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1 Plese note tht this is n uthor-produced PDF of n rticle ccepted for puliction following peer review. The definitive pulisher-uthenticted version is ville on the pulisher We site Journl of Shellfish Reserch April 24 ; Volume 23 (1) : Pges Ntionl Shellfisheries Assocition All rights reserved. Archimer The effect of strvtion on refeeding, digestive enzyme ctivity, oxygen consumption, nd mmoni excretion in juvenile white shrimp Litopeneus vnnmei Lur I. Comoglio 1, *, Griel Gxiol 2, An Roque 3, Gérrd Cuzon 4, Oscr Amin 1 1 Centro Austrl de Investigciones Científics (CADIC CONICET). CC92 (V941BFD) Ushui, Tierr del Fuego, Argentin. 2 Lortorio de Ecologí y Biologí Mrin Experimentl, Fcultd de Ciencis, Sede Ciudd del Crmen, UNAM. Clle 26 N Col Ply Norte, Cd. Del Crmen, Cmpeche, México. 3 Lortorio de Bcteriologí de Crustáceos. Centro de Investigción en Alimentción y Desrrollo. Unidd en Acuicultur y Mnejo Amientl. CIAD, Sálo Cerritos S/N, Apdo postl 711. Mztlán, Sin., México. 4 Centre Océnologique du Pcifique, Thiti, IFREMER, Frnci. *: Corresponding uthor : Lur Comoglio, emil ddress : lcomoglio@hotmil.com Astrct: Juveniles of the white shrimp Litopeneus vnnmei were kept Without food for etween to 15 clys to evlute the impct of strvtion oil physiologic stte (oxygen consumption, poststrvtion refeeding, index, nitrogen excretion, nd O:N rtio) nd digestive enzymes ctivity. Physiologic chnges were found fter 6 dys of fsting, nd refeeding ility declined s result. Nevertheless. the shrimp were le to Survive 16 dys Without food. Strvtion Cused metolism to drop progressively towrd sl level (21 J (.) h(-1.) g(-1)) nd decrese in the rte of mmoni excretion, ecuse of the ctolism of mino cids front solule protein in the heptopncres. This decrese led to n increse in digestive enzymes specific ctivity (U/mg protein). But, expressed s totl U. ll digestive enzyme ctivities decresed in the sence of sustrte from.16 to.7 U/heptopncres (HP) for lph-mylse nd 2.58 to.63 U/HP for totl trypsin. L. vnnmei juveniles showed true physiologic dpttion mechnism to food depiivtion: no chnges in ody weight ut loss in heptosomtic index, no exuvitions, including the utiliztion of HP solule proteins ( drop from 269 to 53 mg/ml). After 1 dys. neoglycogenic pthwy nd the corresponding tissue enzymes ctivities seemed enhnced, nd the nimls derived ll energetic Sustrtes minly from protein (O:N rtio of 17) to cover their metolic costs. Estimtes of sl metolism (Hem) from the routine respirtion rte per cly (from 361 to 725 J (.) g ww(-1) (.) dy(-1) through the 15-dy strvtion period). nd loss of nonfecl energy (HxE) from the nitrogen excretion rte (vrying from 39 to 57 J (.) g ww(-1) (.) dy(-1) during the sme period) were used in ioenergetic prtition model of fsting juvenile. Which indicted tht the energetic requirement to Survive Without feeding ws in the rnge of 418 nd 771 J (.) g ww(-1) (.) dy(-1) during the 15-dy period of strvtion. Keywords: shrimp, Litopeneus vnnmei, strvtion, metolic rte, digestive enzymes. 1

