McMaster University, 1280 Main St West, Hamilton, ON, Canada, L8S 4K1. *Author for correspondence

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1 3925 The Journl of Experimentl Biology 216, Pulishe y The Compny of Biologists Lt oi:1.1242/je RESERCH RTICLE Uptke, hnling n exretion of N + n Cl from the iet in vivo in freshwter- n sewter-limte killifish, Funulus heterolitus, n gstri teleost Crol Buking 1,2, Chris M. Woo 1,2 n Mrtin Grosell 1 1 Rosenstiel Shool of Mrine n tmospheri Sienes, University of Mimi, Mimi, FL 33149, US n 2 Deprtment of Biology, MMster University, 128 Min St West, Hmilton, ON, Cn, L8S 4K1 *uthor for orresponene (roluking@gmil.om) SUMMRY riotrer pproh using iets lelle with 22 N +, 36 Cl n [ 14 C]polyethylene-4 (PEG-4) ws employe to investigte the role of intestinl uptke from the foo in ion homeostsis in the killifish Funulus heterolitus. This euryhline teleost lks oth stomh n the pity for Cl uptke t the gills in freshwter. PEG-4 pperne in the wter ws miniml up to 1 11 h post-feeing, initing the virtul sene of N + n Cl loss in the fees up until this time. Rpi uptke of ietry N + n Cl ourre n more thn 88% of 22 N + n 36 Cl were sore in the intestine y 3 h post-feeing; exretion rtes of N + n Cl originting from the foo were gretest uring this perio. Uptke n exretion of Cl from the iet ws fivefol to sixfol greter thn tht of N + in freshwter, n pproximtely threefol greter in sewter. Exretions of ietry N + n Cl y sewter-limte killifish were fr greter thn y freshwter-limte killifish in this time frme, refleting the muh greter rnhil efflux rtes n turnover rtes of the internl exhngele pools. t oth 3 n 9 h post-feeing, the lrgest frtion of ietry N + ws foun in the rss of freshwter-limte fish, followe y the externl wter, n finlly the igestive trt. However, in sewter-limte fish, more ws exrete to the wter, n less ws retine in the rss. For Cl, whih ws tken up n exrete more rpily thn N +, the mjority of the ietry lo h move to the externl wter y 9 h in oth freshwter n sewter nimls. fter 7 ys trining on low-slt nturl iet (live Lumriulus vriegtus worms; 31.5 mol N + g 1 wet mss) versus high-slt syntheti pellet iet (911 mol N + g 1 ry foo mss), freshwter killifish exhiite lower solute exretion rte of N + from the low-slt iet, ut reltive uptke from the intestine n retention in the rss were virtully ientil from the two iets. Sewter killifish exrete reltively more N + from the low-slt iet. Overll, our results emphsize the importne of ietry N + n Cl in the eletrolyte eonomy of the killifish, prtiulrly in freshwter, n espeilly for Cl. Key wors: intestine, gills, ion flux rtes, hlorie uptke, ionoregultion, feeing, efetion. Reeive 14 Deemer 212; epte 11 July 213 INTRODUCTION The omprison of osmoregultion n ionoregultion in the imetrilly oppose environments of freshwter n sewter hs een the sujet of severl exellent reviews (e.g. Evns et l., 25; Hwng n Lee, 27; Evns, 28). Brnhil n renl proesses re now reltively well unerstoo. Typilly, in freshwter fish, tive uptke of N + n Cl ours t the gills, while the kiney exretes high volume of ilute urine to ompenste for osmoti influx of wter from the hypotoni environment. In ontrst, in sewter teleosts, the gills tively exrete exess N + n Cl gine from the hypertoni environment. Muh of this N + n Cl lo origintes from the gstrointestinl (GI) trt s mrine fish must rink sewter to reple wter lost y osmosis ross the gills n oy surfe. In ft, the role of the intestine in sewter ion n wter lne is muh more pprent ompre with in freshwter fish. gin the sujet of severl reviews (e.g. Wilson et l., 22; Grosell, 26; Grosell, 211), the intestine is essentil for homeostsis in sewter fish through eslintion of ingeste wter, solute-ouple flui sorption n HCO 3 seretion, whih les to more wter uptke through CCO 3 preipittion. The role of the iet in overll pisine ion regultion hs een generlly overlooke, n until reently, there hs een little tivity in this re, with most ionoregultory stuies eing rrie out on fste fish. In generl, the iet is thought to present slt lo, espeilly ommeril iets n slt-rih invertertes, whih my e enefiil for freshwter fish n potentilly ompromising in sewter fish (for review, see Woo n Buking, 211). However, reent stuies on one moel speies, the rinow trout (Onorhynhus mykiss), suggest tht physiologil proesses ssoite with igestion my influene the outome of ietry ion effets. For exmple, iliry seretion of N + in freshwter trout (Grosell et l., 2) ppers to eliminte ny net vntge of ietry N + uptke (Buking n Woo, 26). surprising fining in this speies is tht most of the GI ion uptke ours in the stomh, n not in the intestine (Buking n Woo, 26; Buking n Woo, 26; Buking n Woo, 27). How universl these ptterns re ross the multitue of teleost speies is unknown. However, not ll fish ion- n wter-regulte in the sme mnner. For exmple, nother moel speies, the ommon killifish (Funulus heterolitus) lks stomh (Bkin n Bowie, 1928). Furthermore, the killifish is unusul in tht the gills tively tke up only N +, n not Cl, in freshwter, in ontrst to most other speies (Woo n Mrshll, 1994; Ptrik et l., 1997; Ptrik et l., 1999; Woo n Lurent, 23; Tomsso n Grosell, 25;

2 3926 The Journl of Experimentl Biology 216 (2) Woo, 211). This hs le reserhers to surmise tht killifish must rely on the iet for overll Cl uptke in freshwter. In ft, experiments using in vitro gut s preprtions emonstrte tht intestinl Cl uptke ws upregulte in killifish uring limtion to freshwter (Sott et l., 26), n tht intestinl uptke of Cl ws greter in freshwter-limte killifish thn in sewterlimte speimens (Woo et l., 21). This is in ontrst with the trout (Buking et l., 29) n most other speies (Grosell, 26; Grosell, 211), where intestinl Cl uptke in vitro is inrese with inresing slinity. However, it remins to e proven whether these ptterns our in vivo, where the gut is proessing true hyme rther thn sline. Reently, we hve evelope riotrer metho to stuy the uptke n fte of 22 N from the iet in the killifish in vivo (Woo n Buking, 212). The inert mrker polyethylene glyol-4 ([ 14 C]-PEG-4) is use to etet efetion events, so s to seprte systemi efflux of sore 22 N + into the externl wter from retl efflux vi the fees. In the present stuy, we hve utilize this tehnique to stuy the intestinl uptke, susequent