functions both as a centerof carbohydrate metabolism and a site for gluconeogenesis. PEPCK has been found in

Ž .the hepatopancreas of different species Lallier and Walsh, 1991 and was recentlyŽ .sequenced in Penaeides Van Wormhoudt and Sellos, 1996 . In the crab,Chasmag-

nathus granulata, carbohydrate metabolism changed according to dietary carbohydratewith protein increasing when crab were fed with high protein level and decreasing when

Ž .crab were fed with high carbohydrate levels Kucharski and Da Silva, 1991 . RecentŽ .results of Oliveira and Da Silva 1997 showed that the level of hepatopancreatic

PEPCK was not influenced by high-protein or carbohydrate diets at a salinity of 15‰.Carbohydrate metabolism has been partially studied in shrimp. Several authors have

( )C. Rosas et al.rJ. Exp. Mar. Biol. Ecol. 259 2001 1–22 3

demonstrated that glucose cannot be used directly by shrimp because it produces anegative physiological effect caused by hemolymph glucose saturation resulting from a

Ž .higher rate of absorption across the digestive tract see Shiau, 1998 . For this reason,many researchers have suggested that more complex carbohydrates be used to prepareshrimp 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 glucoseŽ .Pascual et al., 1983; Alava and Pascual, 1987; Shiau, 1998; Shiau and Peng, 1992 .Hexokinase, the first enzyme of glycolysis, has also been studied by a few authorsŽ .Loret, 1990 and although it is suspected to have a low capacity to phosphorylateglucose, it is implicated in the nutritional requirement.

Carbohydrate assimilation efficiency depends on the quality and quantity of thedietary CBH and the regulatory mechanisms of the enzyme activity. According to Le

Ž .Moullac et al. 1994 , the expression of three different amylase genes inLitopenaeusÕannamei is controlled by food composition. This implies shrimps have a high specificcapacity for degradation of carbohydrates in the digestive gland, which, in turn, isregulated by the food characteristics.

Ž .In a recent study Rosas et al., 2001a , we observed that dietary CBH affected energymetabolism, osmotic pressure, and growth ofL. setiferus and L. Õannamei juveniles.Ammonia excretion, hemolymph glucose, and digestive gland glycogen were affected by

Ž .the proteinrenergy PrE ratio, indicating that both shrimp species used carbohydratesand protein according to their availability. In the same study, we observed that at low

Ž .salinity 15‰ , hyperosmotic capacity was affected by thePrE ratio, with low valuesin animals fed with low protein levels. These results indicated that carbohydratemetabolism is closely related to protein metabolism through the release of amino acidsassociated with the maintenance of osmotic pressure.

Based on the saturation ofa-amylase and glycogen concentration from the midgutgland observed inL. stylirostris fed with diets containing between 1% and 33% CBH

Ž .level, Rosas et al. 2000 proposed a maximum limit of 21% dietary CBH level for thisspecies. We also suggested that shrimp can use proteins as a source of energy, producingglucose and glycogen to satisfy their metabolic requirement of carbohydrates when diets

Ž .have very low CBH levels 1% . These results suggested that shrimp had a highplasticity for the use of protein as a source of energy from food because they used thegluconeogenesis pathway to produce CBH.

One way to identify the substrates used by shrimp in a particular diet is through theŽ .ratio of consumed oxygen and ammonia excretion OrN . Using the OrN ratio, proteins

Žwere identified as the main metabolic substrates ofPenaeus esculentus Dall and Smith,. Ž . Ž1986 , Homarus americanus Capuzzo and Lancaster, 1979 ,Crangon crangon Re-

. Ž .gnault, 1981 and different penaeides Rosas et al., 1995 . Moreover, Taboada et al.Ž .1998 showed that the OrN ratio of juveniles was modified by diet protein levels, with

Ž .protein metabolism at high protein levels and mixed protein, lipid, and carbohydratemetabolism at optimal protein levels.

The effect of feeding on the oxygen consumption of shrimp has been well docu-Ž .mented and defined as the apparent heat increment AHI . Evidence obtained withL.

setiferus, L. schmitti, Farfantepenaeus duorarum, and F. notialis postlarvae andL.setiferus juveniles showed proteins ingested through the diet have a great effect on AHI,


Žindicating diets with high protein levels could result in a higher metabolic cost Rosas et. Ž .al., 1996; Taboada et al., 1998 . In a recent study Rosas et al., 2001b , we observed that

Žin L. Õannamei juveniles, the coefficient AHI% apparent heat increment expressed as a.percentage of ingested energy increased as salinity decreased, with the highest values in

shrimps acclimated to 5‰ salinity, demonstrating that AHI and salinity have aninteraction. After a salinity decrease, the AHI% could have increased as a consequenceof an increase in the degradation of the dietary proteins that can be used as a source of

Ž .amino acids used to maintain osmotic pressure Claybrook, 1983 . In such circum-stances, the regulation of internal osmotic pressure in diluted environments is related tothe use of dietary proteins as a source of amino acids to be used as osmotic effectors andis related to the protein level in food.