2 1. Introduction The Pcific white shrimp Litopeneus vnnmei is cultured in extensive, intensive, nd semi-intensive systems nd is, with Litopeneus stylirostris, the most populr shrimp for quculture in Mexico nd Centrl nd South Americ. The stte of Sinlo on the Northwest cost of Mexico, considered the most importnt griculturl producer in the country, hs oserved in recent yers n importnt increse of quculture frms, from pproximtely 1 h of pounds in 1984, to 185 h in 1998, with growth rte of out 15 h/yer. Nowdys, Sinlo hs more thn 2 shrimp frms (out 75% of the ntionl totl) nd produces round 1 T yerly (65% of the ntionl totl) (Hernndez Cornejo & Ruiz Lun 2). The digestive glnd is generlly regrded s mjor storge orgn in decpods crustcens (Huggins & Mundy 1968, Allen 1971). The study of the digestive glnd is of considerle interest ecuse of its role in the ccumultion nd cyclic moiliztion of reserves during the molting process, its contriution of nutrients to the ovry during vitellogenesis, the moiliztion of its reserves during strvtion, nd its role in digestion nd sorption. The level of the digestive enzymes in decpod crustcens does not remin constnt during the developmentl cycles (Vn Wormhoudt 1974) s result of oth externl nd internl fctors. Among the externl fctors, the quntittive nd qulittive vriility of food is poignnt. Physiologicl nd iochemicl effects of strvtion hve een studied in severl decpod species (Cuzon & Ceccldi 1973, Cuzon et l. 198, Anger 1986, Dll & Smith 1986, Wehrtmnn 1991), ut little is known out the effects of prolonged food deprivtion. Chnges in iochemicl composition during strvtion hve een reported in Mrsupeneus jponicus where there ws progressive suppression of metolism compred with normlly fed shrimp (Cuzon et l. 198). The study found tht the shrimps utilized crohydrtes primrily, then lipids to meet their energy requirement; proteins were utilized significntly only during the fourth week of strvtion (Cuzon et l. 198). In L. vnnmei postlrve, tricylglycerol provided energy during short periods of strvtion, wheres protein ws utilized during prolonged strvtion (Stuck et l. 1996). Modifictions in digestive enzyme ctivity hve een found in severl peneid shrimp nd relted to the mount nd qulity of food (Le-Vy et l. 1993, Rodriguez et l. 1994, Le Moullc et l. 1996, Lemos & Rodriguez 1998). Ross et l. (1985) found tht the type nd concentrtion of food influenced the ingestion rte of lrvl L. setiferus, which, in turn, ffected metolic rte. Chnges in digestive enzyme ctivity were found under strvtion conditions in juveniles of Mrsupeneus jponicus (Cuzon et l. 198) nd Metpeneus ensis (Leung et l. 199). Both studies pointed out decrese in digestive enzyme ctivity compred with fed shrimps. Nitrogen excretion nd metolic rtes re influenced y mny fctors, such s moult stge, feeding conditions nd level of ctivity. In this sense, O:N rtio hs een used widely s n index of utilized sustrte for oxidtive metolism (Chu nd Ovsinico- Koulikowsky 1994, Regnult 1981, Dll & Smith 1986, Ross et l. 1995). The ctolism of pure protein produced theoreticl vlues of O:N of etween 3 to 16, wheres the ctolism of equl quntities of proteins nd lipids yield O:N vlues of etween 5 nd 6. Greter vlues of O:N correspond to n increse in lipid nd crohydrte ctolism (Myzud & Conover 1988). The im of this study ws to evlute the effect of strvtion on digestive enzyme ctivity, oxygen consumption, mmoni excretion, nd O:N rtio in Litopeneus vnnmei juveniles. Afterwrds the effect of re-feeding of strved juveniles ws nlyzed to ssess the cpility of shrimp to recover fter prolonged strvtion. 2