internl istriution n exretion of oth 22 N + n 36 Cl from the iet in the killifish. We hve ompre the hnling of 22 N + n 36 Cl from high-slt pellet iet in oth freshwter- n sewterlimte nimls, n we hve further ompre the hnling of 22 N + from low-slt nturl iet (live oligohetes) in oth freshwter n sewter killifish. Riotrer experiments of this nture re omplex, so we hve lso ritilly ssesse the methoology n theory of this pproh. Our working hypotheses were tht Cl woul e tken up from the iet t greter rte thn N +, n tht this ifferene woul e espeilly prominent in freshwter killifish. We lso preite tht retention of oth ions from the iet woul e greter in freshwter nimls thn in sewter nimls, euse the former hve greter nee to quire n onserve ions. Therefore, loss rtes y systemi efflux ross the gills n kiney woul e muh lower in freshwter killifish. Finlly, we postulte tht reltive uptke n retention of N + woul e greter from the low-slt nturl iet in freshwter killifish for the sme resons, ut tht this ifferene woul not our in sewter killifish. MTERILS ND METHODS Experimentl proeures were pprove y institutionl niml re ommittees t the University of Mimi n MMster University. nimls Killifish [Funulus heterolitus (Linneus 1766); oth sexes, 2 6 g] were pture from the wil n hel y quti Reserh Orgnisms (Hmpton, NH, US) in qurntine efore eing sent to the Rosenstiel Shool of Mrine n tmospheri Sienes, where they were limte to lortory onitions for t lest 4 weeks efore experimenttion. nimls were either mintine in flowthrough sewter [N + =485, Cl =569, K + =1.6, C 2+ =1.7, Mg 2+ =59.5 n SO 4 2 =31.6 mmol l 1 ; C, 37.5 ppt, ph 8. (Woo n Grosell, 28)] or freshwter (ehlorinte Mimi City tp wter; N + =1.6, Cl =1.21, K + =.8, C 2+ =.43, Mg 2+ =.13 n SO 4 2 =.14 mmol l 1 ; C, ph 7.4 (Woo n Grosell, 28)] in 5 l glss quri. Fish were fe ommeril pellets (N + =23, Cl =17, C 2+ =628, Mg 2+ =77 n K + =326 mol g 1 ) every 24 h n sujete to 16 h:8 h light:rk yle. Diet preprtion Severl riolelle iets were prepre, with either high or low slt ontent. Both high- n low-slt iets were lso me in n ientil fshion, ut without the inorportion of riotrers, n use for pre-experimentl trining of the fish. High-slt iets (N + =911 mol g 1 ry foo mss; Cl =918 mol g 1 ry foo mss) were me y grining ommeril fish flkes (TetrMin, Spetrum Brns, Blksurg, V, US) into fine power using mortr n pestle. Brine shrimp (Sn Frniso By Brn, Newrk, C, US) were then mixe with the power (4% w/w) to form pste. Susequently, riotrers were e (17.5 Ci of 22 N + or 36 Cl, 1 Ci [ 14 C]PEG-4 per 3 g foo) to the pste. Riotrers ( 22 N, 36 Cl, oth s NCl n [ 14 C]PEG-4) were otine from mershm (Little Chlfont, UK) or NEN-Dupont (Boston, M, US). The isotopes were then thoroughly inorporte y mixing, n the pste ws extrue through 2 ml syringe (without neele tthe) to form long, thin ropes. The iets were then rie in n oven (6 C) for 24 h n rumle y hn to form foo pellets. The iets were store t 2 C efore use. Low-slt iets (live oligohetes; N + =31.5 mol g 1 wet mss) were lso prepre. Cliforni lkworms (Lumriulus vriegtus; quti Foos, Fresno, C, US) were limte to lortory onitions for ~5 ys efore use. Briefly, the lkworms were mintine in high-wlle ontiners with shllow th of ehlorinte Mimi ity tp wter (esrie ove). The th wter ws hnge every 48 h. Before eing use s n experimentl iet, 1 g of worms were ple in 5 ml of wter tht ontine 6 Ci of 22 N +. The worms were inute with the rioisotope for 24 h. The lkworms were riefly rinse in len wter to remove loosely oun surfe riotivity efore eing fe to the killifish. To etermine n pproprite experimentl feeing proeure, eh iet (exept the 36 Cl-lelle high-slt iet, euse of its expense) ws teste for lehing of rioisotope to the wter efore experimenttion egn. Briefly, eh iet (1 g) ws ple in wter (1 ml). The wter ws then smple t vrious time points following the ition of the foo to the wter. There were only smll mounts of 22 N + or [ 14 C]PEG-4 isotope etete in the wter up to 1 h following introution of the rie, high-slt iets. In ontrst, 22 N + rioisotope ws foun in the wter immeitely following trnsfer of live worms. These ftors were tken into ount in the susequent experimentl esigns. Experimentl esign Following lortory limtion, iniviul fish were remove from the 5 l glss quri, weighe n ple in iniviul ontiners (5 ml plsti foo ontiners with lis). Eh experimentl series involve 12 fish, exept in few tests where few fish file to fee stisftorily (N=1 11). Eh series ws onute on seprte set of fish. The ontiners were lkene on one en to provie shelter for the nimls n were supplie with iniviul wter n ir lines to supply freshwter/sewter n oxygen, respetively. The ontiners were lso equippe with removle flp on the top through whih wter smples oul e tken s well s foo elivere. The fish were trine to onsume the high-slt foo pellets or the low-slt lkworms (elivere y hn on foreps through the feeing flp) for ~1 week efore experimenttion egn. The fish generlly te springly uring the first few ys, ut uilt up to the rtion use in the tul experiments y the en of the trining perio. Series 1 One trining ws omplete, the nimls were fste for ~36 h to ler the previous mel from the GI trt. The nimls were then fe high-slt iet with [ 14 C]PEG-4 inorporte

3 Dietry N + n Cl uptke in killifish 3927 (841,738 pm g 1 ry foo mss) until stite. Feeing took ~3 min, n wter flow ws ontinue uring feeing. The mount of foo ingeste y eh fish ws reore n n ttempt to provie similr rtions in ll further series ws unertken. Wter flow ws stoppe t the en of feeing, the wter volume in the hmers ws set to 5 ml, n n initil wter smple ws tken (5 ml), representing time h. Cre ws tken to ensure ll foo ws eten to prevent lehing of the isotope into the wter, n ny uneten foo ws remove within 3 min following the eginning of feeing (i.e. efore time h). Wter smples (5 ml) were then tken every 6 min for 16 h, n then t 24, 28 n 32 h following feeing. Wter ws thoroughly flushe from the hmers t 12 n 24 h following feeing. Susequent to eh wter hnge, wter flow ws gin ese n n initil wter smple ws tken (5 ml). Series 2 The experiment ws then repete with high-slt iet with 22 N + e (287,943 pm g 1 n 911 mol N + g 1 ry foo mss) n with low-slt iet with 22 N + e (66,11 pm g 1 n 31.5 mol N + g 1 wet foo mss). When the low-slt iet (live lkworms) ws fe, fish were trnsferre to new ontiner 3 min fter the eginning of feeing, n initil wter smple ws tken n susequent wter smpling n flushing ourre s efore with the high-slt iets. This ws one s feeing the worms trnsferre signifint mount of isotope in the wter ministere with the worms, ontminting the wter foun in the ontiners s esrie ove. Series 3 similr experiment ws onute with high-slt iet with 36 Cl e (19,38 pm g 1 n 918 mol Cl g 1 ry foo mss). In this experiment, s prior lehing test ws not performe, the fish were trnsferre to len ontiner following 3 min feeing perio with flowing wter. Wter smples were tken every hour s efore. Bse on the [ 14 C]PEG-4 experiment of Series 1, this tril ws terminte 9 h fter feeing euse therefter the pperne of the isotope in the wter ws potentilly onfoune y elimintion of fees. itionlly, it ws oserve tht lrger proportion of the ietry 36 Cl ppere in the wter within the first few hours in omprison with the ietry 22 N +. Out of onern for greter possiility of isotope reyling vi rinking, the experiments were terminte erlier thn those of Series 1 n 2. Series 4 The gol of this series ws to quntify the istriution of the ingeste N + or Cl lo etween the igestive trt, the rss (reminer of the fish) n the externl wter t 3 n 9 h fter feeing i.e. efore ny omplition from efetion oul our. Similr to the previous series, trine fish in iniviul ontiners supplie with either freshwter or sewter were fe to stition with one of the three iets (N=12 for eh iet in eh slinity): low-slt iet with 22 N + e (freshwter=66,11 pm g 1 n 31.5 mol g 1 wet foo mss; sewter=198,286 pm g 1 n 31.5 mol g 1 wet foo mss), high-slt iet with 22 N + e (287,943 pm g 1 n 911 mol N + g 1 ry foo mss for oth slinities) n high-slt iet with 36 Cl e (19,38 pm g 1 n 918 mol Cl g 1 ry foo mss, gin for oth slinities). gin, re ws tken to ensure miniml lehing of the foo into the wter y removing ny uneten foo immeitely following the esstion of feeing. To ensure prllelism etween the 22 N + n the 36 Cl experiments, one the mel h een ingeste (3 min following the eginning of feeing), the fish were trnsferre to new ontiners with len wter n n initil wter smple (5 ml) ws tken. Wter smples were then tken every hour following feeing s efore. However, in this series, fish were euthnize y n overose of nesthesi (MS-222,.2 g l 1 ; Synel Lortories, Vnouver, Cn; neutrlize with NOH) t 3 h (N=6 for eh slinity) n 9 h (N=6 for eh slinity) following feeing. Following euthniztion, the GI trt ws expose through mi-line inision, ligte t oth ens with silk suture, n then remove intt. The mount of rioisotope ws then etermine in the whole oy, the GI trt n the externl wter. nlysis 36 Cl n [ 14 C]PEG in the wter ws etermine y pling 5 ml wter smples into 1 ml of Eolume fluor (MP Biomeils, St Louis, MO, US) n ounting in Trir 21TR liqui sintilltion ounter (Pkr Instruments, Downers Grove, IL, US). 22 N + ws mesure in 1 ml wter smples iretly on Cor II gmm ounter (Pkr Instruments). To etermine the riotivity of the rss, the gut n the foo, ll three were first issolve in five volumes of 1 mol l 1 HNO 3 n hete (6 C) for 24 h in sele tue. The resulting extrt ws then entrifuge, n the superntnt ws teste for riotivity y sintilltion ounting for 36 Cl (1 ml neutrlize extrt plus 4 ml wter plus 1 ml fluor) n y gmm ounting for 22 N + (1 ml iretly). N + n Cl onentrtions in extrts of the iets were mesure using tomi sorption spetrophotometry (Moel 22FS, Vrin ustrli, Mulgrve, VIC, ustrli) n oulometri titrtion (CMT-1; Riometer, Copenhgen, Denmrk), respetively. For quenh orretion of [ 14 C] n [ 36 Cl ] sintilltion smples, ounting effiieny of tissue n foo smples ws orrete k to the sme effiieny s wter smples y internl stnriztion vi the onstnt ition metho (Rogers n Morn, 1966). The mount of rioisotope exrete to the wter (pm w g 1 fish mss) ws then etermine fter ounting for iniviul fish msses n the volume of the ontiner t eh time point. The initil wter smples tken immeitely following feeing (3 min) n wter hnges (12 n 24 h) were sutrte from susequent smples to eliminte kgroun ontmintion. Finlly, the umultive mount of N + or Cl exrete to the wter tht originte from the onsume mel ( mol g 1 fish mss) ws etermine s: Ion exretion = [pm w g 1 fish mss] / S, (1) where S ws the speifi tivity of the foo (pm mol 1 N + or Cl ). Sttistis JMP (version 8, SS Institute, Cry, NC, US) ws use to test the t for signifine. The rw t were first exmine for normlity n heterogeneity of vrine. Dt tht file to meet these onitions s well s ll perentge t were trnsforme s pproprite. In ll series, two-wy (slinity n time) repetemesures NOV ws use to nlyze the t initilly, n this ws ollpse to one-wy repete-mesures NOV n the t were omine in Series 1 only, where slinity h no signifint effet. pproprite post ho tests were then use to exmine the t for signifint hnges: Tukey s test in Series 1, where slinity h no effet, n the Holm Sik test in Series 2, 3 n 4, where oth ftors were signifint. Differenes in rioisotope istriution mong the wter, rss n GI trt were ompre using pire n unpire t-tests, gin s pproprite,

4 3928 The Journl of Experimentl Biology 216 (2) followe y Bonferroni orretions. signifine level of P<.5 ws use throughout. RESULTS Series 1 On verge fish onsume n pproximte 5% oy mss rtion of the [ 14 C]PEG-4-lelle iet (841,738 pm g 1 ry foo mss), regrless of externl slinity. Two-wy NOV emonstrte tht while the influene of time ws signifint, slinity h no effet on the pperne of [ 14 C]PEG-4 in the wter from the ingeste iet, so vlues were omine ross slinities (Fig. 1). During the first hour following ingestion, slightly less thn 1% of the ingeste [14C]- PEG-4 ppere in the wter, therefter inresing y n itionl 4% t 2 h to totl of ~5% of the ingeste lo. Therefter, [ 14 C]PEG- 4 riotivity in the externl wter remine unhnge for 1 h, suggesting negligile itionl exretion. However, t 11 h following mel ingestion, wter riotivity inrese rmtilly, representing ~26% of the ingeste lo (Fig. 1). Exretion to the wter ontinue to inrese for the reminer of the experiment, n y h following feeing, [ 14 C]PEG-4 pperne in the wter h rehe ~65% of the ingeste mount (Fig. 1). Series 2 Both freshwter- n sewter-limte fish onsume n pproximte 5% oy mss rtion of the 22 N + -lelle low-slt iet [31.5 mol N + g 1 wet