RESEARCH ARTICLE Diet influences salinity preference of an estuarine fish, the killifish Fundulus heteroclitus

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1 1965 The Journl of Experimentl Biology 2, Pulished y The Compny of Biologists Ltd doi:.1242/je.06 RESEARCH ARTICLE Diet influences slinity preference of n esturine fish, the killifish Fundulus heteroclitus Crol Bucking 1,2, *, Chris M. Wood 1,2 nd Mrtin Grosell 1 1 Rosenstiel School of Mrine nd Atmospheric Sciences, University of Mimi, Mimi, FL 33149, USA nd 2 Deprtment of Biology, McMster University, 1280 Min Street West, Hmilton, ON, Cnd L8S 4K1 *Author for correspondence t present ddress: Deprtment of Biology, University of Ottw, Mrie Curie, Ottw, ON, Cnd K1N 6N5 (cucking@uottw.c) Accepted 17 Ferury 12 SUMMARY Understnding the interply mong the externl environment, physiology nd dptive ehviour is crucil for understnding how nimls survive in their nturl environments. The externl environment cn hve wide rnging effects on the physiology of nimls, while ehviour determines which environments re encountered. Here, we identified chnges in the ehviourl selection of externl slinity in Fundulus heteroclitus, n esturine teleost, s consequence of digesting mel. Fish tht consumed high levels of dietry clcium exhiited higher preferred slinity compred with unfed fish, n effect tht ws exggerted y elevted dietry sodium chloride. The men swimming speed (clculted s proxy of ctivity level) ws not ffected y consuming diet of ny type. Constrining fish to wter of 22 p.p.t. slinity during the digestion of mel did not lter the mount of clcium tht ws sored cross the intestine. However, when denied the cpcity to increse their surrounding slinity, the compromised ility to excrete clcium to the wter resulted in significntly elevted plsm clcium levels, potentilly hzrdous physiologicl consequence. This study is the first to show tht fish ehviourlly exploit their surroundings to enhnce their ionoregultion during digestion, nd to pinpoint the novel role of dietry clcium nd sodium in shping this ehviour. We conclude tht in order to resolve physiologicl disturnces in ion lnce creted y digestion, fish ctively sense nd select the environment they inhit. Ultimtely, this my result in trnsient diet-dependent ltertion of the ecologicl niches occupied y fishes, with rod implictions for oth physiology nd ecology. Key words: ehvior, digestion, ionoregultion, clcium, Fundulus heteroclitus, feeding. INTRODUCTION Glolly, fish re found in wide rnge of slinities from freshwter (0 p.p.t.) to sltwter (~32 p.p.t.), nd in some cses even hypersline (> p.p.t.) wters. Regrdless, fish mintin reltively uniform plsm ion concentrtions, resulting in significnt environmentlly dependent differences in physiology. In essence, freshwter fish must e le to comt wter gin nd ion loss, while the opposite is true for fish found in sltwter environments. Much is known out the physiologicl processes involved in ion nd wter regultion, from rnchil, intestinl nd renl trnsport (reviewed y Cerd nd Finn, ; Grosell et l., 09; Evns, 08; Ando et l., 03; Krnky, 1998) to hormonl regultion (reviewed y Tkei, 08; Mnzon, 02; McCormick, 01). There re lso mny euryhline species tht re le to trnsition etween the two extremes of freshwter nd sewter. An extreme cse of euryhlinity is seen in fish tht inhit esturies (e.g. killifish, Fundulus heteroclitus) where lrge dily vritions in environmentl slinity my occur with the tidl cycles in the estury (e.g. Elliot nd McLusky, 02; Mrshll, 03; Telesh nd Khleovich, ; Potter et l., ). Euryhlinity is enled y complex chnges in the physiology of fish in response to the opposing osmoregultory nd ionoregultory demnds of