FOOD-DEPRIVATION AFFECTS SEAWATER ACCLIMATION IN TILAPIA: HORMONAL AND METABOLIC CHANGES

The Journl of Experimentl Biology 199, 7 75 (199) Printed in Gret Britin The Compny of Biologists Limited 199 JEB1 7 FOOD-DEPRIVATION AFFECTS SEAWATER ACCLIMATION IN TILAPIA: HORMONAL AND METABOLIC CHANGES MATHILAKATH M. VIJAYAN 1, *, JOHN D. MORGAN 1, TATSUYA SAKAMOTO,, E. GORDON GRAU AND GEORGE K. IWAMA 1 1 Deprtment of Animl Science nd Cndin Bcteril Disese Network, University of British Columbi, Vncouver, BC, Cnd VT 1Z nd Hwii Institute of Mrine Biology, University of Hwii, Kneohe, HI 97, USA Accepted 3 July 199 We tested the hypothesis tht nutritionl stte ffects sewter cclimtion by trnsferring either fed or fooddeprived ( weeks) mle tilpi (Oreochromis mossmbicus) from fresh wter to full-strength se wter. Fooddeprivtion resulted in significnt increse in plsm concentrtions of N +, Cl, cortisol, glucose, totl mino cid, glutmte, serine nd lnine, nd in heptic pyruvte kinse (PK) nd lctte dehydrogense (LDH) ctivities, wheres the prolctin-1 to prolctin-177 rtio (tprl 1 :tprl 177 ) nd plsm prolctin-1 (tprl 1 ), lctte, rginine nd heptic glycogen content nd heptic lnine minotrnsferse (AlAT) nd 3-hydroxycyl- Coenzyme A dehydrogense (HOAD) ctivities were lower thn in the fed group. Sewter trnsfer significntly incresed the tprl 1 :tprl 177 rtio nd plsm concentrtions of N +, Cl, K +, growth hormone (GH), glucose, sprtte, tyrosine, lnine, methionine, phenyllnine, leucine, isoleucine nd vline levels s well s gill N + /K + -ATPse ctivity nd heptic PK nd LDH Summry ctivities, wheres plsm tprl 177, tprl 1, glycine nd lysine concentrtions were significntly lower thn in fish retined in fresh wter. There ws significnt interction between nutritionl stte nd slinity tht ffected the tprl 1 :tprl 177 rtio nd plsm concentrtions of Cl, GH, glucose, sprtte, tyrosine, serine, lnine, glycine, rginine nd heptic PK, LDH, AlAT, sprtte minotrnsferse, glutmte dehydrogense nd HOAD ctivities. These results, tken together, indicte tht fooddeprived fish did not regulte their plsm Cl levels, despite n enhncement of plsm hormonl nd metbolic responses in se wter. Our study lso suggests the possibility tht plsm prolctin nd essentil mino cids my be plying n importnt role in the sewter cclimtion process in tilpi. Key words: Oreochromis mossmbicus, tilpi, stress, metbolism, food-deprivtion, cortisol, growth hormone, prolctin, gill N + /K + - ATPse, ion regultion. Introduction Tilpi (Oreochromis mossmbicus) re commonly found in brckish wter in esturies round the world nd cn tolerte wide rnges of slinity becuse of their efficient ion regultion mechnisms (for review, see Evns, 1993). Gills ply mjor role in ion regultion, nd direct correltion between gill chloride cell density nd N + /K + -ATPse ctivity hs been estblished in this species (Perry nd Wlsh, 199), supporting link between chloride cell function nd ion regultion (for reviews, see Jurss nd Bstrop, 1995; McCormick, 1995). Fish gills re highly oxidtive tissues even in fresh wter (FW) (Mommsen, 19,b), nd the oxygen requirement increses even further when fish re trnsferred to se wter (SW) becuse of the metbolic cost ssocited with ion regultion (Kirschner, 1993). The energy requirement of the gills is thought to be mintined by oxidtion of glucose nd lctte obtined from the circultion (Mommsen, 19,b; Perry nd Wlsh, 199). As the liver is the min site of glucose production in fish (see Surez nd Mommsen, 197), it is likely tht liver metbolism is enhnced in sewter-dpted fish, thereby providing energy substrtes for gill metbolism. However, very little is known bout the reorgniztion of liver metbolism nd the mobiliztion of substrtes during SW cclimtion in fish. Chnges in plsm concentrtions of severl hormones, including cortisol, growth hormone nd prolctin, hve been ssocited with the process of ion regultion nd, consequently with SW cclimtion in fish (for reviews, see Wendelr Bong, 1993; McCormick, 1995). In ddition, these hormones hve been shown to ply role in the mobiliztion of energy substrtes in fish (see Sheridn, 19; Leung et l. 1991; *e-mil: vijyn@unixg.ubc.c. Present ddress: Ntionl Reserch Institute of Fisheries Science, Yokohm 3, Jpn.

