Water calcium concentration modifies whole-body calcium uptake in sea bream larvae during short-term adaptation to altered salinities

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1 The Journl of Experimentl Biology 27, Pulishe y The Compny of Biologists 24 oi:1.1242/je Wter lium onentrtion moifies whole-oy lium uptke in se rem lrve uring short-term pttion to ltere slinities Pero M. Guerreiro 1,2, Jun Fuentes 1, Gert Flik 2, Josep Rotllnt 1, Deorh M. Power 1 n Aelino V. M. Cnrio 1, 1 Centre of Mrine Sienes (CCMAR), University of Algrve, Cmpus e Gmels, Fro, Portugl n 2 Deprtment of Animl Physiology, University of Nijmegen, 6525 ED Nijmegen, The Netherlns Author for orresponene (e-mil: nrio@ulg.pt) Aepte 22 Otoer 23 Whole-oy lium uptke ws stuie in gilthe se rem lrve (9 83 mg) in response to hnging environmentl slinity n [C 2+ ]. Clium uptke inrese with inrese fish size n slinity. Fish expose to lium-enrihe, ilute sewter showe inrese lium uptke ompre with fish in ilute sewter lone. Clium uptke ws unhnge in N + - enrihe, ilute sewter. Overll, [C 2+ ], n not slinity/osmolrity per se, ppers to e the min ftor ontriuting to lium uptke. By ontrst, rinking ws reue y erese in slinity/osmolrity ut ws little ffete y externl [C 2+ ]. Clultions of the mximum Summry ontriution from rinking-ssoite lium uptke showe tht it eme lmost insignifint (less thn 1%) through strong erese in rinking rte t low slinities ( 8 ). Dilute sewter enrihe in lium to the onentrtion present in full-strength sewter (i.e. onstnt lium, eresing slinity) restore intestinl lium uptke to norml. Extr-intestinl lium uptke lso enefite from lium ition ut to lesser extent. Key wors: lium uptke, rinking rte, slinity, environmentl lium, lrve, gilthe se rem, Sprus urtus. Introution Clium is essentil for numer of vitl proesses, from one formtion to the moultion of permeility n exitility of memrnes. As smll hnges in [C 2+ ] hve mrke effet on neurl, riovsulr n musulr funtion, mintenne of irulting [C 2+ ] within nrrow limits is funmentl requirement, n onstnt supply of lium must e ensure to the oy. The ility of ult teleosts to mintin onstnt plsm [C 2+ ] in iverse lium environments is well oumente (Flik et l., 1995; Png et l., 198). Clium uptke eomes of inresing importne uring erly life stges of vertertes, inluing fish, s the skeleton n other ony prts re unergoing rpi evelopment. In the gilthe se rem (Sprus urtus), mrine teleost fish wiely ultivte in southern Europe, the evelopment of the skeleton initites with the formtion of rtilginous templte, followe y grul minerliztion (Fustino n Power, 1998, 1999, 21). The onset of this proess tkes ple few ys fter hthing, n ossifition of the verterl olumn (Fustino n Power, 1998), fins (Fustino n Power, 1999) n rnil omplex (Fustino n Power, 21) ontinues uring the first 7 9 ys posththing (DPH). Sles, eveloping in the hitherto nke fish skin, re forme uring lter stges of lrvl evelopment n mrk the trnsition to the juvenile stge. Bone minerliztion relies on lium vilility in the extrellulr fluis n, unlike terrestril vertertes, whih otin this ion exlusively from the iet, freshwter n mrine fish otin it from the (generlly) lium-rih wter they inhit. The wter-expose gills, skin n intestine epitheli re the primry surfes for lium uptke, while the kiney (n possily the intestine) is involve in exretion (Flik et l., 1995; Flik n Verost, 1993). Internl [C 2+ ] is mintine within nrrow limits espite lrge flututions in environmentl [C 2+ ] tht generlly prllel hnges in wter slinity. The ility to mintin onstnt ion onentrtion n osmollity of oy fluis ppers erly in evelopment. Emryos n lrve hve pity similr to tht of ult fish n regulte ioni grients etween their oy n the mient wter y exreting soium hlorie or soring lium (Alerie, 1988; Guggino, 198). The erly post-emryoni stges of the Europen se ss (Dientrrhus lrx) re le to hypo- n hyperregulte over wie rnge of slinities, this ility eing quire in steps ut lrey eing present t hthing (Vrsmos et l., 21). It hs lso een propose tht the regultion of lium lne in freshwter tilpi lrve (Oreohromis mossmius) limte to low-lium environments is stge epenent n tht moultion of