inhiting freshwter nd sewter, such s ltertions in rnchil trnsport processes (e.g. Krnky, 1998; Wood nd Mrshll, 1994; Mrshll et l., 1999; Mrshll, 02; Ktoh et l., 08; Mdsen et l., 09; Tipsmrk et l., ). While knowledge of the sic physiology of fishes with respect to ion nd wter regultion hs progressed gretly from the first historic investigtions (Smith, 19; Keys, 1931; Krogh, 1937), most studies hve een sed on constrining fish to controlled environments nd in mny cses sujecting them to single step chnges in slinity (e.g. Ktoh et l., 08; Scott et l., 08; Mdsen et l., 09). However, in nture, these nimls re cple of movement throughout their hitts, nd they my rpidly encounter vriety of slinities. Hence, fish ehviour my dictte their physiology over fine temporl scle. Experimentl evidence of preferences for temperture nd slinity exists for vriety of euryhline species (e.g. Fritz nd Grside, 1974; Decon nd Hecht, 1995; Crdon, 00; Serrno et l., ). Interprettion of these studies hs focused on correlting choices with optiml growth (Joling, 1981) or food conversion (Lrsson, 05). Indeed, common snook (Centropomus undecimlis) mintined t pprent optiml slinities invested more energy in processing food thn fish mintined t suoptiml slinities (Grci-Lopez et l., 06). Additionlly, there is evidence tht fish cn lern [e.g. ssocitive lerning (Brtlnd et l., ; Sieeck et l., 09), socil lerning (Strnd et l., )] (reviewed y Kieffer nd Colgn, 1992; Bshry et l., 02) nd rememer (e.g. Hrvey-Girrd et l., ; Nordgreen et l., ). Tken together, evidence of environmentl preferences nd the ility to lern suggests tht fish cn discriminte nd select physiologiclly optiml conditions, nd my ultimtely e le to lern nd rememer which hitts llow for this.

2 1966 C. Bucking, C. M. Wood nd M. Grosell An dditionl fctor tht cn influence ionoregultion nd osmoregultion is feeding. A mel presents the niml not only with energy for growth, reproduction, etc., ut lso with lrge ion lods nd potentil disruptions in cid se lnce. The effect of feeding on vriety of physiologicl prmeters in teleosts hs een investigted recently in sewter [e.g. cid se regultion (Bucking et l., 09; Tylor et l., 07; Tylor nd Grosell, 06; Tylor nd Grosell, 09; Wood et l., ), slt lnce (Bucking et l., 11; Tylor et l., 07; Tylor nd Grosell, 06)] nd freshwter [e.g. cid se regultion (Bucking nd Wood, 08; Cooper nd Wilson, 08; Tylor et l., 07; Wood et l., ), slt lnce (Bucking nd Wood, 06; Bucking nd Wood, 07; Tylor et l., 07), wter lnce (Bucking nd Wood, 06)]. In generl, feeding ppers to result in slinity-dependent ltertions in ion, wter nd cid se trnsport processes in order to llevite digestion-induced disruptions in homeostsis. One gol of the present study ws to mesure the slinity preference of n esturine fish tht would e exposed to chnging slinities in its nturl environment. We employed killifish, Fundulus heteroclitus mcrolepidotus (Wlum 1792), which re uiquitous long the Atlntic cost of Cnd nd the USA nd re frequent inhitnts of intertidl mrshes, creeks nd esturies. Killifish move with ese etween slinities, dpt quickly (Mrshll et l., 1999) nd cn survive direct trnsfer from freshwter to full-strength sewter (Jco nd Tylor, 1983; Zdunisky et l., 1995). However, ionoregultion nd osmoregultion re energeticlly expensive, requiring ATP-driven trnsporters to mintin homeostsis (reviewed y Boeuf nd Pyn, 01), diverting energy wy from growth nd reproduction. Hence, our hypothesis ws tht the killifish would preferentilly select slinity tht would llow them to ionoregulte nd osmoregulte while expending the lest mount of energy i.e. n isotonic slinity of 12 p.p.t. A second gol ws to exmine the effect of feeding nd diet composition on