M. M. VIJAYAN AND OTHERS Vijyn et l. 199,b). Consequently, some of the effects of these hormones on SW cclimtion my be medited indirectly by providing substrtes for gill metbolism. However, no study hs ddressed the effects of prior nutritionl stte on plsm hormonl nd metbolite profiles, heptic metbolic sttus nd the ssocited sewter cclimtion process in fish. In the present study, we deprived mle tilpi of food for weeks with the specific objectives of (i) exmining plsm N +, Cl nd K + concentrtions nd gill N + /K + -ATPse ctivity in FW nd full-strength SW s mesure of ionic regultion in these nimls; (ii) ssessing whether fooddeprivtion modultes plsm cortisol, growth hormone (GH), prolctin-177 (tprl 177 ) nd prolctin-1 (tprl 1 ) concentrtions in FW nd full-strength SW; nd (iii) exmining plsm glucose, lctte, totl nd free mino cid concentrtions nd liver glycogen content, s well s severl liver metbolic enzyme ctivities, in order to shed light on the substrte mobiliztion ssocited with SW cclimtion in this species. Tilpi (Oreochromis mossmbicus) ws used for this study becuse the mechnisms of ion regultion, including the role of hormones, hve been extensively studied in this species (for reviews, see Evns, 1993; McCormick, 1995). Furthermore, it is known tht tilpi cn cclimte to fullstrength SW, s indicted by plsm ion concentrtions nd N + /K + -ATPse ctivity, when fish re held t slinity of 1 for h prior to incresing the slinity to 3 (Hwng et l. 199). Mterils nd methods Animls Mle tilpi (Oreochromis mossmbicus Peters) were mintined in outdoor tnks with flowing fresh wter under nturl photoperiod (1 h:1 h light: drk) t 5 C for t lest month prior to the strt of the experiment. The fish were seprted into two groups; one group ws fed Purin trout chow once dily to stiety, wheres the other group ws deprived of food for weeks prior to experimenttion. Experimentl protocol Groups of ten tilpi from either the fed group or the fooddeprived group were lightly nesthetized with buffered MS (1 p.p.m.; to prevent the stress response ssocited with hndling) nd trnsferred to either FW or brckish wter (1 ; obtined by mixing FW nd SW). Food-deprived tilpi hd significntly lower body mss (15± g) thn fed fish (1±11 g; mens ± S.E.M., N=1). The FW tnks for both the fed nd food-deprived groups cted s the control, while FW inflow ws shut off in the brckish-wter tnks fter h nd the slinity grdully rose to 3 within h. These fish were held t 3 slinity for nother h prior to smpling. This protocol hs previously been shown to cclimte tilpi to full-strength SW within 3 dys (Hwng et l. 199). Both groups were deprived of food during the experiment. Three dys fter trnsfer, eight fish from ech group were quickly smpled (t 1: h) fter giving them n overdose of buffered MS ( p.p.m.). Blood ws removed by cudl puncture nd the plsm obtined fter centrifugtion (1 revs min 1 for min) ws stored frozen t C for hormone nd metbolite nlysis (see below). The liver ws rpidly dissected out nd frozen between blocks of dry ice nd ws then stored t C until glycogen content nd the ctivities of severl metbolic enzymes (see below) could be mesured. Gill filments were removed from the second gill rch, plced in 1 ml of ice-cold SEI buffer (.3 mol l 1 sucrose,. mol l 1 sodium EDTA,.1 mol l 1 imidzole, ph 7.1) nd stored t C until ssyed for N + /K + -ATPse ctivity (see below). Plsm cortisol, growth hormone, tprl 177 nd tprl 1 concentrtions Plsm cortisol concentrtion ws mesured using commercilly vilble rdioimmunossy kit (Incstr Corp., Stillwter, Minnesot, USA) ccording to Iwm et l. (199), while plsm GH, tprl 177 nd tprl 1 concentrtions were mesured using homologous rdioimmunossy ccording to Ayson et l. (1993). Plsm metbolite nd ion concentrtions Plsm glucose nd lctte concentrtions were mesured colorimetriclly using commercilly vilble kits (Sigm, St Louis, Missouri, USA). Plsm N + nd K + concentrtions