foo mss (live worms)] n sttistil testing revele tht oth time n slinity were signifint ftors in N + pperne in the surrouning wter. In fish limte to freshwter, the mount of 22 N in the externl wter flutute etween ~.1 n.2 mol N + g 1 fish mss, or 5 n 13% of the ingeste N + lo, from 1 to 24 h, suggesting no itionl exretion uring this perio (Fig. 2). However, t 28 h following the mel, the mount of N + exrete to the wter inrese shrply (Fig. 2). The umultive efflux of ietry N + to the wter rehe ~.4 mol N + g 1 fish mss, representing 26% of the ingeste N + lo, y 32 h. In ontrst, sewter-limte fish tht ingeste omprle low-slt iet of worms exhiite muh more rpi relese of N + to the wter over the first 3 h following mel ingestion, inresing 1.5-fol from ~.4 to ~.7 mol N + g 1 fish mss, or 27 to 42% of the ietry N + (Fig. 2B). The mount of N + exrete to the wter susequently remine stle until 28 h, when it gin inrese to ~.9 mol N + g 1 fish mss or 58% of ingeste N + for the reminer of the experiment (Fig. 2B). Overll, the mjority of N + exretion ourre uring the first hour following the onsumption of mel in freshwter fish, while the mjority of N + exretion in mrine fish ourre over the first 3 h. The umultive N + efflux for sewter-limte fish over 32 h ws more thn twofol greter thn for freshwter fish fe omprle iet, highly signifint ifferene (Fig. 2B versus 2). In ft, the mount of N + exrete to the wter y sewterlimte fish in the first hour following feeing ws similr to the totl tht ws exrete over 32 h y freshwter-limte fish (Fig. 2,B). itionlly, there ppere to e little eviene of fel loss, s shown y the reltively stle levels of N + exretion 9 h following feeing (see Series 4). Freshwter- n sewter-limte fish gin oth te 5% rtion when presente with the high-slt 22 N + -lelle iet (911 mol N + g 1 ry foo mss). s with the low-slt iet, sttistil testing revele tht oth time n slinity were signifint ftors in N + pperne in the surrouning wter. t lest qulittively, freshwter-limte fish ingesting the high slt iet showe temporl pttern of 22 N + pperne in the wter (Fig. 2C) similr to tht of freshwter fish tht ingeste the low-slt iet (f. Fig. 2). The mount exrete flutute non-signifintly from.2 to.7 mol N + g 1 fish mss (N=12), rnging from.3 to 1.4% of the ingeste N + lo, from 1 to 24 h, one gin suggesting no itionl exretion uring this perio (Fig. 2C). Susequently, the mount of N + exrete to the wter inrese gretly t 28 h, to 2.3 mol N + g 1 fish mss (5% of ingeste N + ), n remine essentilly unhnge t 32 h (Fig. 2C). Sewter-limte fish ingesting the sme 5% rtion of high-slt iet exrete n inresing mount of N + over the first 5 h following mel ingestion, in ontrst with freshwterlimte fish (Fig. 2C), ut qulittively similr to sewterlimte fish tht ingeste low-slt iet (Fig. 2B). Initilly, ~1.6 mol N + g 1 fish mss (3.5% of ingeste N + ) ws exrete 1 h following feeing (Fig. 2D), more thn fivefol greter thn seen in freshwter fish fe the sme mel (f. Fig. 2C). 22 N + efflux then inrese progressively over the next 4 h to pek t ~4.6 mol N + g 1 fish mss (1% of ingeste N + ). Therefter, there ws no further pperne through 24 h (Fig. 2D), with vlues flututing from 8 to 11%. However, t 28 h, N + exretion one gin inrese 1.3-fol to ~6.2 mol N + g 1 fish mss (14% of ingeste N + ) n y 32 h, the umultive exretion of N + to the wter ws threefol higher thn seen with freshwterlimte fish (Fig. 2C) t the sme time. Cumultive [ 14 C]-PEG (pm g 1 fish mss) 4, 3, 2, 1,,,,,,,, Time (h) Fig. 1. Cumultive [ 14 C]-PEG-4 (polyethylene glyol) foun in the wter (pm g 1 fish mss) following feeing from freshwter- or sewter-limte killifish in Series 1. There were no signifint ifferenes etween freshwter or sewter fish so the t hve een omine. Fish onsume 5% rtion ( h). N=24 (N=12 for eh slinity). Different letters represent signifint (P<.5) ifferenes etween time points.

5 Dietry N + n Cl uptke in killifish Nturl low-slt iet Freshwter-limte C 3. Syntheti high-slt iet Cumultive N + efflux (μmol g 1 fish mss) B Sewter-limte D ,e,,,,,,, e e Time (h) Fig. 2. Cumultive N + exrete to the wter ( mol g 1 fish mss) originting from the iet in Series 2 for () freshwter- n (B) sewter-limte killifish fe nturl low-slt iet (66,11 pm g 1 ; 31.5 mol N + g 1 ; 5% rtion), n (C) freshwter- n (D) sewter-limte killifish fe syntheti high-slt iet (287,931 pm g 1 ; 911 mol N + g 1 ; 5% rtion). Feeing ourre t h. Uneten foo ws remove from eh ontiner ut fish were not trnsferre to nother ontiner. N=11 12 for eh slinity. For eh iet, rs tht shre letters re not signifintly ifferent (P>.5). Overll, s with the low-slt iet, the mjority of N + exretion ourre within the first hour following the onsumption of mel y freshwter-limte fish, while sewter-limte fish exhiite grul exretion over the first 4 h. One gin, the mount of N + exrete y sewter-limte fish within the first 1 h following feeing (Fig. 2D) ws similr to the totl mount of N + exrete over 32 h following feeing in freshwter-limte fish fe the sme high-slt mel (Fig. 2C). Series 3 Freshwter- n sewter-limte killifish onsume pproximtely 3.4% rtion of the 36 Cl -lelle high-slt iet (918 mol Cl g 1 ry foo mss). Sttistil nlysis revele tht oth slinity n time were signifint ftors. Speifilly, freshwterlimte fish exrete ~1.6 mol Cl g 1 fish mss to the wter uring the first hour following feeing, or 5% of the totl Cl onsume with the iet (Fig. 3). This exretion ws more thn fivefol higher thn the N + exretion with the sme iet in the first hour (f. Fig. 2C). Cumultive Cl efflux to the wter ontinue to inrese steily over the next 8 h to ~4.9 mol Cl g 1 fish mss (16% of the ingeste Cl lo; Fig. 3), in ontrst to the pttern of stility seen in umultive N + pperne over the sme perio (f. Fig. 2C), suh tht totl Cl efflux ws more thn fivefol greter thn totl N + efflux. Fish limte to sewter tht onsume high-slt iet exrete ~2.8 mol Cl g 1 fish mss (9% of the ingeste 36 Cl ) to the wter within the 1 h following feeing (Fig. 3B). Cl efflux to the wter proeee to inrese over the next 8 h to result in umultive exretion of ~17.8 mol Cl g 1 fish mss (56%; Fig. 3B), vlue tht ws pproximtely threefol higher thn N + exrete to the wter (Fig. 3B versus Fig. 2D) from the sme iet n pproximtely fourfol higher then seen with freshwter-limte fish onsuming the sme mel (Fig. 3).