slinity choice. Killifish re indiscriminte predtors nd hve een vriously clssified s omnivores, detritivores nd piscivores (e.g. Allen et l., 1994; Prinslow nd Vliel, 1974; Ale et l., 07). Being n indiscriminte predtor mkes the killifish n excellent niml to study in the l s this species will redily consume vriety of diets. It lso mkes them importnt memers of the intertidl food we in slt mrshes nd esturies (Knei, 1986). Digestion in killifish does not result in n lkline tide ut rther in n cid tide in freshwter-cclimted killifish (ut not in sewter-cclimted killifish), nor does it result in ltered cid/se excretion to the wter (Wood et l., ). However, feeding does increse Cl nd fluid sorption in the intestine oth in sewter (Mrshll et l., 02) nd in freshwter (Wood et l., ). Consuming mel my present killifish with internl ion regultion chllenges tht depend on the externl slinity. For exmple, consumption of mel of commercil pellets cretes n venue for wter loss nd ion gin in freshwter trout (Bucking nd Wood, 06; Bucking nd Wood, 06; Bucking nd Wood, 07) nd incresed uptke of wter nd certin ions in sewter trout (Bucking et l., 11). Bsed on our initil hypothesis tht killifish would inhit n isotonic slinity, we predicted tht killifish would e more similr physiologiclly to sewter fish in terms of trnsport properties of the gill nd gut (Scott et l., 08). Hence, our second hypothesis ws tht fter mel, killifish would experience n incresed uptke of wter nd ions s seen in sewter trout, cusing them to seek out higher slinity to llevite their wter (y osmosis) nd ion lod (y ctive excretion). MATERIALS AND METHODS Experimentl diets All diets used in the following experiments re presented in Tle 1. Live lckworms (Aqutic Foods, Fresno, CA, USA) were held in ~5 l plstic continers. The worms were supplied with enough freshwter to cover them, nd the wter ws chnged every 48 h. Worms were used for experimentl studies within 7 dys of rrivl in the l. Blckworms were chosen s the se for most diets s they hve low ionic content nd were redily ingested y killifish during preliminry trils. Diet 2 ws mde y king lckworms (t 60 C) for 24 h. The dried lckworms were susequently ground into fine powder using mortr nd pestle. The powder ws then formed into dough y dding ~50% v/w distilled wter nd extruded through ml syringe to mke thin rope of compressed feed. The feed ws then dried overnight nd susequently roken into pellets y hnd. A similr protocol ws used to mke diet 3, with the exception tht ~950 mol g 1 dry mss of CCO 3 ws dded to the worm powder efore it ws formed into dough, while diet 4 ws mde with n dditionl ~900 mol g 1 dry mss NCl in ddition to CCO 3 (see Tle 1). Diet 5 ws prepred y grinding commercil fish flkes (TetrMin fish flkes) into fine powder using mortr nd pestle. Frozen rine shrimp (Sn Frncisco By Brnd, Newrk, CA, USA) were defrosted nd mixed with the powder (40% w/w) to gin form pste, which ws then extruded through ml syringe into long thin ropes of food pste. The food ws dried in n oven (60 C) for 24 h nd then crumled y hnd to form food pellets. With the exception of diet 1, ll diets were stored t C efore use. Tle 1. Composition of diets nd wter used throughout experiments N + Cl C C 2+ Wter content Diet ( mol g 1 dry mss) ( mol g 1 dry mss) ( mol g 1 dry mss) ( Ci g 1 ; series 2.2 only) (%) 1 Live lckworms ±1.4 2 Blckworm pellets ±1 3 High C 2+ lckworm pellets (dded s CCO 3 ) ±1 4 High NCl/high C 2+ lckworm pellets (dded s NCl nd CCO 3 ) ±0.5 5 Fish flkes nd rine shrimp pellets ±0.2 Wter N + (mmol l 1 ) Cl (mmol l 1 ) C 2+ (mmol l 1 ) Mimi sewter ( p.p.t.) Mimi freshwter (dechlorinted) Hmilton rtificil sewter (22 p.p.t.) Hmilton rtificil sewter ( p.p.t.)