2 646 P. M. Guerreiro n others lium fluxes is losely orrelte with levels of oy lium ontent (Chou et l., 22; Hwng et l., 1996). Fish in sewter ten to gin ions suh s soium n hlorie through iffusion n to lose wter y osmosis. The quisition of the pity to ontrol wter lne in reltion to externl slinity through rinking y fish lrve is, therefore, funmentl for osmoregultion (Flik et l., 22; Vrsmos et l., 21), n rinking ours efore the evelopment of funtionl nus n gills (Tytler n Blxter, 1988,). Sine whole-oy lium uptke orreltes positively to environmentl [C 2+ ], slinity (s result of [NCl]) n rinking, the reltive ontriution of eh of these vriles to lium uptke is unertin. In the present stuy, we hve exmine the reltionship etween lium uptke n rinking y sumitting gilthe se rem lrve to rupt short-term hnges of omintions of wter slinity n [C 2+ ]. Mterils n methos Fish n mei Gilthe se rem lrve (Sprus urtus L.) were rere t Instituto e Investig o es Pess e o Mr (IPIMAR; Olhão, Portugl) in 9% reirulting 2-litre onil tnks oring to stnr proeures t 36 slinity n 2 C. Whole-oy lium ontent ws mesure in speimens ollete every 5 ys from 2 DPH to 75 DPH n t 12 DPH (N 1 iniviuls per ge group). Lrve were fste for 24 h efore eing srifie using n exess of 2- phenoxyethnol (.5% v/v; Sigm-Alrih, Mri, Spin), weighe to the nerest.1 mg (Srtorius lne, preision 1 6 g) n igeste with 1 µl of 7% nitri i (v/v; Sigm-Alrih) for 24 h. Iniviul totl oy lium ontent ws etermine y mens of inutively ouple plsm tomi emission spetrometry (ICP-AES; Plsm IL2; Thermo Jrell Ash, Frnklin, MA, USA). For size/ge-relte lium uptke stuies n for ltere slinity experiments, lrve (otine from Timr, Culturs em Águ, Portugl) were 3 6 DPH n 45 DPH, respetively. To otin the esire reutions in slinity, full-strength sewter (SW) ws ilute using istille wter. To otin slinity, [C 2+ ] or [N + ] ientil or ove those of full-strength sewter, mrine slt (Instnt Oen; Aqurium Systems, Srreourg, Frne), lium hlorie (Sigm-Alrih) or soium hlorie (Sigm- Alrih) ws e to full-strength or ilute sewter s require (see Tle 1 for tomi sorption hromtogrphy nlysis of experimentl mei). Determintion of lium influx Fish were nette n trnsferre to 25-ml vessels with erte sewter n, fter 3 min, 45 CCl 2 (NEN, Boston, MA, USA) ws e to the wter (finl tivity of 3.7 kbq ml 1 ). At the en of the experiment (normlly lsting 4 h), wter smples were ollete. The lrve were rinse in trer-free wter, srifie, weighe n igeste s inite ove, neutrlise with n ientil volume of 2 mol l 1 soium hyroxie (Sigm-Alrih) n lehe with 3 µl of 35% hyrogen peroxie (Fluk, Sigm-Alrih) to prevent olour quenhing. Wter lium ontent ws mesure y olourimetri enpoint ssy (Sigm-Alrih, ssy No. 587). Wter n igeste lrvl smples were issolve in OptiPhse HiSfe II liqui sintilltion flui (Wll, Amershm Phrmi Bioteh, Lison, Portugl), n 45 C tivity ws mesure in sintilltion ounter (Moel LS6IC; Bekmn Instruments In., Fullerton, CA, USA). Clium influx (C in ) ws lulte oring to the following eqution: C in = (A f C w )/(A w t), where A f is the totl 45 C tivity in fish (.p.m.), C w is the totl lium onentrtion in wter (nmol l 1 ), A w is the totl 45 C tivity in wter (.p.m. l 1 ), n t is urtion of exposure (h). Clium influx is expresse s nmol h 1. Extrintestinl lium