6 393 The Journl of Experimentl Biology 216 (2) Cumultive Cl efflux (μmol g 1 fish mss) B B Overll, freshwter- n sewter-limte killifish showe similr temporl pttern of Cl exretion following the ingestion of mel; however, it ws quntittively higher in sewter-limte fish. Series 4 In this series, fish were euthnize t 3 n 9 h post-feeing to quntify the istriution of the ingeste N + or Cl lo etween the GI trt, the rss (reminer of the fish) n the externl wter. itionlly, the wter ws exhnge following feeing, llowing the more sutle hnges in rioisotope onentrtion to e oserve (ompre with Series 2). Fish onsume 4.9% rtion of the low-slt iet (31.5 mol N + g 1 wet foo mss, live worms) in oth freshwter n sewter, n one gin, oth slinity n time were signifint ftors. However, in ontrst to freshwterlimte fish fe the low-slt iet in Series 2 (f. Fig. 2), freshwter fish fe the sme iet of worms in Series 4 showe,,e,,e,, Time (h) Fig. 3. Cumultive Cl exrete to the wter ( mol g 1 fish mss) originting from the iet in Series 3 for () freshwter- n (B) sewter-limte killifish fe syntheti high-slt iet (19,38 pm g 1 ; 918 mol Cl g 1 ; 3.4% rtion). Feeing ourre t h n fish were trnsferre to nother ontiner fter.5 h. N=11 12 for eh slinity. Brs tht shre letters re not signifintly ifferent (P>.5).,e,e, e,e,,e C grul inrese in the mount of N + exrete to the wter over the first 3 h (Fig. 4) n 9 h (Fig. 4B) following feeing. In freshwter, 5% of ingeste N + h een exrete 1 h following feeing (~.1 mol N + g 1 fish mss; Fig. 4). This inrese 1.4- fol to 9% t 3 h n to ~11% y 7 h, fter whih it remine stle (~.2 mol N + g 1 fish mss; Fig. 4B). Sewter-limte fish exhiite similr grul exretion of N +. t 1 h following the onsumption of mel, pperne in the externl wter ws ~.4 mol N + g 1 fish mss or 24% of ingeste N + (Fig. 4D), n this h inrese 1.6-fol to 4% y 3 h (~.6 mol N + g 1 fish mss; Fig. 4D) n twofol to 49% y 5 h (~.8 mol N + g 1 fish mss; Fig. 4E). Comprtmentl nlysis revele rpi sorption of ietry 22 N + from the igestive trt in oth freshwter n sewter. In freshwter-limte killifish, y 3 h post-feeing, the frtion foun in the trt ws only 7% of the ingeste 22 N + lo, n this fell to 3% t 9 h. The proportions in the wter inrese from 9% t 3 h to 11% t 9 h, wheres the frtions in the rss erese from 63% t 3 h to 56% t 9 h (Fig. 4C). The unounte-for portions of 22 N + were 22% t 3 h n 29% t 9 h. In sewterlimte killifish, sorption from the igestive trt ws gin rpi, with only 8 n 4% remining t 3 n 9 h post-feeing, respetively (Fig. 4F). The frtion of ietry 22 N + tht h een eliminte to the externl sewter ws 45% y 3 h, n signifintly inrese to 66% y 9 h (Fig. 4F). The proportion of ietry 22 N + in the rss orresponingly erese from 31% t 3 h to 18% t 9 h. The unounte for proportion of 22 N + ws 15% t 3 h n 12% t 9 h (Fig. 4F). Fish fe the high-slt iet (911 mol N + g 1 ry foo mss) in this series onsume only 2.8% rtion. In ontrst to fish fe the low-slt iet (f. Fig. 4), fish fe the high-slt iet showe grul inrese in the mount of 22 N + exrete to the wter in sewter only (Fig. 5D,E), n not in freshwter (Fig. 5,B). Despite this, oth slinity n time were signifint ftors. In freshwter, ~7% of the ingeste lo h een exrete 1 h following feeing (~1.8 mol N + g 1 fish mss; Fig. 5,B), n this remine stle for the urtion of the experiment. In sewter, fish exrete 8% y 1 h (~2.2 mol N + g 1 fish mss); however, this inrese to 2% y 3 h n remine reltively stle therefter (~5.6 mol N + g 1 fish mss; Fig. 5D,E). Despite the 16-fol higher ietry N + lo reltive to the fish fe the low-slt iet, frtionl sorption of ietry 22 N + from the igestive trt ws gin rpi. In freshwter fish, the igestive trt ontine only 7% t 3 h n 11% of ingeste N + t 9 h (Fig. 5C); only the ltter vlue ws signifintly higher thn in the omprle low-slt iet tretment. The mjority of 22 N + ws in the rss, 67% t 3 h n 59% t 9 h, omprle to the perentges in the rss t these times in the fish fe the low-slt nturl iet. Similrly, the reltive mounts exrete to the externl freshwter, 1% t 3 h n 12% t 9 h, were omprle to those seen in killifish fe the low-slt iets (Fig. 5C versus Fig. 4C). This istriution ws not signifintly ifferent etween 3 n 9 h (Fig. 5C). The frtions of N + tht oul not e ounte for were 16% t 3 h n 19% t 9 h. In sewter-limte fish, the frtion of 22 N + foun in the GI trt ws gin low n i not signifintly hnge etween 3 h (12%) n 9 h (8%). The mjority of 22 N + ws foun in the rss espite eresing from 47% t 3 h to 36% t 9 h (Fig. 5F). The mount of 22 N + in the wter i not hnge from 3 h following feeing (22%) to 9 h (2%; Fig. 5F). The frtions of N + tht oul not e ounte for were 21% t 3 h n 37% t 9 h. The low proportion of ietry N + in the intestinl trt within ll ove tretments (rnging from 3 to 12%) my explin the

7 Dietry N + n Cl uptke in killifish 3931 Cumultive N + efflux (μmol g 1 fish mss) B Freshwter, 1 2 3,,,, D E Sewter,, e,e,e,,,, e e Fig. 4. Cumultive N + exrete to the wter ( mol g 1 fish mss) originting from the iet in Series 4 y freshwter-limte killifish fe nturl low-slt iet (66,11 pm g 1 ; 31.5 mol N + g 1 ; 4.9% rtion) over () 3 h post-feeing (N=6) n (B) 9 h postfeeing (N=6), n y sewter-limte killifish fe nturl low-slt iet (198,286 pm g 1 ; 31.5 mol N + g 1 ; 4.9% rtion) over (D) 3 h postfeeing (N=6) n (E) 9 h post-feeing (N=6). Perentge of totl pm foun in the wter, rss or GI trt of (C) freshwter or (F) sewter fish. Feeing ourre t h n fish were trnsferre to nother ontiner fter.5 h. For eh slinity, rs tht shre letters re not signifintly ifferent (P>.5). sterisks inite signifint hnge in the proportion of 22 N + from 3 to 9 h..1.4,.5.2 % of onsume pm C GI trt Crss Wter Unounte F * 3 9 Time (h) 3 9 reltive sene of fel N + exretion oserve in Series 2. s result, no orretions were me for fel losses in Series 2. Fish onsume 3.2% rtion of the 36 Cl -lelle iet in this series, n gin oth slinity n time were signifint ftors. In freshwter-limte killifish, the exretion of 36 Cl inrese from 1% t 1 h following feeing to 27% t 3 h (from ~3. to 7.1 mol Cl g 1 fish mss; Fig. 6), n finlly to ~43% t 9 h (~13.5 mol Cl g 1 fish mss; Fig. 6B). The frtionl mount of Cl exrete y freshwter-limte fish ws higher (1 43%; Fig. 6,B) thn tht seen with N + when fish were fe the sme iet (~7%; Fig. 5,B). In ontrst, fish in sewter exrete ~9.2 mol Cl g 1 fish mss (31% of ingeste) 1 h fter feeing, whih inrese to 6% t 3 h (~17.8 mol Cl g 1 fish mss; Fig. 6D). The umultive efflux of Cl remine unhnge therefter (Fig. 6E), ounting for ~45 8% of the ingeste Cl. By wy of ontrst, fish in sewter exrete etween 2 n 4% of N + from the sme iet over the sme perio (f. Fig. 5E). Frtionl sorption of ietry 36 Cl from the igestive trt ws even more rpi thn the frtionl sorption of ietry 22 N +. In freshwter fish, only 4 n 3% of ingeste 36 Cl were left in the trt t 3 n 9 h fter the mel, respetively (Fig. 6C), signifintly lower thn 7 11% of ietry 22 N + (Fig. 5C). The frtion in the rss ws higher ompre with the wter t 3 h in freshwter-limte fish (46% versus 26%; Fig. 6C). However, y 9 h this pttern ws reverse n the proportion of ingeste 36 Cl in the wter h inrese to 47%, while it h erese in the rss to 25% (Fig. 6C). The unounte-for proportion of 36 Cl ws 25% t 3 h n 26% t 9 h. In sewter-limte fish, the sorption n reistriution of 36 Cl ws exeeingly fst. The mjority of 36 Cl ws foun in the wter in sewter-limte fish t oth 3 h (6%, N=6) n 9 h (79%) post-feeing (Fig. 6F). Negligile frtions were left in the igestive trt: 1% t 3 h n.2% t 9 h (Fig. 6F). The mount in the rss signifintly erese from 7% t 3 h to 1% t 9 h, n the unounte-for proportion of 36 Cl ws 3% t 3 h n 2% t 9 h.