were mesured using n ion chromtogrph (Shimdzu, model HIC-A, Shimdzu Corp., Kyoto, Jpn), while plsm Cl concentrtion ws determined by coulometric titrtion (Hle Buchler Instruments, digitl chloridometer). The free mino cid concentrtion in plsm ws mesured fter reverse-phse high-performnce liquid chromtogrphy (HPLC) using precolumn derivtiztion nd fluorometric detection (Pucht et l. 199). Liver glycogen content nd enzyme ctivities The glycogen content of the liver ws mesured fter myloglucosidse hydrolysis ccording to Keppler nd Decker (197). Mesurements of heptic phosphoenolpyruvte crboxykinse (PEPCK), pyruvte kinse (PK), lctte dehydrogense (LDH), lnine minotrnsferse (AlAT), sprtte minotrnsferse (AspAT), glutmte dehydrogense (GDH), 3-hydroxycyl-Coenzyme A dehydrogense (HOAD) nd mlte dehydrogense (MDH) ctivities were crried out using homogeniztion buffer ccording to Henriksson et l. (19). The enzyme ctivities were mesured ccording to Mommsen et l. (19) t C by continuous spectrophotometry (t 3 nm) on microplte reder (ThermoMx, Moleculr Devices Corp., Menlo Prk, Cliforni, USA). The ctivities re expressed s µmoles of substrte consumed or product liberted per minute per grm wet mss of liver. Gill N + /K + -ATPse ctivity N + /K + -ATPse ctivity (µmoles of ADP liberted per hour per milligrm protein) in crude gill homogentes ws

Food-deprivtion nd sewter cclimtion in tilpi 9 determined t 5 C using the microplte reder mentioned bove ccording to McCormick (1993). The protein content in the gill homogente ws determined using the bicinchoninic cid procedure (Smith et l. 195). Sttisticl nlyses The dt were nlyzed using two-wy nlysis of vrince (ANOVA); where P vlues were significnt (P<.5), multiple comprisons were crried out using the Student Newmn Keuls test. Log-trnsformed dt were used wherever necessry to stisfy homogeneity of vrince, lthough non-trnsformed dt re shown in the tbles nd figures. nd sewter trnsfer on plsm GH concentrtion, but not on plsm tprl 177 or tprl 1 levels, GH levels in the food- Plsm [N + ] (mequiv l 1 ) 1 1 1 1 FD <FD A Results Plsm ion concentrtions nd gill N + /K + -ATPse ctivity Food-deprivtion resulted in significntly higher plsm N + nd Cl concentrtions, but hd no significnt effect on plsm K + concentrtion or gill N + /K + -ATPse ctivity in tilpi (Fig. 1A D; P<.5, two-wy ANOVA, N=7). Sewter trnsfer cused significntly higher plsm N +, Cl nd K + concentrtions nd gill N + /K + -ATPse ctivity regrdless of the nutritionl stte of the niml (Fig. 1A D; two-wy ANOVA, P<.5). There ws significnt interction between nutritionl stte nd sewter trnsfer only for plsm Cl concentrtion, but not for plsm N + nd K + levels or gill N + /K + -ATPse ctivity; plsm Cl concentrtion in the fooddeprived fish in SW ws significntly higher thn tht in ll the other groups (Fig. 1B). Plsm hormonl chnges Food-deprivtion significntly incresed plsm concentrtions of cortisol, but hd no significnt effect on plsm GH or tprl 177 concentrtions, wheres plsm tprl 1 concentrtion nd the tprl 1 :tprl 177 rtio were significntly lower in the food-deprived fish compred with the fed fish (Figs A,B, 3A C; two-wy ANOVA, P<.5). Sewter trnsfer resulted in significntly higher plsm concentrtions of GH nd significntly lower concentrtions of tprl 177 nd tprl 1 ; the tprl 1 : tprl 177 rtio ws significntly higher in the SW fish compred with the FW fish (Figs B, 3A C; two-wy ANOVA, P<.5). There ws no significnt effect of either SW trnsfer or the interction of slinity nd nutritionl stte on plsm cortisol concentrtion in the present study (Fig. A; two-wy ANOVA, P<.5). There were significnt interctions between nutritionl stte Fig. 1. Plsm N + (A), Cl (B) nd K + concentrtions (C) nd gill N + /K + -ATPse ctivity (D) in Oreochromis mossmbicus tht hd been either fed or food-deprived (FD) for weeks nd trnsferred to either fresh wter (FW) or se wter (SW) nd smpled 3 dys lter. Vlues represent mens + S.E.M. (N=7 fish). Tretments tht resulted in significntly different vlues (P<.5, two-wy ANOVA) re noted bove ech histogrm; significnt interction is shown by letters bove brs; vlues with the sme letters re not significntly different (P<.5, two-wy ANOVA). Plsm [Cl ] (mequiv l 1 ) Plsm [K + ] (mequiv l 1 ) Gill N + /K + -ATPse ctivity (µmol ADP h 1 mg 1 protein) 1 1 1 1 1 1. 1.5 1..5 <FD b FW SW FW SW c B C D