Ž .Glutamate dehydrogenase GDH is the key enzyme in the oxidative deamination ofŽ .amino acids during transdeamination Mayzaud and Conover, 1988 . Because of the

control on the net incorporation or removal of ammonia from the free amino-acid poolŽ .FAAP , the regulation properties of GDH have been the subject of extensive study in

Ž .Crustacea see reviews of Claybrook, 1983 . Many euryhaline crustacean speciesrespond to changes in water salinity by altering the FAAP content of their tissue, whilethe inorganic ion content and osmolality of hemolymph are being modified. After asalinity change, a decrease in muscle FAAP followed by an increase in hemolymphFAAP and an increase in ammonia excretion had been documented inP. aztecusŽ . Ž .Schoffeniels, 1970 ,P. chinensis Chen and Lin, 1992 ,Marsupenaeus japonicusŽ . Ž . ŽChen and Chen, 1992 ,P. monodon Lei et al., 1989 , andL. setiferus Rosas et al.,

.1999 . Though the increase of ammonia excretion in low salinity might be partly causedq q Žby an increase in the ionic exchange of NH by Na in the gill Schmitt and Uglow,4

.1997 , it can also reflect an accelerated catabolism of amino acids modifying the GDHactivity. For this reason, the gill has been recognized as the most important tissue related

Ž .to GDH activity Claybrook, 1983 .The present study was designed to assess the relationship of CBH and protein

metabolism in penaeid shrimp. We focussed our investigation on metabolic and bio-Ž .chemical aspects ofL. Õannamei juveniles with special emphasis on a growth and

Ž . Ž .survival, b glucose, proteins, and ammonia in hemolymph, c digestive gland glyco-Ž . Ž . Ž .gen, d osmotic pressure and ammonia in hemolymph, e gill GDH activity, f oxygen

Ž .consumption and ammonia excretion, and g digestive gland PEPCK and hexokinaseactivities. With all this information, we formed a comprehensive schedule of protein–carbohydrate metabolism including enzymatic, energetic, and functional aspects. Theeffect of salinity as a modulator of the protein–carbohydrate metabolism in shrimp wasalso determined.

2. Material and methods

Two experiments were done. The first was designed to evaluate the effect ofCBH–salinity combinations on growth, survival, hemolymph glucose, protein andammonia levels, digestive gland glycogen, and osmotic pressure of shrimp. The secondwas done to evaluate the effect of dietary CBH level on pre- and postprandial oxygen


consumption, ammonia excretion, and the OrN ratio of juvenile of L. Õannamei. Wealso made PECPK and hexokinase activity measurements after a change in dietarycarbohydrate levels at different times during a 10-day period.

2.1. Preparation of diets

Juveniles ofL. Õannamei were fed with artificial diets, prepared with two levels ofŽ .CBH: 1% and 36% Table 1 . The experimental diets were prepared by thoroughly

mixing the dry ingredients with oil and then adding water until a stuff dough resulted.This was then passed through a mincer with a die, and the resulting spaghetti-like stringswere air dried at 608C. After drying, the material was broken up and sieved to aconvenient pellet size and stored aty48C. Three tanks were randomly assigned to eachCBH level.

2.2. First experiment

2.2.1. Growth and surÕiÕal experimentsŽ .A group of 180 shrimps 360"9 mg dry weight were used.L. Õannamei wereŽ .obtained from Pecis Industries Yucatan, Mexico . Shrimps were reared for 30 days in´

Table 1Ž .Percentage composition of five experimental diets containing various CBH levelsL. Õannamei

Ingredients Low CBH High CBH

Fish meal 40 30High quality fish protein 10 10

aconcentratedWheat 0 33Starch 0 7Gluten 15 0Soya bean meal 20 10Cellulose 5 0

b Ž .Rovimix vitamins 2 2Cod liver oil 3 3Lecithin 3 3Na HPO 1 12 4

KH PO 1 12 4Ž .Carbohydrate CBH, % 1 36

Ž .Protein % 50 30cMJrkg 13 15

a Ž .Soluble fish protein concentrate: 90% protein Sopropeche, Boulogne srmer, France .ˆb Ž .Robimix from Hoffman La Roche,a1720: retynil palmitate vitamin A : 8,000,000 UI; Cholacalcyferol

Ž . Ž .vitamin D : 196,000 UI;a-tocopherol acetate vitamin E : 10,000 mgrkg; vitamin K : 800 mgrkg;3 3Ž . Ž . Ž .ascorbyl phosphate vitamin C : 15,000 mgrkg; thiamin vitamin B : 700 mgrkg; rivoflavin vitamin B :1 2

Ž . Ž .2000 mgrkg; pyridoxin vitamin B 1000 mgrkg; Niacine vitamin PP : 10,000 mgrkg; calcium pantothen-6Ž .ate: 5000 mgrkg; cyanocobalamine vitamin B : 50 mgrkg; folic acid: 250 mgrkg; biotin: 30 mgrkg;12

Ž .inositol: 30,000 mgrkg Hofmann La Roche, Bale, Suisse .ˆcCoefficient for energy concentration: 23r35r15 kJ for protein, lipid and carbohydrate, respectively.


Ž .90-l tanks 15 shrimprtank and exposed to 15‰ or 40‰ salinity. Every salinity–CBHcombination had three replicates. The photoperiod was 12:12 h, water temperature was28"18C, dissolved oxygen was)5.0 mgrl, and pH was)8.1. The shrimps were fed

Ž .ad libitum two times a day 0800 and 2000 h . Uneaten food particles and feces wereremoved regularly.

Growth rate was evaluated as the difference between wet weight at the beginning andŽ .end of the experiment. The growth rate was expressed as milligram per day mgrday

Ž .wet weight ww . Survival was calculated as the difference between the number of liveŽ .animals at the beginning and the end of the experiment. An index of performance PI

was estimated for each experimental condition as the product of survival times growthrate. This index indicated the combined effect of CBH–salinity levels on growth andsurvival of shrimp and served as an indicator for the optimum CBH–salinity combina-

Ž .tion Taboada et al., 1998 .

2.2.2. Physiological behaÕiourAfter the growth trial, metabolic measurements were made on living animals ofL.

Ž .Õannamei ns180; between 1.5 and 2.3 g ww . Before the sampling, shrimps wereŽ .placed in chilled 188C and aerated water for 5 min to reduce the effect of manipula-

tion.