uptke ws estimte y sutrting the mount of lium in the wter imie (i.e. rinking rte; see elow) from the totl whole-oy lium uptke. Determintion of rinking rte The proeure use for the etermintion of rinking rte ws the sme s for lium influx ut 3.7 kbq ml 1 of 51 Cr- EDTA (NEN) ws e to the wter. Riotivity ws ounte in gmm ounter (Wll 147 Wizr gmm ounter; Amershm Phrmi Bioteh). In some experiments, lium influx n rinking rte were mesure simultneously. Drinking rte (DR) ws lulte s: DR=A f /(A w t), where A f is the totl tivity of 51 Cr-EDTA in the fish (.p.m.), A w is the trer tivity in wter (.p.m. ml 1 ), n t is the urtion of exposure (h). Results re expresse s nl h 1. Effets of trer exposure time n oy size on whole-oy C 2+ influx The effet of urtion of exposure to 45 CCl 2 on lium uptke ws etermine y smpling lrve in ifferent vessels t 2 h, 4 h, 6 h, 8 h n 16 h fter rioisotope ition. Intt gilthe se rem lrve umulte 45 C t onstnt rte for t lest 16 h n therefore the trer experiments were onute within this time-spn to reflet initil spee of lium influx, thus eliminting possile effets of trer kflow. The effet of oy size ws teste in five ifferent experiments in 1%SW with lrve rnging in mss from 5 mg to 15 mg. Clium influx ws etermine s esrie ove. Time-series of justment of C 2+ influx to ilute sewter Lrve were trnsferre to 25-ml vessels ontining 1%SW (four groups, N 15) or 5%SW (three groups, N 15), n 45 CCl 2 ws immeitely e. Lrve were ollete t 2 h, 4 h, 8 h n 16 h fter trnsfer (HAT; 1%SW) n t 4 HAT, 8 HAT n 16 HAT (5%SW), rinse, srifie, weighe n issolve to etermine lium influx s esrie ove. Effets of [C 2+ ] n [N + ] on C 2+ influx n rinking rtes Gilthe se rem lrve (N=2 4 per group) were

3 Clium uptke n rinking in se rem lrve 647 Tle 1. Ioni omposition n hemil properties of sewter, sewter supplemente with mrine slt, sewter ilute with istille wter, sewter ilute with istille wter supplemente with lium hlorie or soium hlorie n freshwter lone Osmollity Mei (%SW) Slinity ( ) (mosmol kg 1 ) N + (mmol l 1 ) Cl (mmol l 1 ) C 2+ (mmol l 1 ) 15% % % % % % %+C %+C %+C %+N %+N trnsferre to 25-ml vessels ontining the experimentl mei (see Tle 1 for preise ioni omposition): 1%SW, 15%SW,, 1%SW, %SW, 1%SW plus CCl 2 (1%SW+C), plus CCl 2 (+C), plus NCl (+N), 1%SW plus CCl 2 (1%SW+C) n 1%SW plus NCl (1%SW+N) for 4 h n 2 h (%SW only for 4 h) efore ition of 45 CCl 2 n 51 Cr-EDTA. After 4 h in rioisotope-ontining wter, the fish were rinse, srifie, weighe n issolve to etermine lium influx n rinking rte. [C 2+ ] in 1%SW+C ws juste to tht of 15%SW, n +C n 1%SW+C ws juste to tht of 1%SW. [N + ] in +N n 1%SW+N ws juste to tht of 1%SW. Sttistil nlysis The effet of tretments on lium uptke n rinking ws nlyse y one-wy or two-wy ovrine nlysis (ANCOVA) with oy size s ovrite using the Generl Liner Moel moule in the Systt softwre (version 1.2; Systt Softwre In., Rihmon, CA, USA). Contrsts were use to test the reltionship mong ell mens. Lest squres mens n their stnr errors juste for mss re use to report effet of tretments. Multiple liner regression nlysis ws use to ompre the reltive ontriution of lium, soium n osmolrity to lium uptke. Whenever the t were require to e normlise, logrithmi trnsformtion ws use. The signifine level ws estlishe s P<.5 unless otherwise stte. Results Whole-oy minerl ontent The reltionship etween whole-oy lium ontent (Q; µmol) n wet oy mss ws esrie y the eqution Q=.52M (M rnge=.5 5 mg; r =.993, P<.1, N=196). The slope of the regression line (1.249) ws signifintly ifferent from 1, initing tht totl oy lium ws relte to oy mss y slightly positive llometry. Effets of slinity n ition of C 2+ or N + on C 2+ influx Whole-oy lium influx in 1%SW ws strongly epenent on