8 3932 The Journl of Experimentl Biology 216 (2) Cumultive N + efflux (μmol g 1 fish mss) B Freshwter D E Sewter 1 2 3,,,,,, Fig. 5. Cumultive N + exrete to the wter ( mol g 1 fish mss) originting from the iet in Series 4 y freshwter-limte killifish fe syntheti highslt iet (287,931 pm g 1 ; 911 mol N + g 1 ; 2.8% rtion) over () 3 h post-feeing (N=6) n (B) 9 h postfeeing (N=6), n y sewter-limte killifish fe the sme syntheti high-slt iet over (D) 3 h postfeeing (N=6) n (E) 9 h post-feeing (N=6). Perentge of totl pm foun in the wter, rss or GI trt of (C) freshwter or (F) sewter fish. Feeing ourre t h n fish were trnsferre to nother ontiner fter.5 h. ross slinities, rs tht shre letters re not signifintly ifferent (P>.5). sterisk inites signifint hnge in the proportion of 22 N + from 3 to 9 h..5 2 % of onsume pm C GI trt Crss Wter Unounte F * 3 9 Time (h) 3 9 DISCUSSION Overview To our knowlege, this is the first stuy to iretly ompre the uptke n fte of ietry N + n Cl in the sme euryhline speies in freshwter versus sewter. Overll, the present t provie strong support for the importne of ietry N + n espeilly Cl in the eletrolyte eonomy of the killifish, minly in freshwter. This s to the growing eviene tht ietry ions ply key role in ionoregultory homeostsis in fish (reviewe y Woo n Buking, 211). The results provie ler nswers to two of our initil three working hypotheses. Firstly, in orne with preitions, Cl ws tken up from the iet t greter rte thn N +, n the ifferene ws more prominent in freshwter killifish. Thus, in the high-slt iet experiments of Series 2 n 3, the pperne in the wter of Cl from the iet ws pproximtely fivefol fster thn N + in freshwter killifish, n pproximtely threefol fster thn N + in sewter killifish. This ws onfirme y similr sixfol (freshwter) versus threefol (sewter) ifferenes in Series 4. These ifferenes re muh lrger thn n e expline y the ~1.5-fol ifferenes expete s result of exhngele pool sizes lone, s isusse susequently. They were lso onfirme y iret mesurements of unsore riotivity in the intestinl trt t 9 h in Series 4. For exmple, in freshwter killifish, the reltive mount of 36 Cl left in the trt t 9 h post-feeing ws only 2.5% of the ingeste lo, wheres the reltive mount of 22 N + ws 1.5%. Seonly, the hypothesis tht retention of oth ions from the iet woul e greter in freshwter nimls thn in sewter nimls ws onfirme. For exmple, in the high-slt iet experiments of Series 2 n 3, y 9 h only ~1.5% of the 22 N + lo n 16% of the 36 Cl lo h een exrete y freshwter fish, wheres in sewter fish the vlues were 1% ( 22 N + ) n 43% ( 36 Cl ). In Series 4, the reltive vlues for freshwter killifish were 7% ( 22 N + ) n 47% ( 36 Cl ), n for sewter killifish 18% ( 22 N + ) n 79% ( 36 Cl ). Finlly, we

9 Dietry N + n Cl uptke in killifish 3933 Cumultive Cl efflux (μmol g 1 fish mss) B Freshwter D E Sewter, 1 2 3,,,,,, Fig. 6. Cumultive Cl exrete to the wter ( mol g 1 fish mss) originting from the iet in Series 4 y freshwter-limte killifish fe syntheti high-slt iet (19,38 pm g 1 ; 918 mol 36 Cl g 1 ; 3.2% rtion) over () 3 h post-feeing (N=6) n (B) 9 h post-feeing (N=6) n y sewter-limte killifish fe the sme syntheti high-slt iet over (D) 3 h post-feeing (N=6) n (E) 9 h post-feeing (N=6). Perentge of totl pm foun in the wter, rss or GI trt of (C) freshwter or (F) sewter fish. Feeing ourre t h n fish were trnsferre to nother ontiner fter.5 h. ross slinities, rs tht shre letters re not signifintly ifferent (P>.5). sterisks inite signifint hnge in the proportion of 36 Cl from 3 to 9 h GI trt 12 1 C Crss Wter Unounte * 12 1 F * % of onsume pm * Time (h) 3 9 h preite tht reltive uptke n retention of N + woul e greter from the low-slt nturl iet thn from the high-slt syntheti iet in freshwter killifish, ut tht this ifferene woul not our in sewter killifish. s isusse susequently, the hypothesize ifferene ws not etete in freshwter killifish, n it ertinly i not our in sewter killifish. Ptterns of [ 14 C]PEG-4 pperne The pttern of efetion revele y the [ 14 C]PEG-4-lelle iet ws the sme in freshwter n sewter (Fig. 1), n very similr to tht reporte erlier in killifish limte to 1% sewter n fe muh smller rtion (Woo n Buking, 212). Thus signifint efetion i not our until 1 11 h post feeing, s revele y the signifint inreses in 14 C]PEG-4 pm in the wter t this time. Bse on this result, susequent nlyses (Figs 3 6) fouse on the first 9 h post-feeing, so tht the pperne of non-sore 22 N + or 36 Cl riotivity vi fel ishrge woul not onfoun interprettion of true exretion ptterns (i.e. vi gills n urine). Regrless, these lter nlyses (Fig. 4F, Fig. 5F, Fig. 6F) emonstrte tht there ws only very smll perentge (<11%, n often lower) of the ietry lo of 22 N + or 36 Cl riotivity tht remine unsore in the igestive trt t 9 h. The smll mount of 14 C]PEG-4 riotivity ppering in the externl wter prior to 9 h verge ~5% of the totl ingeste lo, n proly hs severl origins suh s lehing from tiny non-eten foo prtiles, regurgittion

10 3934 The Journl of Experimentl Biology 216 (2) n erly smll efetion events; it therefore represents the error in the system. In onsequene, ppernes of 22 N + n 36 Cl over this time frme tht were 5% of the ingeste lo shoul e interprete utiously, s they re lose to kgroun i.e. the resolution limit of the system in quntifying ietry ion exretion to the wter. Riotrer reovery onsiertions In Series 4, the frtion of onsume riotivity tht oul not e ounte for t the time of euthniztion rnge from 12 to 37% in the vrious experiments. In similr experiment, Woo n Buking (Woo n Buking, 212) reporte 15% isrepny. Susequent tests revele tht more thn hlf of this isrepny ws ounte for y loo loss upon issetion, s loo plsm hs high onentrtions of N + n Cl, wheres the reminer likely resulte from errors in ounting, estimte foo onsumption, hmer wter volumes n losses to the wter uring the first 3 min prior to trnsfer. s ll fish were trete the sme, we elete not to orret the t. Speifi tivity n exhngele pool size onsiertions Interprettion of riotrer experiments of this nture n esily e onfoune y speifi tivity issues (i.e. the rtio of hot to totl N + or Cl ) n ifferenes