( )2.2.3. Glycogen concentration in digestiÕe gland DGGŽ .Glycogen was measured in the digestive gland of 15 fasting shrimps 12 h from each

CBH–salinity combination. Glycogen was extracted in the presence of sulfuric acid andŽ .phenol Dubois et al., 1965 . The digestive gland was first homogenized in trichloro-

Ž . Ž .acetic acid TCA, 5% for 2 min at 6000 rpm. After centrifugation 3000 rpm , thesupernatant was quantified. This procedure was done twice. One milliliter of TCA waspipetted into a tube and mixed with five volumes of 95% ethanol. The tubes were placedin an oven at 37–408C for 3 h. After precipitation, the tubes were centrifuged at 3000

Ž .rpm for 15 min. The glycogen pellet was dissolved by addition of 0.5 ml of boilingŽ .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 aspectrophotometer.

( ) ( )2.2.4. Glucose HG and protein HP concentration in the hemolymphGlucose and proteins were measured in hemolymph from the same shrimp used for

the glycogen determination. Before the digestive gland was excised, 100–300ml ofhemolymph was extracted through a needle inserted at the base of the fifth pereiopodafter the shrimp had been dried with a paper towel. A subsample of 20ml was obtainedfrom each one with a syringe containing a 12.5% solution of sodium citrate, to preventclotting. The glucose concentration in the hemolymph was measured with a commercial

Ž .kit for clinical diagnosis Merckotest 3306 . Protein was measured using a microtech-Ž .nique modified from the Bradford method SIGMA 610 .

( )2.2.5. Ammonia concentration in hemolymph HACA subsample of 25ml of hemolymph without anticoagulant was obtained for HAC

Žmeasurements. The sample was diluted four times. The concentration of ammonia total


q . Žammonia: NHqNH was measured using flow injection-gas diffusion Hunter and4 3. Ž .Uglow, 1993 . This technique consists of a carrier stream of NaOH 0.01 M separated

Ž .from an indicator solution Bromothymol blue 0.5 grl by a gas permeable membraneŽ .PTFE . All ammonia in the sample is converted to gaseous NH , which diffuses across3

the membrane and reacts with the indicator to produce a pH-dependent color change thatis detected by a photometer. A calibration curve was made using different concentrations

Ž .of NH SO .4 2 4

( )2.2.6. Osmotic pressure OPOsmotic pressure of the hemolymph was measured from the same shrimp used for the

glycogen and glucose determinations. Osmotic pressure of the hemolymph and waterŽwere measured in a microosmometer with 20ml of sample per titration 3 MO-PLUS;

.Advanced Instruments, USA .

( )2.2.7. Glutamate dehydrogenase actiÕity GDHGills from experimental shrimps were immediately dissected and quickly frozen in

liquid nitrogen then kept aty258C until subsequent analysis. Enzyme assays wereperformed individually on crude homogenates of gill tissue following the method

Ž . Ž .proposed by King et al. 1985 and Regnault 1993 . Conditions of these assays were 50mM imidazole.HCl buffer, pH 8.0, made with 0.5 mM PMFS, 5 mM mercapthoethanol,and 750 mM ammonium acetate. We used 40ml gill extract and 155 mMa-keto-

Ž . Ž .glutarate final volumes1 ml . Enzyme activity was determined from the slopeV8 ofŽ y3.NADH oxidation recorded at 320 nm s6.22=10 at room temperature using a

Shimadzu PR-1 spectrophotometer. Supernatant protein was estimated by the FolinŽmethod using bovine albumin as a standard. Results were expressed as mIUmmol

y1 y1 .NADH formed min mg protein .

2.3. Second experiment

Ž . Ž .We used 90 shrimps 2.6"0.3 g ww maintained in four fiberglass tanks 1000 lconnected to a flow-through seawater system with filtered and aerated water of 40‰salinity, pH)8.1, and ammonia-0.01 mgrl. These shrimps were fed ad libitum andacclimated 18 days to each diet. After this time, a group of shrimps was used todetermine oxygen consumption and ammonia excretion and another group was used toevaluate PECPK and hexokinase activity, and glucose and glycogen levels in thedigestive gland. Pre- and postprandial measurements of oxygen consumption andammonia excretion were done to determine the effect of dietary carbohydrate levels on

Ž .AHI, PPNE postprandial nitrogen excretion , and the oxygen consumedrnitrogenŽ .excretion ratio OrN . The measurements of enzyme activity and glycogen concentra-

tions in the digestive gland were done with animals acclimated during 18 days to eachdiet and after a CBH level change. After acclimation, shrimps acclimated at high CBHlevels were fed with low CBH levels and the reverse. PECPK and hexokinase activitywere recorded in shrimps at time 0, 4, 6 and 8 days after this diet change. Digestive


glands from experimental shrimps sampled were immediately dissected and quicklyfrozen in liquid nitrogen, then kept aty258C until subsequent analysis. Enzyme assayswere made individually on crude homogenates of digestive glands. Conditions of these

Ž .assays were 2.4ml pyruvate kinase 740 Urml . Hepatopancreas were grounded in aPotter apparatus and the extract centrifuged at 20,000=g during 30 min. The buffercontained 6 mM benzamidine, 2 mM PMSF, 10 mM caproic acid, 10 mM EDTA, 10mM iodoacetate, and 10 mM mercaptoethanol in 50 mM HEPES buffer adjusted to pH7.8.

2.3.1. PEPCK measurementEnzyme assays were done individually on crude homogenates of the digestive gland

of shrimp. The tissue was homogenized in Tris buffer and centrifuged at 20,000 rpm for30 min at 58C.

Ž .The formation of PEP phosphoenolpyruvate from oxalate was measured in aŽ . Ž .reaction mixture which included per ml : Tris acetate pH 7.5 , 65mM; MnCl , 1.72

mM; MgCl , 1.7mM; ITP, 0.8mM; NADH, 0.17mM; oxalacetate, 0.6 M; glutathione,2Ž .2.35mM; and 2.4ml of a mixture of pyruvate kinase and LDH 740 and 1030 Urml

Ž . ŽSigma . A control value was obtained by omitting ITP Chang and Lane, 1966; Noce.and Utter, 1975 . Activity was expressed as micromoles of NADH per mg of protein

consumed per minute at 258C.