oy mss (C in =2588M.496 ; Fig. 1A), even fter normlistion to unit of oy mss (C in,m =2588M 54 ; Fig. 1B). The sme reltionship ws oserve 8 HAT to mei of other slinities; the slope ws similr ut the y-interept ws proportionl to the slinity (t not shown). Beuse the epeneny of lium influx on oy mss ws highly signifint ross the mss rnge of the lrve use in the experiments, susequent t nlysis onsiere mss s ovrite to remove its effet. When fish were trnsferre from 1%SW to 5%SW, justment to lower lium influx ws notiele etween 4 h n 8 h fter the onset of exposure (Fig. 2). By 8 HAT, the influx rte t 5%SW ws 3% signifintly lower thn t 1%SW n ws 65% lower y 16 HAT. Trnsfer of lrve from 1%SW to 15%SW le to 5% inrese in lium influx y 8 HAT (Fig. 3A) n 1% y 24 HAT (Fig. 3B). Trnsfer to lower slinities lowere lium influx 8 HAT y 5% t n y 9% t %SW (Fig. 3A). A similr reution ws lso foun 24 HAT. To estlish whether slinity or mient [C 2+ ] etermine the rte of lium uptke, lrve were expose to ltere slinities n to ifferent [C 2+ ] n [N + ]. Lrve expose to 1%SW to whih [C 2+ ] ws juste to 17.6 mmol l 1 (similr to tht of 15%SW) h lium influx rtes t 8 HAT n 24 HAT similr to those in 15%SW (Fig. 3A,B). Similrly, trnsfer to 25% n 1% ilute sewter in whih [C 2+ ] h een juste to levels of 1%SW resulte in inrese lium influx rtes. At 8 HAT, the inrese ws only signifint t 1%SW+C (lthough influx rtes i not reh those of 1%SW), ut t 24 HAT oth +C n 1%SW+C h lium influx rtes similr to those foun in 1%SW (Fig. 3B). Pling lrve in ilute meium supplemente with NCl

4 648 P. M. Guerreiro n others Clium influx (pmol h 1 ) A r =.685 N=355 P<.1 B r =.69 N=355 P< Speifi lium influx (pmol mg 1 h 1 ) Boy mss (mg) 1 1 Fig. 1. (A) Reltionship etween whole-oy lium influx n wet oy mss. (B) The sme reltionship with lium influx ivie y oy mss, referre to s speifi lium influx. Inite re the regression lines (mile lines) n their 95% onfiene limits, orreltion oeffients (r ), sttistil signifine levels (P) n numer of fish use (N). to levels similr to 1%SW resulte in high mortlity (Tle 2), whih ws most severe in the 1%SW+N group: ll fish ie within 2 HAT. Nevertheless, the surviving +N group h lium influx rtes similr to their respetive ontrols () t oth 8 HAT n 24 HAT. Interestingly, fish trnsferre iretly to %SW were le to just etter thn fish in 1%SW+N, with 5% of the initil group surviving to the en of the 8-h perio. To onfirm tht Clium influx (nmol h 1 ) %SW 5%SW Time (h) Fig. 2. Time-ourse of whole-oy lium influx rte in fish mintine t 1%SW (fille irles) n in fish trnsferre to 5%SW (open irles) for 16-h perio. Eh point is the men lest squres juste for oy mss (1 5 mg), n the vertil lines re their stnr errors. Asterisks represent sttistilly signifint ifferenes to 1%SW (P<.1; P<.1) t given time. lium influx ws relte only to fish size n to wter [C 2+ ], multiple regression nlysis, whih lso inlue wter osmolrity n [N + ] s inepenent vriles, ws rrie out. We foun strong olinerity etween meium [C 2+ ], [N + ] n osmolrity, implying some unertinty s to the reltive ontriutions etween the three vriles to lium influx. The multiple liner regression eqution, pooling t from 8 HAT n 24 HAT, ws: log C in = [N + ].1Os+.41[C 2+ ]+.679 log M, where Os is osmolrity. As n e oserve, oring to this moel, the effet of [C 2+ ] on C 2+ influx is t lest one orer of mgnitue higher thn tht of [N + ] or osmolrity, suggesting tht mient [C 2+ ] is mjor moifier of C 2+ influx. The reltionships Tle 2. Cumultive mortlity, M (%), n numer of surviving fish (N) reore 8 h n 24 h fter trnsfer to the mei shown in Tle 1 8 HAT 24 HAT Mei (%SW) M (%) N M (%) N 15% % % % % NA NA 1%+C %+C %+C %+N %+N 1 1 NA, not teste; HAT, hours fter trnsfer.