3 Diet influences slinity preference 1967 Effect of feeding on slinity preference series 1 The experiments descried here nd elow were pproved y institutionl niml cre committees t the University of Mimi nd McMster University. Wild-cught (New Hmpshire, USA) F. heteroclitus mcrolepidotus (oth sexes; 2 6 g) were purchsed from Aqutic Reserch Orgnisms Ltd (Hmpton, NH, USA), nd held t the Rosenstiel School of Mrine nd Atmospheric Sciences (University of Mimi, FL, USA) in 50 l quri supplied with running sewter (22 24 C, 37.5 p.p.t.; mmol l 1 : N + 485, Cl 569, K +.6, C 2+.7, Mg , SO ) (Wood nd Grosell, 08). The nimls were fed dily 1.5% rtion of commercil pellets (mmol kg 1 : N + 2, Cl 170, K + 326, C , Mg 2+ 77) (Wood et l., ) during lortory cclimtion nd holding. To determine the slinity preference of the killifish, shuttle ox system (Loligo Systems, Tjele, Denmrk) ws used, which hs een descried in detil elsewhere (Serrno et l., ). Briefly, the system consisted of two lrge tnks connected y smll tunnel, which llowed the niml to shuttle etween the two comprtments. One comprtment ws connected to sewter source (composition ove; referred to s the sewter comprtment) while the other comprtment ws connected to dechlorinted freshwter source [Mimi City tp wter; mmol l 1 : N , Cl 1.21, K , C , Mg , SO (Wood nd Grosell, 08), referred to s the freshwter comprtment]. However, the wter flow (i.e. which wter source, either sewter or freshwter, ws turned on) ws not continuous, ut ws determined y the position of the niml within the system. Pumps connecting the two chmers mintined the slinity differentil etween the two tnks t 5 p.p.t. t ll times. The position of the niml ws continuously recorded (ueye cmer with infr-red cpilities, IDS Imging Development Systems, Oersulm, Germny) nd the imge ws converted into X,Y-coordintes y LoliTRACK softwre (Lolitrck Lite 1.1, Loligo Systems) once every second. The X,Y-coordintes were relyed to the Ltech Noteook progrm (Omeg Engineering Inc., Stmford, CT, USA), which nlysed the coordintes in rel time to determine whether the fish ws locted in the high slinity comprtment or the low slinity comprtment of the system. When the fish ws locted within the high slinity comprtment, the Ltech Noteook progrm ctivted the sewter source to deliver sewter to the comprtment (nd susequently incresed the slinity of oth chmers in the system). Correspondingly, the Ltech Noteook progrm ctivted the freshwter source when the fish ws locted within the low slinity comprtment (susequently decresing the slinity of the system). Hence, y selectively delivering either sewter or freshwter to the system, the slinity ws controlled y the loction of the niml with constnt offset 5 p.p.t. etween the two comprtments. Throughout ech experiment, the slinity of ech comprtment ws lso continuously mesured using two conductivity proes (TetrCon 3 conductivity proe, WTW, Weilheim, Germny) nd meters (WTW conductivity meter, WTW). The slinity of ech comprtment ws lso relyed every second to the Ltech Noteook progrm for lter nlysis long with the X,Ycoordintes (nd corresponding time stmp) descried ove. At the eginning of ech tril, the system ws filled with 0% sewter in oth chmers to mtch the cclimtion slinity of the nimls. Once fish ws plced in the system, the trcking softwre ws ctivted nd the fish could control the delivery of wter depending on which chmer it chose to occupy. The entire system ws housed in n environmentl chmer to mintin constnt temperture (22 24 C) s well s to control the light:drk cycle, which ws set t 14 h: h. During the drk cycle, infrred lights plced eneth the system llowed the fish to e visile to the cmer, which