2.3.2. Hexokinase measurementThe measure of activity was done in HEPES buffer 50 mM, pH 7.8, containing 0.5

mM KCl, MgCl , bovine serum albumin 1 mgrml, 10 mM aminocaproic acid, 3.2 mM2

DTT, and 0.6 mM NAD. After stabilization of the curve, 50ml 1 M glucose, and 5mlŽ . Žof G-6PDH 250 Urml were added. The reaction was started using 50ml ATP 100

.mM and 50ml of crude extract. Activity is expressed as moles of NADH formed perminute at 258C per mg of protein.

2.3.3. Protein measurementProteins were measured with the Lowry method using serum bovine albumin as a

standard and specific activity estimated as micromoles of NADH per milligram protein.

2.3.4. DigestiÕe gland glycogen and glucose concentrationŽ .Glycogen was measured in digestive gland of six fasting shrimps 12 h from each

CBH level at 0, 4, 6 and 8 days after a dietary CBH change level. Glycogen wasŽ .extracted in the presence of KOH, ethanol, and saturated Na SO Van Handel, 1965 .2 4

Ž .The digestive gland tissue 10 mg was heated with 0.5 ml 30% KOH for 15 min.Portions of 0.4 ml of the digest were pipetted into centrifuged tubes; 0.05 ml Na SO2 4

and 1 ml 80% ethanol were added. The tubes were centrifuged at 2000 rpm for 20 min.. Ž .The pellet was dispersed in 0.5 ml water and anthrone HCl was added 3 ml . Tubes

were heated at 908C for 20 min, chilled and read at 620 nm using a Shimadzu PR-1spectrophotometer. Digestive gland glucose was measured in the digestive gland ho-


Ž .mogenate with a commercial kit for clinical diagnosis Merckotest 3306 . This informa-tion was used to correlate the variations of digestive gland glucose with hexokinaseactivity.

2.3.5. Oxygen consumption and ammonia excretionOxygen consumption was measured in 10 shrimps from each diet. Oxygen consump-

tion was determined individually by a continuous flow respirometer in closed systemŽ .Rosas et al., 1998 . Oxygen consumption was calculated as VOsO yO =Fr,2 2e 2ex

Ž y1 y1.where VO is oxygen consumption mg O h animal , O indicates oxygen2 2 2eŽ .concentration at the entrance to the chamber mgrl , O is oxygen concentration at the2ex

Ž . Ž y1.exit mgrl and Fr is the flow rate ml h . Oxygen concentration was measured usingŽ . Ž .a digital oximeter YSI 50B digital, USA with a polarographic sensor"0.01 mgrl ,

previously calibrated with oxygen-saturated seawater at 288C. The shrimps were after-wards fed food pellet fragments of 0.06"0.002 g each in the respirometric chambers.The same amount of food was placed in a control chamber without organisms toestimate the oxygen lost by food decomposition. Oxygen consumption of fed shrimpwas measured every hour for a 4-h period, between 0800 and 1300. Once the experiment

Ž y1 y1.was concluded, the shrimps were weighed. Specific rateR mg g h wasrout

estimated from the VO of the unfed shrimp. The specific rate of the apparent heat2Ž y1 y1.increase R ; J g h was estimated from the difference between VO of theAHI 2

unfed shrimp and the maximum value attained after feeding. A 14.3 J mgy1 conversionfactor of oxygen consumption was used to transform the unfed and fed VO to J gy1 dry2

Ž . Ž .weight dw Lucas, 1993 .At the same time, as the measurements of oxygen uptake were made, we also

Ž .obtained samples of water whose concentration of N–NH mgrl was measured. The3

ammonia excretion was determined from the differences between the ammonia concen-tration at the entrance and the exit of each chamber and multiplying that by the rate of

Ž q .water flow. The concentration of ammonia total ammonia; NHqNH was measured4 3Ž .using a flow injection–gas diffusion system Hunter and Uglow, 1993 . The ammonia

Ž .excretion of unfed and fed shrimp postprandial nitrogen excretion; PPNE was relatedto the ww of the shrimp. The AHI and PPNE were converted to AHI and PPNE

Ž .coefficients percentage of ingested energy . To determine the role of nitrogenmetabolism in the AHI, the PPNErAHI ratio was calculated. This ratio was expressedin percentage and was obtained individually.

The atomic ratio of the OrN was estimated for both fasting and feeding shrimp andused values of oxygen consumption and ammonia excretion transformed to units ofmgat gy1 ww hy1. Feeding OrN was obtained using the maximum oxygen consumptionand nitrogen excretion obtained during the experimental period.

2.3.6. Statistical analysisThe effect of dietary carbohydrate–salinity combinations was analyzed separately

using ANOVA. Homogeneity of variances was verified with Cochran’s test. Meansobtained during the treatment were compared by using Duncan’s multiple range testŽ .Zar, 1974 .


3. Results

3.1. First experiment

3.1.1. Growth rate and surÕiÕalŽ .The final weight and growth rate 65.7 mgrday of L. Õannamei at 15‰ salinity and

Ž .low 1% dietary CBH level was significantly higher than that obtained for shrimpsŽ . Žmaintained at 15‰ and 40‰ salinity and high 36% dietary CBH levels mean of 53.7

. Ž .mgrday Table 2 . The lowest growth rate was obtained in shrimps maintained at 40‰Ž .salinity and low dietary CBH level P-0.05 . There were no differences between

Ž .survival obtained between treatments with values between 82% and 93% Table 2 . Themaximum PI was obtained in shrimps maintained at 15‰ salinity and the low CBH

Ž .level Table 2 .

( )3.1.2. DigestiÕe gland glycogen concentration DGGŽ .A higher DGG 6.7 mgrg tissue was obtained in shrimps fed the high-CBH diet and

Ž . Ž .regardless of salinity Fig. 1 . A lower DGG 3.4 mgrg tissue was obtained in shrimpsŽ .fed diets containing low CBH level at 15‰ salinityP-0.05 . An intermediate value

was recorded in shrimps fed diets containing low CBH level and maintained in 40‰Ž . Ž .salinity 4.68 mgrg tissue P-0.05 .