in exhngele pool sizes. The first of these issues is whether signifint reyling of rioisotope (i.e. kflux ) ours etween omprtments, n the seon is the interprettion of riotrer pperne in the wter from the iet, when the riotrer hs move through n intermeite omprtment (the exhngele whole-oy N + or Cl pool), whih ws initilly unlelle. The thir is the effet tht possile ifferenes in pool size will hve on riotrer pperne rtes in the wter. With respet to the first issue (possile kflux), lultions were rrie out se on mesure riotivity levels in the iet, rss n wter, mesure N + n Cl onentrtions in the wter n iet, n previous etermintions of exhngele whole-oy pool sizes for N + n Cl in freshwter- n sewter-limte F. heterolitus (Woo n Lurent, 23). The norml riterion for riotrer flux mesurements is tht speifi tivity on the fluxgenerting sie shoul e kept 1-fol higher thn on the fluxreeiving sie so s to voi signifint reyling of the riotrer (Kirshner, 197). This riterion ws met for fluxes from iet to oy n oy to wter up to 9 h in ll experiments with the lowslt nturl iet. However, in trils with the high-slt syntheti iet, while this riterion ws met for fluxes from oy to wter throughout the experiment, it ws not met for fluxes from iet to oy fter ~3 h, n y 9 h the speifi tivity rtio ws only twofol to fivefol. Thus signifint riotrer kflux likely ourre from the oy pool to the igestive trt ontents fter 3 h, resulting in n overestimte of the mount of N + or Cl remining in the trt y the en of the experiment. Nevertheless, s y 3 h, more thn 88% of the ietry N + n Cl h lrey een sore from the trt in ll experimentl onitions, the influene of this prolem on the overll onlusions of this stuy were negligile. With respet to the seon n thir issues, interprettion of riotrer pperne in the wter from the iet, it shoul e emphsize tht the mesure flux represents only the N + or Cl tht originte from the iet, euse it is se on the speifi tivity of the iet. It oes not represent the totl flux of N + or Cl out of the niml, whih will epen on the speifi tivity of the exhngele N + or Cl pool of the niml. For exmple, for given onstnt whole-oy N + efflux rte n given onstnt N + sorption rte from the iet, less of the totl efflux rte will originte from the iet if the internl pool size is ig thn if it is smll. Fortuntely, these pool sizes were mesure in n erlier stuy on F. heterolitus (Woo n Lurent, 23), n they re similr in freshwter versus sewter nimls for oth N + (tully 18% lower in sewter nimls fter 7 ys limtion) n Cl (no ifferene), whih simplifies interprettion of freshwter versus sewter ifferenes. Nevertheless, the exhngele whole-oy Cl pool is only 65% of the exhngele whole-oy N + pool (Woo n Lurent, 23), so ll other ftors eing equl, we woul expet ietry Cl efflux into the wter to our t 1.53 times the rte of ietry N + efflux. The tul ifferenes oserve were fr greter (threefol to sixfol), reinforing the onlusion tht ietry Cl is sore from the iet n exrete to the wter t higher rte thn ietry N +, espeilly in freshwter-limte killifish. The importne of ietry N + n Cl in F. heterolitus Despite the lk of stomh (Bkin n Bowie, 1928), whih ppers to e the mjor ion-sorptive site in rinow trout (Buking n Woo, 26; Woo n Buking, 27), killifish lerly sor most ( 88%) of the ietry N + n Cl through their igestive trt within 3 h post-feeing, regrless of whether the iet is high or low in slt. Inee, the rpi sorption of ions speks to highpity sorptive epithelium in the proximl portion of the igestive trt, lthough this requires further investigtion. Furthermore, muh of this sore N + n Cl enters the exhngele whole-oy pools. Woo n Lurent (Woo n Lurent, 23) mesure the rte onstnts for turnover (K; the perentge per hour of the exhngele whole-oy pool tht is lost to the externl wter) in freshwter- n sewter-limte F. heterolitus. K vlues were ~2% for oth N + n Cl in freshwter nimls, versus 34% (N + ) n 45% (Cl ) in sewter nimls, in or with the lrge freshwter versus sewter ifferenes seen in the present stuy. It is possile to mke rough estimte of the frtions of totl effluxes to the wter tht originte from the foo. Using vlues verge over the first 3 h fter the mel when efflux rtes were gretest, N + efflux originting from the mel in freshwter rnge from ~5 mol kg 1 h 1 from the lowslt nturl iet to ~7 mol kg 1 from the high-slt syntheti iet, n ietry Cl efflux (mesure only from the high-slt iet) rnge from 9 to 23 mol kg 1 h 1. To put this in perspetive, reporte uniiretionl N + n Cl efflux rtes originting from the whole oy of freshwter-limte killifish verge ~8 mol kg 1 h 1 [2 to 15 mol kg 1 h 1 in vrious stuies (Potts n Evns, 1966; Potts n Evns, 1967; Metz et l., 1967; Ptrik et l., 1997; Ptrik n Woo, 1999; Woo n Lurent, 23; Woo, 211)]. Thus ietry N + n ount for 6 87% of uniiretionl whole-oy N + efflux, n ietry Cl n ount for up to 1% of uniiretionl whole-oy Cl efflux. In sewter-limte killifish, N + efflux rtes from the iet rnge from ~2 (low-slt iet) to 18 mol kg 1 h 1 (high-slt iet) n Cl efflux rtes from 4 to 6 mol kg 1 h 1 (highslt iet only). Uniiretionl whole-oy N + n Cl efflux rtes hve een mesure in severl stuies on F. heterolitus (Motis et l., 1966; Metz et l., 1967; Potts n Evns, 1967; Pi, 1978; Woo n Lurent, 23; Woo, 211), with reporte vlues rnging from 17, to 45, mol kg 1 h 1. Thus t most, ietry N + ounts for ~1% n ietry Cl ounts for 35% of uniiretionl whole-oy efflux rtes. gin, we emphsize tht these figures pply to uniiretionl N + n Cl flux rtes originting from the iet, not the totl (net) flux rtes from the whole