enled the system to e opertionl over complete 24 h cycle. Ech tril consisted of monitoring single fish for 48 h within the system. Before experimenttion, feeding ws suspended for ll nimls for 48 h, to encourge feeding when food ws presented to the fish within the system s well s to ensure n initil unfed stte. A single fish ws then indiscrimintely chosen nd plced in the shuttle ox system following weighing. For 24 h the position of the fish within the system s well s the slinity of ech chmer were recorded. Following 24 h, the fish were fed to stition with one of the diets presented in Tle 1, or shm fed. Shm feeding consisted of feigning ll ctions involved in feeding (entering the chmer, moving n rm over the tnk s one would during feeding, etc.) ut without ctully providing ny food for the fish. Susequently, the loction of the fish nd slinity of the system were recorded for n dditionl 24 h. Excess food ws siphoned from the tnks (tnks with shm-fed fish were lso siphoned) upon stition. Cre ws tken to rndomize which chmer the food ws presented in, s well s to egin ech tril nd feed t stndrd time (16:00 h ws chosen for oth). At the end of the experiment, fish were lightly nesthetized (MS-222, tricine methnesulphonte, Syndel Lortories, Qulicum Bech, BC, Cnd; uffered to ph 7.00) nd their ody length (BL, cm) mesured. Overll, 7 fish were used for ech dietry tretment. Cre ws tken to expose only nive fish to the system (i.e. fish were not reused etween tretments). All fish chosen for the experiments utilized the system nd none were excluded. An ttempt ws mde to ensure n even numer of mles nd femles for ech tril ( totl of 16 mle fish nd 19 femle fish were used). To determine the slinity preference of individuls, the overll slinity of the system ws clculted s the men slinity recorded etween the two chmers t ech time point (every second of the 48 h tril). The men slinity ws chosen ecuse the fish must trvel ck nd forth etween the two chmers to estlish n overll preferred slinity, given the nture of the set-up, nd thus we cnnot e certin tht the chmer the fish ws occupying t ny prticulr time point ws chosen ecuse the slinity in tht chmer ws the more preferred slinity or ecuse the slinity in the other ws the less preferred slinity. The mode slinity of the system ws then clculted over min time periods. As the mode represents the most commonly mesured slinity over the min, this reflects the most commonly chosen slinity of the fish (i.e. men preferred slinity). To determine the vriility in the preferred slinity during ech tretment, the men (±s.e.m.) slinity preference of individuls (N 7) ws then clculted t ech min time point. The overll preferred slinity of fish in ech tretment ws lso clculted s the men preferred slinity over ech 24 h period, s well s for ech light nd drk cycle, nd for selected post-feeding time intervls. Finlly, swimming speed (BL s 1 ) ws clculted y mesuring the distnce moved (in cm) y the fish every second using the X,Y-coordintes of the fish recorded y the Ltech Noteook progrm (Serrno et l., ). The swimming speed of ech fish t every second ws then verged over min time period excluding periods of inctivity (i.e. when swimming speed ws 0). The men length of inctive periods during the trils did not vry significntly etween tretments (see Results). The first 4 h of ech tril ws excluded from sttisticl nlysis s trining period. Physiologicl effects of externl environment during feeding series 2.1 Fundulus heteroclitus mcrolepidotus were otined s ove for the slinity choice experiments, nd mintined in lrge fireglss tnk