( )3.1.3. Hemolymph glucose concentration HGŽ .The higher values of HG mean of 0.43 mgrml were recorded in shrimps fed diets

Ž .containing high CBH levels independent of the salinityP-0.05; Fig. 1 . The lowerŽ .values mean of 0.35 mgrml were obtained in shrimps fed diets containing low CBH

levels in both salinities.

Table 2Effect of salinity in growth rate, survival and performance index of white shrimpL. Õannamei juveniles fed

Ž . Ž .diets containing high 30% and low 1% CBH levels and exposed at 15‰ and 40‰

Ž .Salinity ‰

15 40

High CBH Low CBH High CBH Low CHBa a a aŽ .Initial weight mg 360"9 360"9 360"9 360"9

b a b cŽ .Final weight mg 2025"150 2331"162 1917"130 1584"90Ž .Time day 30 30 30 30

b a b cŽ .Growth rate mgrday 55.5"4.1 65.7"4.5 51.9"3.5 40.8"2.3a a a aŽ .Survival % 84"4 82"5 93"5 93"4

Performance index 46.9 53.9 45.7 38.1Ž .mgrday

Mean"S.E.Ž .Entries with different letter are significantly differentP-0.05 .


Ž . Ž .Fig. 1. Effect of dietary CBH level as glycogen mgrg tissue and glucose mgrml concentration of whiteshrimp L. Õannamei juveniles exposed at different salinities. Mean"S.E.

( )3.1.4. Hemolymph protein concentration HPC and gill GDH actiÕityŽ .HPC was higher at the low dietary CBH levels in both salinities Fig. 2 . A

consistently lower value of HPC was obtained in shrimps fed diets containing high CBHŽ . Ž .levels in both salinities 210 mgrml P-0.05 . In shrimps maintained at 15‰ salinity,

Ž .a high-GDH activity 27.8 mUIrmg protein was recorded in shrimps fed the low CBHŽdiet. In shrimps maintained at 40‰ salinity, a higher GDH activity 39.5 mUIrmg

.protein was recorded in shrimps fed the low CBH diet in comparison to that obtained inŽ .shrimps fed with a high CBH level 25.4 mUIrmg protein . That GDH activity was the

Ž . Žhighest activity obtained in all treatmentsP-0.05 . The lowest GDH activity 11.03.mUIrmg protein was obtained in shrimps fed a diet containing a high CBH level and

Ž .maintained at 15‰ salinityP-0.05 .

( ) ( )3.1.5. Osmotic pressure OP , hemolymph ammonia concentration HACŽ .Osmotic pressure Table 3 was affected by salinity, with low values in shrimps

Ž .maintained at 15‰ salinity 692 mosMrkg and higher values in shrimps maintained inŽ . Ž .40‰ salinity 813 mosMrkg P-0.05 . There were no differences between type of

Ž .food in each salinity P)0.05 .

Ž .Fig. 2. Effect of dietary CBH levels on gill GDH activity mUIrmg protein and protein hemolymphconcentration of white shrimpL. Õannamei juveniles exposed at different salinities. Mean"S.E.


Table 3Ž .Effect of carbohydrate levels CBH on physiological and biochemical responses of white shrimpL. Õannamei

juveniles exposed to different salinities

15‰ 40‰

High CBH Low CBH High CBH Low CBHlevel level level level

a a b bAmmonia in hemolymph 2.9"0.25 4.4"0.30 5.3"0.45 7.2"0.41Ž .mgrl

a a b bOsmotic pressure 692"12 708"9 813"8 798"13Ž .mosMrkg hemolymph

a a b bExternal osmotic pressure 497"4 491"3 1163"7 1162"9Ž .mosMrkg

Mean"S.E.Ž .Entries with different letter are significantly differentP-0.05 .

ŽThe HAC varied with dietary protein–salinity combinations with high values 7.2.mgrl in shrimps fed diet containing low CBH levels and maintained in 40‰ salinity,

Ž .and low values 2.9 mgrl in shrimps fed diet containing high CBH levels andŽ .maintained in 15‰ salinityP-0.05 . In both salinities, a higher HAC was observed in

Ž .shrimps fed with low CBH levelsP-0.05 .

3.2. Second experiment

3.2.1. Oxygen consumptionOxygen consumption varied directly with CBH level. The oxygen consumption rate

Ž . Ž y1 y1of fasting shrimps time 0 fed a diet containing a high CBH level 0.35 mg O h g2. Ž y1ww was 42% less than shrimps fed a diet containing a low CBH level 0.60 mg O h2

y1 . Ž .g ww Fig. 3 . Oxygen consumption rate increased after feeding in each of thetreatments, starting 1 h after feeding and returning to the prefeeding rate 4 h later.Shrimp fed with a low CBH level did not return to the fasting oxygen consumption. The

Ž .Fig. 3. Oxygen consumption mgrhrg of fasting and feeding white shrimpL. Õannamei juveniles fed dietscontaining different CBH levels. Mean"S.E.


maximum oxygen consumption as a percentage of fasting rate was obtained in shrimpsŽ .fed a diet containing a low CBH level 174% in comparison with that obtained in

Ž . Ž .shrimps fed a diet containing a high CBH level 121% Table 4 .In shrimps fed with a high CBH level, the AHI was 244% higher than that obtained

Ž .in shrimps fed with diet containing a low CBHP-0.05; Table 4 . In shrimps fed withboth diets, the time to reach the peak was 2 h.

3.3. Ammonia excretion

The ammonia excretion increased after shrimp from both treatments were fed,Ž .starting 2 h after feeding and returning to prefeeding rates 3–4 h later Fig. 4 . A higher

ammonia excretion was observed in shrimps fed a diet containing a low CBH than thatŽ .observed in shrimps fed a diet containing a high CBHP-0.05 .