5 Clium uptke n rinking in se rem lrve 649 Clium influx (nmol h 1 ) %SW 1%SW e 1%SW %SW 1%SW+C +C 1%SW+C +N 1%SW+N A Meium omposition 15%SW 1%SW NA 1%SW %SW 1%SW+C +C 1%SW+C +N 1%SW+N Fig. 3. Effet of slinity (fille rs) n of ition of lium (rs with igonl lines) or soium (rs with horizontl lines) on se rem lrve whole-oy lium influx fter 8 h (A) or 24 h (B) exposure. At 1%SW n, lium n soium were e to mth levels t 1%SW. At 1%SW, lium levels were e to mth those t 15%SW (see Mterils n methos n Tle 1). Eh r represents the men lest squres juste for oy mss n respetive S.E.M. Ientil letters inite groups tht o not iffer sttistilly t P=.5. The ggers inite 1% mortlity; NA, not teste. B etween lium influx, oy mss n meium [C 2+ ] t 8 HAT n 24 HAT were s follows: C in,8 =283+M [C 2+ ].618 (juste r 2 =.511, N=213, P<.1; rnge [C 2+ ] mmol l 1 ; rnge M mg) n C in,24 =813+ M.575 +[C 2+ ].424 (juste r 2 =.511, N=128, P<.1; rnge [C 2+ ] mmol l 1 ; rnge M mg). Effet of slinity n ition of [C 2+ ] or [N + ] on rinking As with lium uptke, rinking rte ws highly positively orrelte to fish mss (t 1%SW DR=46.6M.785 ; r =.677, P<.1, N=78). A mrke slinity-epenent reution in rinking ws oserve t 8 HAT (Fig. 4A). Fish tht were expose to mei t lower slinities exhiite rinking rtes Drinking rte (nl h 1 ) %SW 1%SW 1%SW %SW 1%SW+C +C 1%SW+C +N 1%SW+N A %SW 1%SW Meium omposition NA 1%SW %SW 1%SW+C +C 1%SW+C +N 1%SW+N Fig. 4. Effet of slinity (fille rs) n of ition of lium (rs with igonl lines) or soium (rs with horizontl lines) on se rem lrve rinking rte fter 8 h (A) n 24 h (B) exposure. See legen to Fig. 3 for further etils. B

6 65 P. M. Guerreiro n others tht were signifintly lower thn those of ontrol 1%SW fish. Fish expose to 15%SW h rinking rtes tht were slightly higher ut not sttistilly ifferent from those kept t 1%SW. Drinking t %SW ws signifintly reue in reltion to ll other teste mei. After 24 HAT, similr reltionships to tht of 8 HAT were oserve (Fig. 4B). Aition of lium or soium h no signifint effets on rinking, lthough there ws n inition tht ition of lium n espeilly soium might stimulte rinking fter 24 h. However, the lrge vriility n low numer of surviving fish ment this tren oul not e onfirme. Clium imie ws estimte from the rinking rte, n the extr-intestinl ontriution to the whole-lium uptke ws etermine (Fig. 5). Extr-intestinl ontriutions were higher in mei with lower slinity n less lium, ounting for ~93% of whole-oy lium entry t 1%SW n %SW n for ~85% t. At 1%SW n ove, the extr-intestinl ontriution ws only 45 5%. The effet of ing lium to the wter t lower slinities ws to inrese the reltive ontriutions to levels lose to those of 1%SW. However, ition of N h little effet. The hnge in the reltive ontriution of the two routes of lium uptke is summrize in Fig. 6. The t further emphsise tht inrese lium uptke t higher slinities is intimtely ssoite with rinking ut it is lso epenent on environmentl [C 2+ ], sine extr-intestinl uptke is lso enhne. It ws not possile to seprte the reltive importne of rinking n [C 2+ ] euse of olinerity in the multiple regression. Disussion Gilthe se rem grown in full-strength sewter show liner inrese in oy lium ontent, initing onstnt lium umultion relte to skeletl growth. The reltionship is llometri, possily refleting the higher ensity of the lifie skeleton. These requirements in lium re met y n exponentil inrese in lium uptke s the fish grows, n the positive reltionship etween fish size n lium influx is mintine in lrve expose to ifferent