11 Dietry N + n Cl uptke in killifish 3935 niml, whih re thought to e pproximtely equl y the lssi Silv moel (Silv et l., 1977) s elorte elow. Furthermore, the present stuy provies iret in vivo eviene of the ritil importne of Cl uptke from the iet in freshwter killifish, to support onlusions se previously on iniret eviene. Erlier gill ion flux mesurements in F. heterolitus h shown tht Cl uptke vi the gills ws negligile in freshwter wheres Cl loss rtes to the wter were not (Woo n Mrshll, 1994; Ptrik et l., 1997; Ptrik n Woo, 1999; Woo n Lurent, 23; Woo, 211), espite lower gill permeility to Cl thn to N +, s revele y trnsepithelil potentil experiments (Woo n Grosell, 28). This ontrsts with N + flux rte mesurements in the sme stuies, where vigorous tive N + uptke t the gills n pssive N + loss ourre t similr rtes. Furthermore, erlier in vitro gut s experiments (Sott et l., 26; Woo et l., 21) h inite tht Cl uptke ross the gut ws upregulte in freshwter killifish. The present t onfirm tht the rte of Cl uptke from the iet is greter thn tht of N +, n tht this ietry Cl is lost to the externl wter t fster rte, espeilly in freshwter nimls. This is most likely similr in other fish lking tive rnhil Cl uptke in freshwter suh s the eel n luegill (e.g. Tomsso n Grosell, 25); however, this remins to e onfirme. The mehnisms of intestinl uptke n trnsepithelil exretion of N + n Cl were not exmine in the present stuy. With respet to the ltter, in sewter nimls, the gills re unoutely the mjor route of efflux, n the lssi Silv moel (Silv et l., 1977) of equimolr trnsellulr Cl extrusion n prellulr N + extrusion energize y solterl N + /K + -TPse proly pplies (Evns et l., 25; Hwng n Lee, 27; Evns, 28). In freshwter nimls, the rnhil efflux mehnisms re less ler, though rnhil losses re generlly thought to e pssive, vi hnnels n tight juntions, n N + n Cl exretion in the urine eomes minor ut signifint omponent [see lultions in Woo n Lurent (Woo n Lurent, 23) n Woo (Woo, 211)]. Interestingly, rnhil i-se regultion in freshwter killifish seems to rely minly on ifferentil moultion of N + n Cl efflux rtes, i.e. strong ion ifferene (SID) effets (Stewrt, 1981), rther thn on mnipultion of N + (versus H + ) n Cl (versus HCO 3 ) uptke rtes, in view of the sene of the ltter (Woo n Mrshll, 1994; Ptrik et l., 1997). With respet to intestinl uptke mehnisms, erlier we reporte tht Cl uptke in vitro ws greter in freshwter killifish gut s preprtions thn sewter preprtions, ws prtilly ouple to greter HCO 3 seretion, n ws upregulte y feeing (Woo et l., 21). Net Cl uptke lso tene to e greter thn net N + uptke (Sott et l., 26). This fits with the generl view tht intestinl Cl uptke ours prtilly in exhnge for HCO 3 n prtilly ouple to N + uptke (Grosell, 26; Grosell, 211; Grosell, 211), ut the killifish is unusul in hving greter intestinl Cl uptke in freshwter thn sewter. Nevertheless, this oinies with the unusul sene of n tive Cl uptke mehnism t the gills in this euryhline speies when it is in freshwter, n the ft tht rnhil i-se regultion epens on the ifferentil regultion of N + versus Cl efflux, s isusse erlier. In turn, this lso oinies with the lk of HCl seretion in the igestive trt euse the stomh is sent (Bkin n Bowie, 1928). Inee, euse of the elevte intestinl HCO 3 seretion following feeing, the freshwter-limte killifish is the only teleost known to exhiit postprnil ii tie (Woo et l., 21) rther thn the stnr lkline tie seen in most fish (Woo et l., 25; Buking n Woo, 28; Cooper n Wilson, 28; Buking et l., 29; Li et l., 21). n inrese in rnhil Cl exretion (representing strong nion) over N + exretion (representing strong tion) woul help to relieve the i lo in the loo through the SID theory (Stewrt, 1981), s suggeste y Woo n Mrshll (Woo n Mrshll, 1994) n Ptrik et l. (Ptrik et l., 1997). Low-slt versus high-slt iets The fish h een trine on the low-slt nturl iet n the highslt syntheti iet for ~7 ys prior to the experiment. We h therefore preite tht in the freshwter killifish, the reltive uptke n retention of N + woul e greter from the former, refleting the srity of N + in freshwter, ut tht this ifferene woul not our in sewter killifish, euse of the exess of N + in the mrine environment. This hypothesis ws se on previous stuies showing tht high-slt iets inrese rnhil N + efflux ross the gills in freshwter fish (Smith et l., 1995; Niyogi et l., 26), n tht limtion to ion-poor wters erese rnhil N + efflux rte (MDonl n Rogno, 1986). However, the results i not etet the preite effet in freshwter killifish, n onfirme tht it ws sent in sewter killifish. Thus, in Series 4, for freshwter killifish, exretion of N + from the low-slt iet up to 9 h ws muh lower on n solute sis thn from the high-slt iet, ut y this time, lmost ientil perentges (~11%) h een exrete to the wter n retine (~56%) in the rss, regrless of the ietry slt lo. Perentges remining in the igestive trt t 3 h (efore riotrer reyling in the high-slt iet eme potentil prolem) were lso similr (~7%). In sewter killifish, muh more of the ietry N + lo ws exrete to the wter n muh less ws retine in the rss from the low-slt iet, wheres the reltive mounts remining in the igestive trt were similr. The explntion for the mrginl effet in freshwter killifish my e mtter of threshol. In ritilly reviewing numer of sltfeeing ppers on slmonis (Smith et l., 1995; Kmune et l., 23; Pyle et l., 23; Niyogi et l., 26), Woo n Buking (Woo n Buking, 211) onlue tht the threshol for inreses in rnhil N + efflux rte ly ove slt onentrtion of 127 mol N + g 1 ry foo mss. The high-slt iet use in the present experiments ontine only 911 mol N + g 1 ry foo mss. It woul e interesting to test whether the sme onsiertions pply to uptke n retention of Cl from high- n low-slt iets in the freshwter killifish. However, ost onsiertions prelue repeting the experiment with low-slt iets lelle with 36 Cl. Conluing remrks Reltive to the immense mount of knowlege on ionoregultion in reltion to wterorne ions, our unerstning of ionoregultion with respet to ietry ions remins in its infny. The present stuy hs highlighte the ifferentil hnling of ietry N + n Cl, n the impt of externl slinity, ut mny other ftors remin unexplore, suh s the intertive impts of rinking, exerise, environmentl O 2 n other ions in the iet. One prtiulrly importnt ftor my e ehviourl hnges ssoite with ifferent ioni omposition of the iet. For exmple, the high-slt syntheti iet is lso high in C 2+, n in ompnion stuy using the sme high-slt syntheti iet, we foun tht killifish voluntrily hoose higher slinity fter eting this iet, pprently to help their C 2+ homeostsis (Buking et l., 212). The intertion mong wterorne, ietorne n ehviourl spets of ionoregultion is rih re for future investigtion.