In shrimps fed a diet containing low CBH, the PPNE was 175% higher than thatŽ .obtained in shrimps fed a diet containing high CBHP-0.05; Table 4 . The maximum

ammonia excretion as a percentage of fasting rate was obtained in shrimps fed a dietŽ .containing low CBH 170% in comparison with that obtained in shrimps fed dietŽ . Ž .containing high CBH 157% P-0.01 . The time to reach the peak was 2 h in shrimps

from both treatments.

3.3.1. O:N and PPNErAHI ratiosŽShrimp fed diets containing both CBH levels had a similar fasting O:N ratio mean

. Ž .value 5.72 indicating a protein metabolism Fig. 5 . After feeding, shrimps fed a dietcontaining low CBH had their OrN ratio decrease to between 3 and 4, maintaining the

Ž .protein metabolism P-0.05 . In contrast, the OrN values from shrimps fed a dietcontaining high CBH had the OrN ratio increase after 3 h to a maximum of 19.5,indicating a change from protein metabolism to protein, lipid, and carbohydrate mixed

Ž . Ž .metabolism Fig. 5 . The role of PPNE in AHI PPNE-AHI, % was 92% in shrimps fed

Table 4Ž . Ž .Effect of dietary CBH level on postprandial nitrogen excretion PPNE , apparent heat increase AHI and

PPNErAHI ratio of white shrimpL. Õannamei juveniles maintained in 40‰

Type of food

Low CBH level High CBH level)Ž .Apparent heat increase Jrhrg ww 1.57q0.33 3.52"0.17

Ž .Time to reach the pike h 2 2Ž .Oxygen consumption % increment after fed 174 121

)Ž .Postprandial nitrogen excretion Jrhrg ww 1.44q0.15 0.82"0.09Ž .Time to reach the pike h 2 2

Ž .Ammonia excretion % increment after fed 170 157Ž .PPNErAHI % 92 22

Mean"S.E.)Means statistical differencesP-0.05.


Ž .Fig. 4. Ammonia excretion mg N–NHrhrg of fasting and feeding white shrimpL. Õannamei juveniles fed3

diets containing different CBH levels. Mean"S.E.

Ža diet containing low CBH and 22% in shrimps fed a diet containing a high CBH Table.4 .

3.3.2. PECPK actiÕity, hexokinase actiÕity, and digestiÕe gland glucose and glycogenconcentration

The PECPK digestive gland activity was affected by the dietary CBH level. TheŽ .high-PECPK activity 0.72 UIrmg protein was recorded in shrimps fed a diet contain-

ing low CBH. This value was significantly higher than that obtained in shrimps fed aŽ . Ž . Ž .diet containing high CBH 0.40 UIrmg protein P-0.05 Table 5 . In contrast, a

Ž .higher glycogen and glucose concentration 9.1 mgrg tissue and 22.6mgrmg proteinswas observed in shrimps fed a diet containing high CBH than those measured in shrimps

Ž .fed a diet containing low CBH P-0.05 . Hexokinase activity is low and is notinfluenced by the level of dietary carbohydrates. An inversion of the diet led to an

Fig. 5. Effect of dietary CBH levels on NrO ratio on fasting and feeding white shrimpL. Õannamei juveniles.Mean"S.E.

()

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osaset

al.rJ.E

xp.Mar.B

iol.Ecol.259

20011

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15

Table 5Variation of glucose, glycogen, hexokinase and PEPCK in the hepatopancreas of white shrimpL. Õannamei juveniles fed on different carbohydrate diet contents

Low to high CBH High to low CBH

Ž . Ž .Time days Time days

0 2 4 6 8 0 2 4 6 8a a b b c b b b a dŽ .Glycogen mgrg 6.0"0.3 6.2"0.8 10.4"1.5 9.9"1.2 10.7"1.9 9.1"0.6 8.4"0.6 9.7"0.6 6.5"0.3 5.9"0.2a a b b b a a aŽGlucose mgrmg 12.4"2.4 13.0"2 22.0"1.6 26.1"2.5 22.6"1.4 11.4"1.6 14.2"2.2 11.1"2.1

.proteinsa a b c b c b cŽPECPK UIrmg 0.72"0.1 0.78"0.2 0.40"0.01 0.16"0.01 0.40"0.11 0.13"0.03 0.40"0.05 0.13"0.01

.proteinsa a b c a a c bŽHexokinase mUIrmg 12.8"1.7 9.6"1.1 3.2"0.6 6.4"0.5 11.2"1.4 9.6"1.6 6.4"0.9 3.2"0.25

.proteins

Ž .A mean of eight digestive glands were analyzed per each diet and mean standard error S.E.M. are given.Different letters means statistical differences,P-0.05.


increase of glucose and glycogen with high carbohydrate adaptation, and a decrease ofglucose and glycogen with low carbohydrate adaptation after 4 days. Between day 0 and6, no change for PEPCK or hexokinase activity was detected; however, we observed a

Ž .consistent reduction of activity of both enzymes after the change of diet Table 5 . Atday 8, a significant increase of hexokinase activity was measured in shrimps after a

Ž .change of diet from low to high CBH levelsP-0.05 .

4. Discussion

CBH metabolism ofL. Õannamei is limited and governed by protein metabolism. InŽ .both experimental salinities, the high dietary CBH level 36% caused a lower growth

Ž .rate than that in shrimps fed with a low CBH diet. Carbohydrate CBH metabolism isgoverned by protein metabolism because shrimp can produce enough HG and DGG with

Ž .almost no CBH in the diet 1% , showing the important role of protein in thegluconeogenic pathway. The maximum growth rate was in shrimps maintained at 15‰

Ž . Ž . Žsalinity and with a low CBH diet 1% . This diet had a high protein level 50% Table.2 .