slinities. The higher speifi lium uptke (i.e. expresse s influx per oy mss) in lrve thn in ults my e, t lest A Clium influx (nmol h 1 ) B Fig. 5. Effet of slinity n of ition of lium or soium on estimte se rem lrve intestinl (fille rs) n extrintestinl (hthe rs) lium uptke fter 8 h (A) or 24 h (B) exposure. Moifition in lium n soium in 1%SW, n 1%SW is esrie in the legen to Fig. 3. Asterisks inite sttistilly signifint ifferene etween intestinl n extr-intestinl fluxes t P=.5. Dggers inite 1% mortlity; NA, not teste %SW 1%SW 1%SW NA %SW 1%SW+C Meium omposition +C 1%SW+C +N 1%SW+N

7 Clium uptke n rinking in se rem lrve 651 prtilly, ue to the lrger surfe-to-volume rtio of lrve n/or the ifferene in epithelium permeility for lium etween lrve n ults (Hwng et l., 1994). The results lso emonstrte tht, s suggeste previously (Flik et l., 1996), liner extrpoltions from lrge to smll fish re not eptle. Sine extrellulr lium is mintine within tight limits, this inites tht gilthe se rem lrve re le to regulte their lium uptke (n loss) in orer to omply with the lium emns of their norml physiologil proesses s well s the extr emn impose y the intense lifition perio. This is lso supporte y the oservtion tht fish pte to low-lium environments re ple of moulting trnsport mehnisms to sustin similr or greter umultion rtes of oy lium ontent uring growth (Chou et l., 22; Flik et l., 1986; Hwng et l., 1996; Mol et l., 1999; Vonk et l., 1998). Gilthe se rem lrve trnsferre from 1%SW to hyposmoti environment (5%SW) require t lest 16 h to pt lium trnsport mehnisms to the new environmentl onitions. In juveniles of silver se rem (Sprus sr), rupt hyposmoti exposure (from 33 to 6 ) resulte in eline in serum totl lium t 24 HAT n return to preexposure levels y 12 HAT (Kelly n Woo, 1999). A similr effet ws reporte y Mner et l. (1993) for the gilthe se rem, with omplete reovery ourring fter 3 ys. In gilthe se rem lrve, it ws not possile to mesure serum lium, ut strong reltionship etween lium influx n wter slinity ws foun oth t 8 HAT Clium influx (nmol h 1 ) Intestinl Extr-intestinl Whole-oy Clium (mmol l 1 ) Fig. 6. Reltionship etween juste mens of whole-oy (squres, roken line), intestinl (irles, ontinuous line) n extr-intestinl (inverte tringles, otte line) lium uptke n environmentl lium onentrtion for the t from Fig. 5. Regression lines re shown, n lose n open symols inite, respetively, 8 h n 24 h fter trnsfer from 1%SW to other experimentl slinities. n 24 HAT. Furthermore, [C 2+ ] ppers to e the min environmentl ftor etermining lium influx, s shown y the ft tht fish expose to CCl 2 -enrihe sewter h whole-oy influx rtes similr to those of ontrol (nonenrihe) sewter of similr lium ontent. By ontrst, slinity itself ppere to hve little effet on lium influx sine NCl-enrihe ilute sewter h no effet on lium influx. However, n effet of NCl on lium influx nnot e ompletely exlue sine the low numer of surviving lrve in the NCl-trete group my not hve llowe more thorough nlysis. Few stuies hve esrie the effets of environmentl lium on lium uptke in fish lrve. Tilpi lrve limte to freshwter with high or low lium ontent h ifferentil lium influx when expose to iverse lium onentrtions (Hwng et l., 1994). Tilpi lrve, in ommon with the se rem lrve, h signifintly lower lium influxes immeitely fter trnsfer to wter ontining less lium. Moreover, effluxes eme progressively lower in the low-lium group, initing lium retention to mintin positive lne (Chou et l., 22; Hwng et l., 1996). These oservtions further