3 This informtion my contriute to etter understnding on the physiology of this commercilly importnt shrimp species nd will hve n ppliction in ssisting the mngement in frms. 2. Mterils nd methods Experimentl conditions nd design. Juveniles of Litopeneus vnnmei were provided y commercil frm, Mricultur del Pcífico, locted in Mztlán, Sinlo, Mexico, during Mrch 21. The orgnisms were trnsported to the lortory culture system of the Centro de Investigción en Alimentción y Desrrollo (CIAD) nd fed with commercil food during the cclimtizing period in 1-L tnks. They were then trnsported to the lortory, nd groups of 2 orgnisms (wet weight =.998±.213 g) were mintined for different strvtion periods (-control group-, 3, 6, 9, 12, nd 15 dys) in 1-L glss quri. Experimentl conditions were: 34o/oo slinity, 24 C wter temperture nd 12D:12L. Wter ws completely renewed dily nd molts nd ded orgnisms were removed. Oxygen consumption, mmoni excretion, nd tomic rtio (O:N). After ech strvtion period groups of 5 individuls were plced in individul 5-mL respirometer chmers in flow-through system using continuous pump flow (ISMATEC, 12 ml/min flow). All mesured were done t the sme dy time in order to otin comprle dt. Orgnisms were cclimted for 2 h in the ekers nd smple of wter from ech chmer ws tken to determine the initil concentrtion of oxygen nd mmoni; flsks were seled for 3 min, fter which new smples were tken to mesure finl concentrtions. The flow-through nd the seled periods were djusted to void depletion of oxygen concentrtion y more thn.5 mg/l. Smples for mmoni excretion were filtered nd fixed with H2SO4 (ph 2) nd then frozen until processing. The concentrtion of oxygen ws mesured using polrogrphic oxygen electrode (YSI 59) nd mmoni ws determined y the indophenol technique (Prsons et l. 1984). Consumed oxygen nd excreted mmoni were tken s eing the net difference etween the strt nd end of the seled period. One out of every six chmers ws left without shrimp nd mesured s control. The tomic O:N rtio ws estimted ccording Tod et l. (1998) using the individul vlues of oxygen consumption nd mmoni excretion trnsformed to µg At / g/ h s follows: oxygen vlues were multiplied y 62.5 (1 to convert the milligrms into microgrms divided y the tomic weight of oxygen, 16), mmoni vlues were multiplied y 58.9 (otined through dividing 1 y the product of the tomic weight of nitrogen (14) nd the frction of nitrogen in NH3 (.824)). Digestive enzyme ctivities After physiologicl mesurements, shrimps were dissected nd the heptopncres stored t 7 C in individul 1-mL microtues until enzyme ssys were done. Frozen smples were homogenized in 1 ml ice-cold pure wter. Homogentes were centrifuged (t 14 x g for 6 min t 4 C) nd the queous superntnt, crude or diluted (1:1 v/v), ws immeditely used for enzyme nlysis. The solule-protein content ws mesured y the method of Brdford (1976), using micro-plte reder t 495 nm. 3

4 Duplicte ssys for ech smple were mde. Trypsin ctivity ws mesured y the method of Erlnger et l. (1961) with Nα-enzoyl-DL-Arg-p-nitronilide (BAPNA) s sustrte. Chymotrypsin ctivity ws ssyed y the method of Delmer et l. (1979) using Nα-succinyl-L-lnyl-L-prolyl-L-phenyl-lnine-4-nitronilide (SAPNA) s sustrte. Hydrolysis for oth enzyme ctivities ws mde in.1 M Tris-uffer, ph 8 t 25 C nd the sornce mesured t 45 nm. One unit of enzyme ctivity ws defined s 1 µmole of p-nitronilide lierted in 1 min t 25 C. α-mylse ctivity ws ssyed ccording to Bernfeld (1955) with 1,5% oyster glycogen s sustrte in 1 mm phosphte uffer, ph 7. Asornce mesurements were mde t 52 nm. For this method, one unit of enzymtic ctivity ws defined s 1 mg of mltose lierted in 1 min t 37 C. Generl protese ctivity ws estimted in homogentes using zocoll s sustrte in phosphte uffer, ph 7.5 (Todd, 1949). Asornce ws mesured in spectrophotometer t 52 nm. For this method, one unit ws defined s the mount of enzyme tht ctlyzes the relese of zo dye cusing A/ t=.1min (Wlter, 1988). α-glycosidse ctivity ws estimted using p-nitrophenyl- α-d-glycopyrnoside s sustrte in 5 mm phosphte uffer, ph 6. Asornce ws mesured t 41 nm. One unit of enzymtic ctivity ws defined s the mount of enzyme tht hydrolyzes 1 µm of sustrte per minute. Post-strvtion re-feeding index (PSRFI) Other sugroups of orgnisms for ech tretment (n=6) were kept in individul chmers nd exposed to known mount of food previously lyophilized. The proximte composition of the food ws 35% protein, 3.5% oil, 3% fier, 16% sh, 12% humidity; verge weight of ech lyophilized pellet ws 15.14±2.28 mg; clorific content, 5.5 cl/mg of food. After 2 h 3 min the non consumed food ws removed nd lyophilized gin. The consumed food ws clculted to mesure the post-strvtion re-feeding index, defined s PSRFI (Food consumed W/Body W). Bioenergetics model From ioenergetic point of view, in order to integrte model of energy prtition for strved shrimp, we followed n eqution (Bureu et l. 2): DE requirement = (RE+Hem+HiE+HxE (UE+ZE)+SE), This model originlly included prmeters where DE is digestile energy requirement; RE is energy gin; HEm is mintennce of energy requirement; HiE is het increment of feeding; HxE is non-fecl energy losses; nd SE surfce loss or exuvie. With this model generl energy lnce model for strvtion conditions in shrimp cn e clculted. However, in strved shrimps, the following prmeters cn e considered s : DE= ( no feeding) HiE= (no het increment of feeding) SE = ( no exuvi), Hence, model for strving shrimp could e: 4