4 1968 C. Bucking, C. M. Wood nd M. Grosell (~500 l; re-circulting p.p.t. rtificil sewter; Instnt Ocen, Atlnt, GA, USA) t McMster University (Hmilton, ON, Cnd) for severl months. The nimls were then trnsferred into two lrge quri for 1 month cclimtion period, nd supplied with chrcolfiltered re-circulting rtificil sewter ( p.p.t.; Instnt Ocen). The nimls were then grdully cclimted to 22 p.p.t. over 24 h period sed on the results seen when the nimls ctively controlled the environmentl slinity (series 1). Fish were susequently fed diet 2 (Tle 1) until stition, nd one qurium ws mintined t 22 p.p.t., while nother qurium ws sujected to slinity increse to p.p.t. over 2 h (gin sed on oservtions from series 1). Animls were terminlly nesthetized in MS-222 fter 4 h ( totl of 6 h following feeding; N 7 for ech tretment). This time frme ws chosen sed on dt collected during series 1 tht suggested this ws the time of gretest chnge in slinity preference during digestion. A cudl lood smple ws then otined with n ice-cold, heprinized cpillry tue (Fisher Scientific Cnd, ON, Cnd) nd centrifuged to otin plsm frction, which ws susequently plced in liquid nitrogen nd stored t 80 C for lter nlysis. The gstrointestinl (GI) trct ws quickly removed nd the entire contents were collected, plced in liquid nitrogen, nd stored t 80 C for lter nlysis. The experiment ws then repeted with fish fed two dditionl diets (diet 3 nd diet 4; Tle 1). The GI trct contents were digested with cid (5 volumes of 1 mol l 1 HNO 3 ) nd het (60 C) for 48 h. The plsm nd GI trct contents were nlysed for N + nd C 2+ y tomic sorption spectrophotometry (AAS; Vrin Austrli Model 2FS, Mulgrve, Vic, Austrli) using certified stndrds (N +, Rdiometer, Copenhgen, Denmrk; C 2+, Fisher Scientific); interference y other ions during mesurement ws corrected y dding Cs 2+ nd L 3+ to the smples, respectively. Physiologicl effects of externl environment during feeding series 2.2 Animls were cclimted to rtificil full-strength sewter for 1 month period nd then sujected to grdul decrese in slinity (from to 22 p.p.t.) over 24 h period, s in series 2.1. The nimls were susequently fed to stition with diet 2, 3 or 5 (Tle 1). However, diets 2, 3 nd 5 were spiked with 45 C 2+ ( Ci CCl 2 g 1 dry food mss; PerkinElmer, Wlthm, MA, USA). Immeditely following feeding, individul fish were removed from the quri nd plced into individul erted chmers (500 ml), which were initilly filled with 22 p.p.t. rtificil sewter. Susequently, over 2 h the wter in ech chmer ws either grdully replced until the slinity ws p.p.t. or kept t 22 p.p.t. s in series 2.1. Once the finl slinity ws reched, n initil wter smple ws tken, the fish were held stticlly for 4 h, nd finl wter smple ws tken. Animls were then terminlly nesthesized in MS-222 s ove, the GI trct ws removed nd the whole ody ws reserved for lter nlysis of 45 C 2+ content. 45 C 2+ content ws mesured in the initil nd finl wter smples (1 ml smples), cid-digested chyme nd intestine, diet (digestion protocol s for series 2.1; 0 l of cid-digested smple) nd the ody of the whole niml (cid-digested s for the chyme nd intestine; 0 l cid-digested smples). ACS fluor (Aqueous Counting Scintillnt, Amershm, Little Chlfont, UK) ws dded to ech wter smple, while Ultim Gold scintilltion fluor (Pckrd Bioscience, Meriden, CT, USA) ws dded to the cid-digested smples, nd oth were stored in the drk overnight efore mesurement to reduce chemiluminescence. The ctivity of 45 C 2+ ws then mesured using liquid scintilltion nlyser (Tri-Cr 2900TR; PerkinElmer). Quench curves were determined for ech smple nd fluor type. The distriution of 45 C 2+ mong the wter, the ody nd the GI trct (including the chyme) ws determined s percentge of the whole 45 C 