emphsise the ility of lrvl fish to ommote their trnsporting mehnisms in response to hnges in lium. An unexpete oservtion in the present stuy ws tht NCl supplementtion of ilute SW to otin 36 g l 1 resulte in very high mortlities. This suggests tht ion-exhnge mehnisms my hve een ffete n tht the rtio etween N + n/or Cl n the other ioni omponents in the wter re essentil for mintenne of norml physiology in lrve. Potentil explntions for the effets oserve with exess N + oul e (1) N + /K + imlne tht woul ompromise the N + /K + pump or (2) slowing own or even reversl in iretion of the N + /C 2+ exhnger in the seros sie leing to n umultion of intrellulr N +, whih woul e ggrvte y the mentione K + imlne. However, further work will e require to lrify whih mehnisms re ffete y NCl supplementtion. Drinking n lium uptke The results from the present stuy with gilthe se rem lrve show ler epenene of rinking rtes on externl osmolrity uring the pttion of gilthe se rem to low-slinity environments. This is expete sine the ehyrting strength of the meium etermines the mount of wter require to reple osmoti losses through oy surfes (Fuentes n Ey, 1997). Other stuies with mrine n euryhline fish lrve herring (Clupe hrengus), plie (Pleuronetes pltess), o (Gus morhu; Tytler n Blxter, 1988), se ss (Flik et l., 22) n tilpi (Lin et l., 21; Miyzki et l., 1998)

8 652 P. M. Guerreiro n others hve similrly shown erese or inrese in rinking when expose to lower or higher slinities, respetively. In the se rem lrve, exposure to 15%SW i not inrese rinking rtes signifintly ove those of fish in 1%SW, possily euse slt los woul e too high, s inite y the higher mortlity t 15%SW. Reution in rinking t 55, ompre with 34, hs een oserve in ult Aphnius ispr in the De Se, proly s mens to reue slt intke (Skhug n Lotn, 1974). In sewter fish, the mount of lium tht enters the intestinl trt s onsequene of rinking is rther lrge, oth ue to high rinking rtes n the elevte lium onentrtion in wter (Flik et l., 1995; Flik n Verost, 1993; Sunell n Björnsson, 1988). There re no ville t on lium sorption from the intestine in lrve n only few stuies hve onsiere this prmeter in ult fish. Estimtes of the ontriution of intestinl lium uptke re vrile n rnge from lmost zero to 2% in freshwter n sewter tilpi (Shoenmkers et l., 1993) to 4% in sewter o (Sunell n Björnsson, 1988) n 7% in the euryhline flouner Prlihthys lethostigm (Hikmn, 1968). However, it is likely tht intestinl C 2+ sorption rtes vry with mient slinity, C 2+ or ironte (Wilson et l., 22) onentrtions. Estimtes in gilthe se rem juveniles in 1%SW inite tht the intestinl lium sorption rte n reh 9% (Guerreiro et l., 22). Assuming n ientil sorption rte in gilthe se rem lrve, intestinl lium trnsport oul ount for s muh s 4% of the totl lium uptke. In the gilthe se rem, the epenene of whole-oy lium uptke n environmentl [C 2+ ] is minly ue to high epenene of the intestinl route on environmentl onitions. The erese in the intestinl ontriution with the erese in slinity most proly ws result of limittion impose y the mount of rinking, whih n e enhne or reue in proportion to environmentl [C 2+ ]. At lower environmentl [C 2+ ], lium influx is lrgely epenent on extr-intestinl trnsport. These results re in greement with those otine y Vonk et l. (1998) for rnhil lium influxes in juvenile tilpi rise t ifferent slinities n wter lium ontent. The lk of prllelism in the reltionship etween extr-intestinl n intestinl lium uptke s funtion of [C 2+ ] n the smller slope