5 RE = -(Hem+HxE (UE+ZE) in which RE cn e understood s endogenous energy needed to survive (Ross, pers. comm.) nd it is negtive ecuse in sence of energy input it is not possile to hve energy gin sensu stricto. It is clled lso the scope for growth (SFG). Using this model, we clculted the endogenous energy (RE) for strving shrimp. Sttisticl nlysis To determine significnt differences mong strvtion periods one wy ANOVA nd Tuckey rnge test were used, when the dt were norml. Those results with no normlity were evluted through the Kruskl Wllis test nd Dunn s multiple comprisons (Dniels 1978). For oth nlyses, p ws set t Results Weight nd Survivl No significnt differences were oserved (p>.5) in reltion to individul wet weight mong tretments. However, survivl decresed the longer the period of strvtion (Tle 1). The percentge of molts were pproximtely 1% for control, 3 nd 6 dy tretments, fter tht no molt were recorded. Oxygen consumption, mmoni excretion, nd O:N rtio Oxygen consumption incresed significntly t 3, 6, nd 9 dys in strved nimls (men vlue 1.75 mg O 2 /h /g) compred with the control. At 12 nd 15 dys vlues were not significntly different from the control group (men vlue 1.13 mg O 2 /h /g). (Kruskl-Wllis, H=14.1; p=.15) (Figure 1). There were no significnt differences in the mount of mmoni excreted etween strved nd control nimls (men vlue.8 mg N-NH 3 /h /g, Kruskl-Wllis, H=1.74, p=.88) (Figure 2). This my e ecuse high stndrd devitions were oserved, especilly in the 6 nd 12 dys strvtion groups (.3 nd.67 mg N-NH 3 /h /g, respectively). No significnt differences etween tretments were oserved with regrds O:N tomic rtios (Kruskl-Wllis, H=1.14, p=.71) (Figure 3). However, there ws trend towrds incresed rtios etween dys 3 nd 9, suggesting tht lipid ws eing ctolised. By dy 12 the drop in the rtio to 1-2 suggests tht protein ws once gin the min source of energetic fuel. Enzyme ctivity Significnt differences were found in the heptosomtic index fter different periods of strvtion (Kruskl Wllis, H=25.6, p=.1). The index ws significntly lower fter nimls were strved eyond 9 dys. Beyond the 9 th dy, index decresed further 5% until reching the end of the experiment (Tle 1). Heptopncretic totl solule protein ws significntly ffected y the period of strvtion (ANOVA, p<.5). The highest vlue 5