7 M. M. VIJAYAN AND OTHERS 3 FD A 1 FD A Plsm [cortisol] (ng ml 1 ) 5 15 75 <FD Plsm [tprl177] (ng ml 1 ) FW>SW Plsm [growth hormone] (ng ml 1 ) 1 1 b c B Plsm [tprl1] (ng ml 1 ) 1 >FD FW>SW B FW SW FW SW Fig.. Plsm cortisol (A) nd growth hormone (GH) (B) concentrtion in Oreochromis mossmbicus tht hd been either fed or food-deprived (FD) for weeks nd trnsferred to either FW or SW nd smpled 3 dys lter. Vlues represent mens + S.E.M. (N=7 for cortisol; N=5 for GH). Tretments resulting in significntly different vlues (P<.5, two-wy ANOVA) re noted bove ech histogrm; significnt interction is shown by letters bove brs; vlues with the sme letters re not significntly different (P<.5, two-wy ANOVA). Plsm tprl1:tprl177 rtio 1 1 b >FD C deprived fish in SW being significntly higher thn in ll the other groups (Fig. B). The tprl 1 :tprl 177 rtio, however, ws significntly higher in SW thn in FW in the fed fish; this effect ws bolished in the food-deprived fish (Fig. 3C). Plsm metbolite, liver glycogen nd heptic enzyme ctivities Food-deprivtion resulted in significntly higher plsm glucose nd totl mino cid concentrtions, while plsm lctte nd liver glycogen concentrtions were significntly lower compred with the fed fish (Fig. A D; two-wy ANOVA, P<.5). Sewter trnsfer resulted in significntly higher plsm glucose concentrtion, but hd no significnt effect on plsm lctte, totl mino cid concentrtions or liver glycogen content compred with the FW fish (Fig. A D; two-wy ANOVA, P<.5). Plsm glucose concentrtion ws significntly higher in the food-deprived fish in SW compred with ll the other groups, wheres there were no interctive FW SW FW SW Fig. 3. Plsm prolctin 177 (tprl 177; A) nd prolctin 1 (tprl 1; B) concentrtions nd plsm prolctin 1:prolctin 177 rtio (tprl 1:tPRL 177; C) in Oreochromis mossmbicus tht hd been either fed or food-deprived (FD) for weeks nd trnsferred to either FW or SW nd smpled 3 dys lter. Vlues represent mens + S.E.M. (N= for tprl 177; N=7 for tprl 1). Tretments resulting in significntly different vlues (P<.5, two-wy ANOVA) re noted bove ech histogrm; significnt interction is shown by letters bove brs; vlues with the sme letters re not significntly different (P<.5, two-wy ANOVA). effects between nutritionl stte nd slinity for lctte, totl mino cids or liver glycogen content. With the exception of histidine nd threonine, the concentrtion of free mino cids in the plsm showed significnt tretment effects (Tble 1). Food-deprivtion lone

Food-deprivtion nd sewter cclimtion in tilpi 71 Plsm [mino cid] (µmol l 1 ) Liver [glycogen] (µmol g 1 ) Plsm [glucose] (mmol l 1 ) Plsm [lctte] (mmol l 1 ) 3 1 5 15 1 5 FD <FD >FD <FD >FD b A B C D Fig.. Plsm glucose (A), lctte (B) nd totl mino cid (C) concentrtions nd liver glycogen content (D) in Oreochromis mossmbicus tht hd been either fed or food-deprived (FD) for weeks nd trnsferred to either FW or SW nd smpled 3 dys lter. Vlues represent mens + S.E.M. (N= fish). Tretments resulting in significntly different vlues (P<.5, two-wy ANOVA) re noted bove ech histogrm; significnt interction is shown by letters bove brs; vlues with the sme letters re not significntly different (P<.5, two-wy ANOVA). (regrdless of the medium slinity) significntly incresed plsm glutmte, serine nd lnine concentrtions, wheres rginine concentrtion ws significntly lower compred with the fed fish; none of the other free mino cids showed ny significnt effects of food-deprivtion compred with fed