for extr-intestinl uptke (Fig. 6) suggest tht the ominnt uptke trnsport mehnisms t the two sites re ifferent, perhps with tive trnsport eing n importnt omponent extr-intestinlly n more pssive or exhnge mehnisms fvoure intestinlly when lium grients re fvourle. In support for this hypothesis, regulte low-ffinity, high-pity trnsport, possily meite y rrier protein, hve een esrie (Klren et l., 1993). The question s to whether intestinl trnsport is just onsequene of rinking or provies signifint ontriution to lium lne remins to e lrifie. In fish lrve, the mitohonri-rih hlorie ells, the mjor site for C 2+ n NCl exhnge in the externl epitheli (Flik et l., 1995, 1996; Perry n Flik, 1988), re lote throughout the oy integument, nmely in res where the interfe etween the thing wter n the extrellulr fluis is thin. Sewter-pte lrve hve more hlorie ells thn those in freshwter, presumly to exrete exess NCl (Flik et l., 22; vn er Heijen et l., 1999). As fish lrve evelop, the istriution of the hlorie ells hnges n they eome restrite to the gills in juveniles (see review y Kneko et l., 22; vn er Heijen et l., 1999; Wles n Tytler, 1996). In 3-y-ol gilthe se rem lrve pte to sewter, the highest ensity of hlorie ells (MR-ells) is present in regions with high lium requirements suh s the jw n fin epitheli, whih re unergoing minerlistion (P.M.G., J.F., G.F., J.R., D.M.P. n A.V.M.C., unpulishe results). The ientifition of extr-rnhil MR-ells in these regions, s well s in the trunk n operulr surfe, suggests tht in gilthe se rem lrve they my ply n importnt role in extr-intestinl lium uptke, proviing for iret uptke in res of high lium emn, s previously suggeste (Flik et l., 1995; Hwng et l., 1994; Vrsmos et l., 22). In onlusion, the present stuy shows tht gilthe se rem lrve re le to ommote their liumtrnsporting mehnism oring to the environmentl onitions n tht rinking n the intestine provie the min route for the inrese lium influx t higher slinities. The ontriution of the intestine to the overll lium uptke in ilute sewter n freshwter is negligile, n extrintestinl mehnisms ensure equte lium uptke in lium-eplete environments. By ontrst, in lium-rih wters n in sewter, the intestinl route eomes inresingly importnt. The mehnism y whih lium is tken up in the intestine remins to e eluite ut the strong uptke in response to e lium n the reltionship with slinity suggests tht lium trnsport is ssoite with or epenent on tht of other ions. This reserh hs een rrie out with the finnil support of the Commission of the Europen Union, Qulity of Life n Mngement of Living Resoures speifi RTD progrmme (Q5RS- Q5RS ). P.M.G., J.F. n J.R. were in reeipt of fellowships PRAXIS/BD/927/96, PRAXIS/BPD/2233/99 n SFRH/BPD/1524/2, respetively, from Função pr Ciêni e Tenologi (Portugl). Referenes Alerie, D. F. (1988). Osmoti n ioni regultion in teleost eggs n lrve. In The Physiology of Developing Fish, vol. XI A (e. W. S. Hor n D. J. Rnll), pp Lonon: Aemi Press. Chou, M.-Y., Yng, C.-H., Lu, F.-I., Lin, H.-C. n Hwng, P.-P. (22). Moultion of lium lne in tilpi lrve (Oreohromis mossmius) limte to low-lium environments. J. Comp. Physiol. B 172, Fustino, M. n Power, D. M. (1998). Development of osteologil strutures in the se rem: verterl olumn n ul fin omplex. J. Fish Biol. 52, Fustino, M. n Power, D. M. (1999). Development of the petorl, pelvi, orsl n nl fins in ulture se rem. J. Fish Biol. 54, Fustino, M. n Power, D. M. (21). Osteologi evelopment of the

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