6 (268.7 ± mg/ml) ws otined in the control, followed y the 3 nd 6 dy strvtion experiments (men vlue mg/ml). The lowest significnt vlues were found in the 9 to 15 dy experiments (53.23 mg/ml) (Figure 4). Digestive enzyme ctivity ws, in generl, significntly ffected y different periods of strvtion. Heptopncretic digestive crohydrses results re shown in figure 5. Totl mylse ctivity ws significntly decresed y strvtion (ANOVA, p<.5), from the highest vlue in fed juveniles (control) (.16 ±.2 totl U), followed y the 3 nd 6 dy experiments (men vlue.11 totl U), nd the lowest vlue in the 9, 12, nd15 dys strved groups (men vlue.7 totl U) (Figure 5). However, the specific ctivity incresed s the solule protein content decresed (Figure 5). Glycosidse ctivity ws significntly decresed (men vlue 1.47 totl U) y ll strvtion periods s compred to control fed nimls (3.6 ± 1.5 totl U ) (Figure 5c). For this enzyme, the specific ctivity lso incresed s the solule protein content in the heptopncres decresed (Figure 5d). Proteinse ctivity ws significntly ffected y the strvtion periods (ANOVA, p<.5) (Figure 6). The highest vlue (5.3 ± 2.4 totl U) ws oserved in the control, which ws not significntly different from 3 nd 6 dy experiments, wheres the lowest vlue of totl proteinse ctivity (1 ±.12 totl U) ws oserved in the shrimps strved for 15 dys (Figure 6). As with the crohydrses, the specific ctivity of totl proteinses ws highest in shrimps strved for 12 dys (Figure 6). Totl trypsin ctivity ws significntly lower from the 9th dy to the end of strvtion (men vlue.63 totl U). Fed shrimp nd those deprived for 3 nd 6 dys yielded men trypsin ctivity vlue of 2.58 totl U (Figure 6c). Totl chymotrypsin ctivity diminished long the strvtion dys. Significnt differences were found mong the fed shrimps (45.41 ± totl U), the 3 nd 6 dys strvtion groups (men vlue 25.2 totl U) nd the 9, 12 nd 15 dys strvtion groups (men vlue 7.87 totl U) (Figure 6e). The specific ctivities of these ltter two endoproteinses were not significntly ffected y strvtion (Figure 6d nd 6f). Post-strvtion re-feeding index (PSRFI) The PSRFI ws no significntly different mong control, 3 nd 6 dy strvtion experiments, conforming n homogenous group (5.9%; 4.71% nd 4.75%, respectively), wheres the mount of food consumed decresed for the 9, 12 nd 15 dy strvtion groups eing nother homogenous group (3.72%, 3.3%, nd 2.97% respectively, Figure 7) (Kruskl-Wllis, H= 21.5; p=.6). Bioenergetics model Estimtion of RE vlues indictes tht t the eginning s well s t the end of the strvtion period, shrimp needed the sme endogenous energy to survive, while etween 3 nd 9 dys they needed more energy, used essentilly in respirtion to mintin sl metolism (Tle 2). However, the routine respirtion rte mesured ws 61% higher t dy 3 of strvtion thn in fed shrimp, nd remined in the sme high trend until dy 9 of strvtion. After then, vlues diminished to the sme vlue s in controls. 6

7 4. Discussion In the present study the wet weight did not chnge with strvtion, ut survivl rte decresed. Under strvtion conditions, first event commonly oserved is weight loss in reltion with energy expenditure for sl metolism; fter few dys, shrimp in premolt stges will not evolve further nd refrin exuvition, sving round 1.4 kj (Red & Culton 198), which is the energy expenditure t molt. This mens tht shrimp hve the dpttion to tolerte strvtion sving energy from exuvi, including the energy chllenged to moilize reserves, chitin digestion, nd exuvition. In this wy shrimp could compenste the weight loss, mintining ody weight without significnt chnges s oserved in the present study. Homeostsis in shrimp cn chnge to help nimls sustin severe food deprivtion nd survive; it is not instntneous s the post-strvtion nd re-feeding showed; however not ll strved shrimps styed live (Tle 1). It mens tht under the experimentl conditions tested, there ws kind of discrimintion mong shrimp cple or not to dpt nd survive. Prt of the explntion could come from previous life history efore nimls were smpled in the frm nd plced under lortory conditions. Also, individuls will resist food deprivtion for severl dys without significnt moridity nd chnge in some metolic routes s n dpttion to the severe trophic conditions is hypothesized: roust nd fstest growing individuls would hve een selected through frming. L. vnnmei juveniles s mny species of crustcens showed iochemicl dpttion response to n sence of food (decrese of digestive enzyme ctivities) using their own reserves (heptopncretic glycogen, protein nd proly lipids, estimted through O:N rtio vritions) for homeostsis nd to chnnel enough energy for sl metolism in tht period. According to these results, high rte of moiliztion of reserves ws oserved etween dy 3 nd 9 of strvtion, when the recovered energy (RE) ws mximl. Shrimp reserves re minly limited to lipids stored in the digestive glnd. When shrimp re strving, they will use those reserves, incresing the energy det, reducing the digestive glnd weight nd its components. Reduction of enzyme ctivity nd solule protein during strvtion underlined the chnge. It mens the shrimp re iochemiclly nd energeticlly well dpted to fsting ecuse they could moilize their reserves to e used s energetic sources, t the sme time, the enzyme ctivity in the digestive glnd ws mintined. This type of strtegy hs een oserved in other shrimp species. Cuzon et l. (198) showed tht M. jponicus used protein to otin energy though the ctolism of mino cids present in digestive glnd cells during prolonged strvtion period. Similr response ws oserved in Peneus esculentus y Smith nd Dll (1991), evidencing tht shrimp cn moilize their own energetic reserves through ctolizing lipids (fter 3 to 6 dys of strvtion) or protein to sustin t food deprivtion. Otherwise negtive correltion ws oserved in strvtion condition etween totl heptopncretic solule protein nd totl ctivity for ll mesured digestive enzymes. This correltes well with the lower vlues of post-strvtion re-feeding rte. In generl terms, digestive enzymes follow the presence or sence of food. Smin et l. (1983) found tht mylse increses in cse of food deprivtion with pek nd then the enzyme production decreses s n dpttion to low nutrition sttus nd to sve energy. Digestive enzymes of M. jponicus showed similr trend tht supports previous results (Cuzon et l. 198). However, results otined fter re-feeding indicte tht the necessry time to recover the digestive glnd integrity depends on the fsting period. These results show tht lthough L. 7