fish (Tble 1). Sewter trnsfer (regrdless of the nutritionl stte) significntly incresed plsm sprtte, tyrosine, lnine, methionine, phenyllnine, leucine, isoleucine nd vline concentrtions, while plsm glycine nd lysine concentrtions were significntly lower compred with the FW fish, regrdless of the nutritionl stte of the niml (Tble 1). Fooddeprivtion, however, resulted in significntly higher concentrtions of sprtte, tyrosine, serine nd lnine, nd significntly lower concentrtion of rginine in SW compred with the other groups (Tble 1; interctive effects). Plsm glycine concentrtion ws significntly lower in SW in the fed group nd not in the food-deprived group, while none of the other free mino cids showed ny interctive effects (Tble 1). There were no significnt tretment effects on heptic PEPCK or MDH ctivities in the present study (Tble ). Food-deprivtion (regrdless of the medium slinity) significntly incresed heptic PK nd LDH ctivities, wheres heptic AlAT nd HOAD ctivities were significntly lower compred with the fed group (Tble ). Sewter trnsfer (regrdless of the nutritionl stte of the niml) resulted in significntly higher heptic PK nd LDH ctivities; the high vlues in SW were due to the significntly higher ctivity of these two enzymes in the food-deprived fish (Tble ; interctive effects). There ws no significnt effect of either food-deprivtion or SW trnsfer on ny of the other heptic enzyme ctivities (Tble ). Heptic AlAT, AspAT, GDH nd HOAD ctivities in FW were significntly lower in the food-deprived fish compred with the fed fish. Sewter trnsfer, however, resulted in significntly higher heptic AlAT, AspAT nd GDH ctivities only in the food-deprived fish, while the fed fish showed either no chnge (AlAT, AspAT) or hd significntly lower heptic GDH nd HOAD ctivities (Tble ). FW SW FW SW Discussion Ion regultion Our results clerly demonstrte tht food-deprivtion ffects SW cclimtion processes in tilpi. Regultion of plsm N + nd Cl concentrtion is importnt for successful cclimtion

7 M. M. VIJAYAN AND OTHERS Tble 1. Plsm free mino cid concentrtions in Oreochromis mossmbicus tht hd been either fed or food-deprived for weeks nd trnsferred to either fresh wter (FW) or se wter (SW) nd smpled 3 dys lter Food-deprived Amino cid FW SW FW SW P<.5* Asprtte 3±.9, ND 9.±.9 b, 1.3±. c Tyrosine 5.±3..±1.9 3.1±. b 3.3±9.7 c Glutmte 1±1.7 7.3±1.7 1.9±3.3 17±3. <FD Serine.5±7.,b 5±. 5.3±..5±1.3 b <FD Alnine 1±1 19± 1±11 335± b <FD; Glycine 5±9 1±1 b 393±77 3±7 FW>SW Arginine 7.±3.,b 7.1±7.5.±5.9 b 33.7±5. c >FD Methionine.1±. 3.±3..9±3.9 3.5±. Phenyllnine 5.5±.5 1±.3 5.7±5.7 7.±13.5 Leucine 1.±1.1 171±1.3±7. 1±1 Isoleucine 3.5±..±.9 3.5±3.9 99±1. Vline.5±9. 15±9.7±9 15±3 Lysine 1±13 13±15 1±37 19±3 FW>SW Histidine 3±9 1±9 3±3 5±1 Threonine 1±5 1±7 13±1 115±19 Amino cid concentrtions (in µmol l 1 ) represent mens ± S.E.M. (N= fish). 3 vlues below detectble levels; ND, vlues below detectble levels. *Significnt tretment effects nd the interction re shown s superscripts (two-wy ANOVA); vlues with the sme superscripts re not significntly different between tretments (P<.5, two-wy ANOVA). Tble. Liver enzyme ctivities in Oreochromis mossmbicus tht hd been either fed or food-deprived for weeks nd trnsferred to either fresh wter (FW) or se wter (SW) nd smpled 3 dys lter Food-deprived Enzymes FW SW FW SW P<.5* PEPCK.39±.7.3±.1.5±.1.39±.1 PK.±.15.±..±.1 3.7±.5 b <FD; LDH 17.±1.,b 1.3±3.3 1.±1.1 b 3.5±5. c <FD; AlAT 3.± 3.5±1.1 17.1±1. b 3.±1. c >FD AspAT.1±3. 5.±.,b.3±5 b.± GDH.±. 9.5±1.9 b 5.±5.1 b 55.±3.,b HOAD.3±.5 3.±.17 b.9±.1 c 3.3±.1 b,c >FD MDH 11±.3 11±3 17±. 