To explain the preceding, we need to consider the role of dietary protein both ingrowth and in the physiological adjustments associated with the maintenance of thehomeostasis in low salinity.

In low salinity, shrimp need to use protein as source of amino acids to maintain theŽ .osmotic pressure and for growth Claybrook, 1983 . When shrimps are fed with a low

Ž .CBH level 1% , protein can be used as a source of energy also. During salinityŽacclimation, a very rapid change in free amino-acid content occurs Gerard and Gilles,

.1972 , suggesting that the regulation of cell volume after a hypoosmotic change is arapid process in crustaceans. Although inL. Õannamei, the final free amino-acid poolŽ .FAA level is reached 24 h after a hypoosmotic shock from 37.5‰ to 28‰ salinityŽ .Richard et al., 1975 ; in the present study, there was a decrease in blood osmoticpressure in shrimps acclimated for 30 days to 15‰ salinity, indicating that extracellularregulation is not powerful enough to ensure homeosmoticity, and the tissue will, in

Ž .consequence, undergo osmotic stress. A high dietary protein level 50% , as used inŽ .shrimps fed with a low CBH diet 1% , will be necessary to ensure the FAA are

supplied at low salinity without affecting the proteins used for growth or as a source ofŽ .metabolic energy. Recently, Shiau 1998 showed that protein requirements ofP.

Ž .monodon reared at 16‰ salinity was higher 44% than that observed in shrimpsŽ . Ž .maintained in 40‰ salinity 40% . Although Shiau 1998 did not explain why the

protein requirement increased in low salinity, we can hypothesize that as inL.Õannamei, L. monodon use more protein as a source of FAA, which is the basis forallowing it to compensate its homeosmoticity.

In the present study, we observed that protein in hemolymph is related to the lowCBH level in the diet and, in consequence, to the high protein levels. This diet produced

Ž .a high protein concentration in hemolymph in both salinities Fig. 2 , showing thatprotein metabolism, in general, is enhanced when shrimps are fed with high protein

Ž .levels. Marangos et al. 1989 suggested that high-hemolymph protein concentration


indicates that hemolymph, through hemocyanin, is able to store proteins after salinityacclimation. After a salinity change, the loss of FAAs from muscle results from theirexcretion into the blood. This must impose an additional osmotic load on the blood,which would increase water inflow from an external medium. Transfer of FAAs to thegastric fluids into the digestive gland provides a means of minimizing this additional

Ž .load Dall, 1975 . The digestive gland is thought to be an important synthesizing organof hemocyanin, and products of this synthesis might cause an increase in hemolymph

Ž .protein content when the FAAs are transferred from blood Gellisen et al., 1991 . Theincrease in digestive gland weight reported inM. japonicus after a salinity change can

Ž .be used to confirm that mechanism Marangos et al., 1989 . In whatever form theosmotically active nitrogenous components of the tissues are excreted inL. Õannamei, itis clear that adaptation to lowered salinity involves a loss from the body of organicmolecules. Although production of these substances by the tissue appears to beintracellular, ultimately they have to be supplied by the food. We think that in lowsalinity, the high growth rate observed in shrimps fed diets low in CBH and, inconsequence, high protein levels, resulted from the use of amino acids provided by foodas osmotic effectors, which reduced the loss of amino acids from muscle, and promotedgrowth. In such circumstances, the loss of muscle weight reported in other shrimp

Ž .species after a salinity change Marangos et al., 1989 might be compensated through anincrease in dietary protein level. The hemolymph protein content observed in the presentstudy was independent of salinity and controlled by dietary protein levels; high-hemo-lymph protein levels were measured in shrimps fed with diets containing 50% proteinŽ .Fig. 2 . After digestion and absorption, the amino acids are transported by the blood

Žand carried through the body to the various tissues, where they are absorbed Smith and.Dall, 1991 . Because hemocyanin is the most abundant protein in hemolymph and it can

Ž .be used by shrimp as a protein store Marangos et al., 1989 , we suggest that the proteinhemolymph increase observed inL. Õannamei juveniles was hemocyanin, which wasused to accumulate proteins. Depending on salinity, this hemocyanin could be used as a

Ž .source of osmotic effectors or as metabolic energy Dall and Smith, 1986. Recently,Ž .Condo et al. 1991 showed that crustacean hemocyanin could be useful as an adaptive`

molecule to environmental changes, because crustaceans can manufacture one or morehemocyanin types, allowing adaptation to their own particular ecological, behavioural,and physiological milieu. From our results, as with hemoglobin in mammals, a nutri-tional role can be included in the crustaceans’ hemocyanin. Recently, an increase ofprotein content in hemolymph related with hemocyanin had been observed inP.

Ž . Ž .monodon Chen and Cheng, 1995 andM. japonicus Chen and Cheng, 1993 .The low growth rate observed in the high salinity–dietary protein combination could

be caused by theAgrowth-depression effectB observed in other shrimp species fed withŽ .high protein diets Millamema et al., 1998 . Although thisAeffectB has not been fully

established, results obtained in this study suggest that the toxicity of hemolymphammonia in shrimps maintained in salinity of 40‰ and fed a low CBH diet had animportant role. Ammonia-N may affect various metabolic process, such as oxygentransport and osmotic pressure, which may compromise the normal functioning of the

Ž .shrimp affecting the growth rate and survival Schmitt and Santos, 1999 . According toŽ .Claybrook 1983 , this ammonia is the product of both oxidation of hemolymph proteins


Žand the amino acid pool lost from the muscle. In shrimp with lower growth rate 40‰.salinity, low CBH diet and high dietary protein , the hemolymph ammonia concentration

Žwas significantly higher than that observed in shrimp with higher growth rate 15‰. Ž .salinity and high CBH level , which suggests that this level 7.2 mgrl of HAC might