2+ ctivity for ech fish. The solute mount of dietry C 2+ found in the GI trct, the wter nd the ody ws clculted sed on the ctivity level of ech comprtment (c.p.m. g 1 fish mss) nd the specific ctivity of the food (cpm mol 1 C 2+ ). Sttistics Dt re expressed s mens ± 1 s.e.m. (N). For series 1, chnges in slinity preference nd swimming speed t ech min time point over the 48 h experiment nd with tretment (unfed, diet 1, diet 2, diet 3 nd diet 5) were exmined with two-wy repeted mesures ANOVA followed y post hoc testing (Holm Sidk) s pproprite. Chnges in men, overll slinity preferences (i.e. Tle 2, diel effects, sex effects nd tretment dy effects) were exmined using pired nd unpired t-tests s pproprite, followed y pproprite Bonferroni corrections. For series 2.1, plsm nd chyme N + nd C 2+ concentrtions, long with wter, GI trct nd ody C 2+ concentrtions were exmined using two-wy ANOVA with slinity (22 vs p.p.t.) nd diet (unfed, diet 1, diet 2, diet 3, diet 4 nd diet 5) s fctors followed y Holm Sidk post hoc testing. And, finlly, for series 2.2, the 45 C 2+ percentge distriution ws trnsformed (rc sine trnsformtion) nd two-wy ANOVA (with slinity nd diet s fctors s in series 2.1) ws used to exmine the dt, followed y Holm Sidk post hoc testing. When two-wy ANOVA nlyses were employed for dt nlysis the significnce of the interction etween the fctors is reported in the text. Where the interction effect etween fctors ws significnt, ech fctor ws exmined individully. Where the interction effect etween the min fctors ws not significnt, ut min fctor itself ws significnt, multiple comprisons (Holm Sidk post hoc tests) were employed to determine specific significnt differences. SigmStt 3.1 ws used to nlyse the dt. RESULTS Series 1 There were no significnt differences in the preferred slinity of killifish over the first 24 h period of ech 48 h tril (interction term P 0.998; diet P 0.741; time P 0.067) nd therefore the preferred slinity over this period ws verged for ll tretments (Fig. 1A). Excluding the first 4 h during which the men slinity in the system decresed from the full-strength sewter where ech tril ws egun, the men slinity over the first h for ll tretments ws 21.5±0.4 p.p.t. (N ; Fig. 1A). For the second 24 h period of ech tril, the tretments were nlysed individully s follows ecuse sttisticl nlysis reveled significnt interction of diet nd time (P<0.001) s well s significnt effects of diet itself (P 0.011) nd time (P<0.001). For the unfed tretment, the men slinity preference over the second 24 h of the 48 h tril ws 21.3±0.3 p.p.t. (N 7; Tle 2, Fig. 1B), identicl to tht in the first 24 h (Fig. 1A). As with unfed fish, fish tht were fed diet 1 (live lckworms) showed no significnt chnges in slinity preference over the second 24 h tril period despite trnsient fluctutions (Fig. 1C). The men preferred slinity ws 21.0±0.3 p.p.t. (N 7; Tle 2). There were lso no significnt differences etween fish fed diet 1 nd unfed fish t ny time point (Fig. 1B,C). In comprison, for fish tht consumed diet 2 (dried lckworm pellets) the preferred slinity in the second 24 h ws significntly lower (17.3±0.5 p.p.t., N 7) reltive to unfed fish or fish fed diet 1 (Tle 2). When exmining individul time points, there were no significnt effects of diet or time compred with unfed fish or fish fed diet 1 (Fig. 1B D). When fish were fed diet 3 (high C 2+ lckworm pellets) nd slinity preference ws