1±5 Vlues represent mens ± S.E.M. (N= fish). *Significnt tretment effects nd the interction re shown s superscripts (two-wy ANOVA); vlues with the sme superscripts re not significntly different (P<.5, two-wy ANOVA). Phosphoenolpyruvte crboxykinse (PEPCK), pyruvte kinse (PK), lctte dehydrogense (LDH), lnine minotrnsferse (AlAT), sprtte minotrnsferse (AspAT), glutmte dehydrogense (GDH), 3-hydrocyl-Coenzyme A dehydrogense (HOAD) nd mlte dehydrogense (MDH). Enzyme ctivities re expressed s µmol min 1 g 1 wet mss 1. to SW, nd the plsm ion concentrtions in the fed fish (Fig. 1A C) were within the rnges reported for SWcclimted O. mossmbicus (Hwng et l. 199; Yd et l. 199). The higher plsm Cl concentrtion in the fooddeprived fish in SW my be due to severl fctors including higher drinking rte, incresed brnchil permebility nd/or decresed ion excretion. Previous studies, however, hve shown tht food-deprivtion decresed the drinking rte in Atlntic slmon (Slmo slr) (Usher et l. 19) nd lso the gill permebility to ions in rinbow trout (Oncorhynchus mykiss) (Nnce et l. 197), thereby implicting modultions in Cl excretion processes s the possible cuse for the ionic imblnce. Although body size hs been shown to ffect slinity-tolernce in fish, ll the food-deprived fish in this study were lrger thn the size t which fish hve been shown to rech mximum slinity tolernce (Wtnbe et l. 199), suggesting tht the chnges in body mss (3 % lower in the food-deprived fish) were probbly not the cuse of the impired Cl regultion. NCl excretion in SW is thought to be crried out

Food-deprivtion nd sewter cclimtion in tilpi 73 exclusively by the chloride cells, nd the present model for ion regultion underscores the importnce of N + /K + -ATPse ctivity (or the sodium pump) for the excretion of N + nd Cl (for review, see Jurss nd Bstrop, 1995). The higher gill N + /K + -ATPse ctivity in the SW fish, however, rgues ginst the inctivity of the sodium pump s likely cuse of the poor Cl regultion in food-deprived fish. Previous studies hve shown tht food-deprivtion decresed both the size nd numbers of chloride cells in SW-cclimted Oreochromis mossmbicus (Kultz nd Jurss, 1991). In our study, the plsm Cl concentrtion in the food-deprived fish in SW ws higher thn the plsm N + concentrtion, which lso suggests tht food-deprivtion my be modifying the trnsepithelil brnchil Cl trnsport mechnism(s). Further work is necessry before this process cn be clerly explined. Hormonl chnges Plsm cortisol is considered to be n importnt hormone for SW cclimtion becuse cortisol hs been shown to increse both gill chloride cell prolifertion nd N + /K + - ATPse ctivity in fish (for review, see McCormick, 1995). In the present study, the bsence of n increse in plsm cortisol concentrtion in the fed fish in SW is not surprising s previous studies hve shown tht plsm cortisol concentrtion is trnsient fter SW entry, peking t bout h, fter which it drops to bseline levels (Assem nd Hnke, 191), perhps due to the fster clernce of cortisol in SW (Redding et l. 19). As our fish were in SW for 3 dys, it is likely tht plsm cortisol vlues hd returned to the FW levels in these fish. The trnsient increse in plsm cortisol concentrtion is thought to ssist in the SW cclimtion process directly by enhncing the ion regultion process nd/or indirectly by providing energy substrtes for ion regultion (Assem nd Hnke, 191). Plsm cortisol concentrtion ws higher in the fooddeprived fish nd ppered to increse in SW (Fig. A), suggesting tht these fish were subjected to n osmotic stress s shown by the higher plsm Cl concentrtion (Fig. 1B). The higher plsm cortisol concentrtion my be directly ssisting in mintining the gill N + /K + -ATPse ctivity (McCormick, 1995) nd/or indirectly ssisting in the ion regultion process by mobilizing energy substrtes for gill metbolism (Assem nd Hnke, 191; Vijyn et l. 199,b; see below). The increse in plsm GH nd decrese in tprl 177 nd tprl 1 concentrtions in SW re consistent with those reported in the literture for O. mossmbicus (Yd et l. 199) nd further underscore the role of these hormones in ion regultion in fish (see Wendelr Bong, 1993; McCormick, 1995). The higher growth hormone