8 vnnmei hve n dpttion mechnism to tolerte fsting conditions, recovery cnnot e chieved if strvtion is long enough to produce physicl dmge in the digestive glnd nd loss of enzyme synthesis. A pttern of vrition for digestive enzymes presented y L. vnnmei during shorter experimentl period differed from the one of M. jponicus. At dy 15, for exmple, specific enzymes ctivity incresed s sort of dpttion to sor the minute mount of food, ut s energy expenditure incresed, the pek disppered shortly. Both mylses nd proteses exhiited the sme trend. Myzud nd Conover (1988) descried strvtion condition regrding the use of energetic sustrtes estimted through O N: rtio. It cn e ssumed tht cetyl CoA, which is the finl product of β-oxidtion of ftty cids. Such oxidtion required oxygen toms wheres, in norml condition, β-oxidtion of neutrl lipids required oxygen toms to produce cetyl CoA. Increse in O 2 consumption of shrimps etween dys 3 nd 9 of strvtion with no increse of nitrogen excretion, rought high O:N rtio. Then, β-oxidtion of heptopncretic lipid reserves could e occurring in this period. An explntion for chnge cn e found in the O:N rtio in which the use of lipid s energetic sustrte ws cler followed y protein s the min energy source. Ammoni excretion vlues provided indiction on sustrte oxidtion; lthough nitrogen excretion ws not significntly different from the control long the strvtion period, O:N rtio vlues showed trend to use lipids s energetic sustrte etween dys 3 nd 9 of strvtion. Such trend hs lso een evidenced in M. rosenergii (Clifford & Brick 1983). After 12 strvtion dys, shrimp returned to the use of protein to derive energy. Likewise, s Myzud nd Conover (1988) reported for plnktonic crustcens, decrese in O:N rtio with time of strvtion seemed to e common to ll species with predominntly proteinsed metolism. O:N rtio vrition cn e relted to glucose homeostsis, through the regultion of glyconeogenesis (Cuzon et l. 21) nd glycolysis (Hochchk et l. 1988). These results re not in contrdiction to those reported y Dll nd Smith (1986) for Peneus esculentus. These uthors pointed out reduction in metolic rte with strvtion providing mechnism for prolonged survivl. O:N rtios gve n explntion for successive fuel sustrtes s strvtion incresed in intensity (Tle 3). Glycogen in the heptopncres is ffected first, s in Crngon crngon (Regnult 1972), then neutrl lipids re hydrolyzed, nd towrds the end of the strvtion period, protein re utilized similrly s in M. jponicus juveniles (Cuzon et l.198). By nd lrge, it ppers tht tropicl species L. vnnmei living t 24 up to 27 C sewter would hrdly sustin food deprivtion over 15 dys; on the contrry, species such s M. jponicus under temperte conditions (2 C) cn stnd on 4-week strvtion period. They possess similr level of digestive enzyme ctivities; then, there re good resons, ccording to vlues of metolism rtes otined in this study to think tht tropicl species present higher sl metolism nd ctivity tht leves them more dependent on regulr food supply for their development. Acknowledgments This study ws funded y CONACYT- project N (A. Roque), UNAM-DGAPA-IN (G. Gxiol), CONICET-Argentin (L.Comoglio nd O. Amin) nd IOC (O. Amin). We thnk very especilly Dr. Crlos Ross for criticlly reviewing the mnuscript nd Mrs. Ingrid Mscher for editoril ssistnce. 8