Ž .be toxic for juvenileL. Õannamei Table 3 . This high HAC is close to that reported toŽ .affect the growth and survival ofP. monodon Chen and Lin, 1992, 1995 , and the

oxygen consumption and oxyhemocyanin, acylglycerol, and cholesterol ofL. ÕannameiŽ .juveniles Racotta and Hernandez-Herrera, 2000 .´

Our results suggest that GDH activity regulated both HAC and hemolymph proteinŽ .levels, with high values in shrimps fed with a low CBH diet and high protein diet and

maintained in 40‰ salinity and lower in shrimps fed with high CBH and maintained inŽ .15‰ salinity Fig. 2 . These results mean that shrimp with a high-gill GDH activity

might waste more energy in oxidizing excess amino acids, reducing the energy forgrowth. Although we only made measurements in the gill GDH activity, we can expectthat GDH from muscle, digestive gland, and heart increased with the hemolymph

Ž .proteins, affecting the energy available for growth. Regnault 1993 showed that muscleGDH activity was affected by nutritional condition of the crabCancer pagurus.

In shrimp, the use of proteins as a source of energy is well documented. Dall andŽ .Smith 1986 showed in shrimp that a significant part of the dietary protein must be

metabolized for energy. In the present study, the metabolic substrate used byL.Ž .Õannamei juveniles was affected by dietary CBH levels Fig. 5 . Using diets containing

a low CBH level, shrimp used only proteins as a source of energy, both during fastingŽ .and feeding OrN between 6 and 3 . Although in shrimps fed diets containing high

CBH levels, the fasting OrN ratio showed a protein metabolism; after feeding, shrimpsŽchanged their metabolic substrate to a protein–carbohydrate–lipid mixed substrate Fig.

.5 . The effect of dietary protein levels and proteinrenergy ratio on OrN ratio has beenŽ .well documented. Rosas et al. 1995 showed that the OrN ratio of L. setiferus, L.

schmitti, P. duorarum, and P. notialis postlarvae was modified by diet protein levels,indicating protein substrates at high protein levels and mixed substrates at optimal

Ž .protein levels. Similarly, Taboada et al. 1998 showed the OrN ratio of L. setiferusjuveniles changed in relation to dietary protein levels. These authors reported thatshrimp consuming 30% protein used a mixture of lipids and protein as a metabolicsubstrate with OrN values of between 32 and 45.

AHI has been associated with the caloric effect of food. This is a measurement ofŽmetabolic activity of post-absorptive processes following food ingestion Beamish and

.Trippel, 1990 . AHI in crustaceans depends on the quality, quantity, and energeticŽ .component balance of the food Du-Preez et al., 1992; Rosas et al., 1996 . In the present

study, we observed that variations of the dietary CBH levels affected the AHI and PPNEof L. Õannamei juveniles reflecting the effect of food proteins. According to Rosas et al.Ž .1996 , the role of nitrogen metabolism in the overall metabolism of shrimp is a keyfactor in the AHI because the deamination and synthesis of protein are probably thegreatest contributors. Production of ammonia results mainly from the catabolism ofamino acids of both alimentary and metabolic origin, affecting the quantity of proteinsthat can be used for growth. Because PPNE is a measure of excreted ammonia of

Ž .alimentary origin Gibson and Barker, 1979 , it can be associated with AHI through


PPNErAHI ratio. It can be seen in Table 4 that the role played by PPNE in AHIŽ . Ž .PPNErAHI ratio is lower 22% in shrimps fed diets containing high CBH in

Ž .comparison with shrimps fed diets containing low CBH levels 92% . These resultsnewly confirm that the metabolism ofL. Õannamei juveniles is controlled by dietaryprotein levels, affecting the processes involved in the mechanical and biochemicaltransformations of ingested food. Similar regulation of dietary protein levels on

Ž . Ž .PPNErAHI ratios were observed by Taboada et al. 1998 and Rosas et al. 1996 withL. setiferus juveniles-1 g ww fed with purified diets.

It was evident from the results obtained in the present study thatL. Õannamei canconvert protein to glycogen by the gluconeogenic pathway, which permitted shrimp tomaintain a minimum circulating glucose concentration of 0.34 mgrml in the hemolymph.A high-PECPK activity was measured in shrimps fed the low CBH diet, indicating that

Ž .the gluconeogenic pathway is activated, as in vertebrates Peret et al., 1981 by a lowdietary CBH level. The crabC. granulata, like L. Õannamei, is able to grow in differentsalinities and with a high-glucose synthesis capacity from alanine-14C competes with theuse of amino acids for osmoregulation. The relation between the gluconeogenic pathway

Ž .and the adaptation to hypoosmotic stress inC. granulata Da Silva and Kucharski, 1992has been demonstrated. The low DGG concentration obtained in low salinity and with alow CBH diet suggests that inL. Õannamei, like C. granulata, the glycogen synthesisdepends on the intensity of the use of amino acids in osmotic regulation, which, in turn,

Ž . Ž .was enhanced by the low salinity 15‰ and high dietary protein level 50% .After a change of diet, the change of PEPCK activity took 6 days, showing that the

Ž .adjustments related to the adaptation to a new diet are longer than in vertebrates 2 daysŽ .Moon, 1988 .

The crustaceans tissue FAA pool, which is 10 times greater than in vertebratesŽ .Munday and Poat, 1971 , should be taken in consideration to understand the role of theFAAs in carbohydrate biosynthesis.

Acknowledgements

This project was partially supported by an ECOS and ANUIES program of collabora-Ž .tion between France and Mexico no. M97B04 , by Direccion General de Atencion al´ ´

Personal Academico-UNAM, CONACYT-FOSISIERRA and IMP-FIES 96F49VI. Spe-´cial thanks are given to Industrias Pecis, SA de CV and Ocean. Ramon Mendez Lanz,´ ´President of Fideicomiso para estudios y proyectos from Fisheries Secretary of Campeche

[ ]State Goberment. Thanks to Dr. Ellis Glazier for editing the English-language text.SS

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