response in the fooddeprived fish in se wter, where there ws evidence of impired ion regultion, clerly supports the proposl tht GH plys role in SW dpttion in the tilpi (Yd et l. 199). Prolctin plys n importnt role in the ion regultion process in FW, but the role of this hormone in the cclimtion process to SW is not cler. However, studies hve shown tht SW-cclimted fish injected with prolctin hve high plsm N + levels, indicting tht prolctin ttenutes N + efflux in these nimls (see McCormick, 1995). Thus, the decrese in plsm prolctin concentrtions in SW (Fig. 3A,B) is probbly required for the efficient regultion of plsm N + concentrtion in SW. The significntly lower tprl 1, but not tprl 177, level in food-deprived compred with fed fish suggests tht nutritionl stte my differentilly modulte the plsm concentrtion of the two prolctins. Our results show tht the plsm tprl 1 :tprl 177 rtio in the fed fish increses in SW (Fig. 3C), nd this is likely to be due to the higher relese of tprl 1 reltive to tprl 177 in SW. Interestingly, the bolition of this incresed tprl 1 :tprl 177 rtio in the fooddeprived fish coincides with impired Cl regultion in tht group, rising the possibility tht the rtio of the two prolctins my be relted to the SW-cclimtion process in tilpi. The chnges in plsm prolctin concentrtion nd tprl 1 :tprl 177 rtio, however, support the contention tht tprl 1 nd tprl 177 re differentilly regulted, s proposed erlier from studies either in vivo (Yd et l. 199) or in vitro using pituitry preprtions (Yoshikw-Ebesu et l. 1995). Chnges in metbolite levels The plsm metbolite concentrtions (Fig. A C; Tble 1), liver glycogen content (Fig. D) nd liver enzyme ctivities (Tble ) in the fed fish indicte tht there ws no excessive substrte mobiliztion for energy purposes in SW fish. The decresed heptic GDH nd HOAD ctivities in the fed fish in SW suggest lower heptic potentil for mino cid nd ftty cid ctbolism, implying lower energy demnd. This heptic metbolic orgniztion is consistent with the observtion tht the metbolic rte of long-term SW-cclimted tilpi is lower thn tht of FW fish (Ron et l. 1995) nd tht, s result, tilpi my hve higher growth rtes in SW thn in FW (Kultz nd Jurss, 1991; Kuwye et l. 1993; Ron et l. 1995). Food-deprivtion decresed the heptic cpcity for mino cid (AlAT, AspAT, GDH) nd ftty cid (HOAD) ctbolism in FW (Tble ), nd this my represent generl decrese in heptic metbolism s shown before in fsted fish (Foster nd Moon, 1991). The mintennce of plsm glucose concentrtion under these circumstnces my hve been chieved by incresed glycogenolysis nd/or gluconeogenesis s shown by lower liver glycogen nd plsm lctte concentrtions, nd higher levels of plsm totl mino cids in food-deprived compred with fed fish (Fig. B D). In support of this rgument, previous studies hve shown tht mino cids nd lctte re preferred substrtes for heptocyte gluconeogenesis in fish (Surez nd Mommsen, 197). The ppernce of lower heptic potentil for protein ctbolism in food-deprived fish in FW is certinly reduced in SW, implying n incresed energy demnd. The higher plsm free mino cid concentrtions, especilly of lnine nd serine (importnt energy substrtes in fish; French et l. 191, 193), with the concomitnt increse in heptic AlAT nd AspAT ctivities suggest peripherl proteolysis nd mino cid ctbolism in food-deprived fish in SW. A recent study hs shown tht cortisol mobilizes mino cids nd increses mino