9 Tles Tle 1. Wet weight (g), survivl (%) nd heptosomtic index (%) of the food deprived shrimps L. vnnmei. Men ± SD. STARVATION DAYS wet weight (g) 1.7±.1.86±.2.97±.23.99±.3.85±.1 1.4±.12 survivl (%) HSI (%) 4.1±.7 3.3±.4 2.9± ±.4 2.1± ±.9 9

10 Tle 2. Energetic lnce of strved juveniles of L. vnnmei t different strvtion dys. HEm (mintennce of energy requirement); HxE is non-fecl energy losses; RE (the endogenous energy). Men ± SD. Different letters indicte significnt differences (p<.5). Strvtion dys HEm (joules/d/g ww) HxE (joules/d/g ww) RE (joules/d/g ww) 449 ± 94 53±5-473± ± 68 46± ± ±187 39±22-696± ± ±15-655± ± 167 c 57±39-418± ± ±16-473±152 Tle 3. Theoreticl limits of the O:N rtion ccording to Chrmntier (com pers) dys of J 1 J 3 J 6 J 1 J 15 fsting O:N sustrte glycogen triglycerids mino cids 1

11 Figures mg O2 /h /g 3 2,5 2 1,5 1,5 dys of strvtion Figure 1. Routine Oxygen consumption of juveniles of Litopeneus vnnmei fter ech strvtion period. Men± SD. Different letters indicte significnt differences (p<.5). 11

12 mg N-NH3 /h /g,2,15,1,5 dys of strvtion Figure 2. Routine Nitrogen excretion of juveniles of Litopeneus vnnmei fter different strvtion periods. Men ± SD. 12

13 O:N rtio dys of strvtion Figure 3. O:N rtio of food deprived juveniles of Litopeneus vnnmei. 13

14 Solule protein of heptopncres (mg/ml) c c c dys of strvtion Figure 4. Heptopncretic totl protein of the juveniles of Litopeneus vnnmei food deprived. Men ± SD. Different letters indicte significnt differences (p<.5) 14

15 lf Amylse (Totl U/HP),3,25,2,15,1,5 c c c lf Amylse U/mg protein,4,3,2,1 dys of strvtion dys of strvtion A B lf Glycosidse (Totl U/HP) lf Glycosidse U/mg protein,1,8,6,4,2 -,2 dys of strvtion dys of strvtion C D Figure 5. Totl nd specific citivities of α-mylse (A nd B) nd α-glycosidse (C nd D) of food deprived juveniles of Litopeneus vnnmei. Men ± SD. Different letters indicte significnt differences (p<.5) 15

16 Proteinse (Totl U/HP) c c c Proteinse U/mg protein,25,2,15,1,5 -,5 dys of strvtion dys of strvtion A B Trypsin (Totl U/HP) Trypsin U/mg protein,5,4,3,2,1 dys of strvtion dys of strvtion C D Chymotrypsin (Totl U/HP) c c c Chymotrypsin U/mg protein,8,6,4,2 dys of strvtion dys of strvtion E F Figure 6. Totl nd specific digestive proteinses ctivities of juveniles of Litopeneus vnnmei fter different strvtion periods. Men ± SD. Different letters indicte significnt differences (p<.5). 16

17 8 PSRFI (%) dys of strvtion Figure 7. Ingestion rte of juveniles of Litopeneus vnnmei fter the different strvtion periods (PSRFI %). Men ± SD. Different letters indicte significnt differences (p<.5). 17

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