7 M. M. VIJAYAN AND OTHERS cid ctbolism in O. mossmbicus (Vijyn et l. 199b). Tht observtion coupled with higher plsm cortisol concentrtion in food-deprived fish in se wter in the present study suggests significnt role for cortisol in the mino cid mobiliztion process (Fig. A; Tbles 1, ). The C3 precursors rising from mino cid brekdown my be chnnelled to gluconeogenesis in fish (Vijyn et l. 199) nd my prtly ccount for the higher glucose concentrtion in the food-deprived fish in SW (Fig. A), perhps medited by cortisol (Vijyn et l. 199b). The elevted plsm glucose concentrtion of food-deprived fish in SW my lso hve been mintined by glycogenolysis, lthough the bsence of further decrese in heptic glycogen content in SW could be due to the concurrent chnnelling of C3 precursors for glycogen repletion, s suggested by Pereir et l. (1995). This glucose my be utilized to fuel gill metbolism, including the energy required for ion regultion, since previous studies hve shown glucose to be the preferred oxidtive substrte for gill metbolism (Mommsen, 19,b; Perry nd Wlsh, 199). The reduction of liver glycogen content in the food-deprived fish together with the elevtion of PK nd LDH ctivity, my imply higher heptic glycolytic potentil in these nimls in order to fuel liver metbolism (Vijyn et l. 199b). It is interesting tht the plsm concentrtions of severl essentil mino cid (methionine, phenyllnine, leucine, isoleucine nd vline) incresed in the SW fish regrdless of the nutritionl stte of the niml (Tble 1). The significnce of this increse in SW is not known, lthough we cn speculte tht some of these mino cids my ply role in the SW cclimtion process in fish. These essentil mino cids re required for the synthesis of peptides nd proteins nd, therefore, their vilbility my regulte the synthesis of hormones tht re importnt in the ion regultion process. In support of this rgument, in vitro studies using tilpi pituitry glnd hve shown tht both GH- nd prolctin-secreting cells respond to vritions in mino cid levels in the medium (Rodgers et l. 199). Furthermore, mmmlin studies hve shown tht brnched-chin mino cids (isoleucine, leucine nd vline) increse plsm GH levels (Stewrt et l. 19). Since levels of these three mino cids were higher in the SW fish (which hd higher GH concentrtion), it is possible tht mechnism similr to tht operting in mmmls my regulte the secretion of GH in the tilpi. In conclusion, our results support the possibility tht fooddeprivtion ffects the SW cclimtion process by impiring Cl regultion in O. mossmbicus. This effect does not pper to be due to the inctivtion of gill N + /K + -ATPse since the ctivity of this enzyme ws higher in SW thn in FW fish. Furthermore, the impirment ws not due to n inbility to relese cortisol or GH s levels of both these hormones were lso higher in the food-deprived fish. Furthermore, mino cid nd glycogen mobiliztion nd plsm glucose concentrtion were higher in food-deprived fish, clerly indicting tht energy substrte vilbility ws not fctor in the poor Cl regultion. We conclude either tht the N + /K + -ATPse ctivity ws not functioning to its full cpcity or tht the N + /K + -ATPse levels chieved were not sufficient to mintin ion blnce. It is lso possible tht some other fctor(s) independent of this ion trnsporter contributed significntly to the impired Cl excretion. Kultz nd Jurss (1991) hve shown tht food-deprivtion decreses the size nd number of chloride cells in tilpi. This, together with our finding tht plsm Cl concentrtion is elevted reltive to plsm N + concentrtion in the food-deprived fish, suggests tht nutritionl stte cn modulte Cl trnsport in tilpi. We hypothesize tht food-deprivtion, directly or indirectly, ffects trnsepithelil brnchil Cl trnsport mechnism(s). The results lso suggest tht plsm prolctin nd essentil mino cids my ply n importnt role in the SW cclimtion process in tilpi. Future studies will exmine the possible interction(s) between nutritionl sttes nd hormones, especilly GH nd insulin-like growth fctors, on osmoregultion in the tilpi. The uthors grtefully cknowledge the help of Benny Ron nd others t the Hwiin Institute of Mrine Biology, Coconut Islnd, Hwii. We extend our thnks to Shnnon Blfry nd Ellen Teng for help during the experiment nd Cristin Pereir for her ssistnce with enzyme mesurements. 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