5 Diet influences slinity preference 1969 Men slinity preference (p.p.t.) Men slinity preference (p.p.t.) B D A C E Fig. 1. (A) The men preferred slinity of killifish over ll tretments over the first 24 h of ech tril. Feeding (or shm feeding) occurred immeditely following this initil 24 h period. (B F) Men preferred slinity of killifish tht were (B) unfed, (C) fed diet 1, (D) fed diet 2, (E) fed diet 3 or (F) fed diet 5 (see Tle 1) following the initil 24 h tril period (see A). The htched res represent the drk portion of the light:drk cycle of the chmer. N 7 for ech diet. *Significnt effect (P<0.05) of time within ech diet tretment. Significnt difference from unfed fish t the sme time point. Significnt difference from unfed fish nd fish fed diet 2 t the sme time point. Significnt difference from unfed fish, fish fed diet 1 nd fish fed diet 2 t the sme time point. Significnt difference from unfed fish, fish fed diet 1, fish fed diet 2 nd fish fed diet 3 t the sme time point Men slinity preference (p.p.t.) 40 F ** * ** Time (h) verged over the susequent 24 h, there ws no significnt effect of tretment on the preferred slinity when compred with unfed fish (overll men preferred slinity 23.4±0.3 p.p.t., N 7; Tle 2). However, when exmining individul time points there were significnt increses in the preferred slinity of fish fed diet 3, when compred with fish tht were not fed, or were fed either diet 1 or diet 2 over rief period following feeding (Fig. 1E). Notly, when exmined on this finer time scle (one tht more ccurtely represented the time frme of digestion), the overll men slinity preference of fish fed diet 3 incresed t 0 12 h postfeeding (i.e h) nd incresed even further t 4 12 h postfeeding (i.e h; Tle 2). Fish tht consumed diet 5 (fish flkes nd rine shrimp pellets tht were high in oth C 2+ nd NCl) exhiited n even more pronounced effect of tretment when compred with fish tht were fed diet 3, nd the difference ws highly significnt reltive to unfed fish, or fish fed either diet 1 or diet 2. On verge, the preferred slinity of fish fed diet 5 during the second 24 h tril ws 28.4±0.4 p.p.t. (N 7; Tle 2). Agin, when exmined on finer time scle the slinity preference of fish fed diet 5 ws much lrger, 1.5-fold higher t 0 12 h postfeeding (24 36 h) nd 1.6-fold higher t 4 12 h post-feeding (28 36 h) thn in unfed fish (Tle 2). Specificlly, there ws n increse in the preferred slinity 2 h fter feeding, significntly rising ove tht of fish fed diet 1 nd 2 (Fig. 1F). The preferred slinity continued to increse for nother 2 h until full-strength slinity ws reched (Fig. 1F). The preferred slinity of fish fed diet 5 then fell to ecome not significntly different from the other tretments, 9 h following feeding (33 h; Fig. 1F). Overll, there were no significnt diel effects or sex effects on the preferred slinity of nimls during ech tril (exmined using pired t-tests compring the men slinity for ech condition within ech tretment) with two exceptions. Fish tht were fed diets 3 nd 5 showed significnt increse in the men drk cycle slinity preference during the second 24 h tril period over the light cycle slinity preference (i.e. men night vs dy slinity preference during second 24 h of the tril). The increse in slinity preference in the

6 1970 C. Bucking, C. M. Wood nd M. Grosell Tle 2. Summry of men slinity preference (p.p.t.) following feeding or shm feeding t 24 h Time period Unfed Diet 1 Diet 2 Diet 3 Diet h 21.3± ± ±0.5* 23.4± ±0.4* h.8± ± ±0.6* 24.9±0.9* 31.6±0.7* h.9± ± ±0.4* 26.6±0.3* 32.5±0.4* N 7 for ech diet. *Significnt difference from the unfed group over the sme time period. drk ws confounded y feeding occurring 6 h prior to the lighting trnsition. In contrst with the men preferred slinity, there were no effects of tretment or time on men swimming speed (interction effect P 0.991; diet P 0.336; time P 0.798; Fig. 2), nd s such ll tretments were comined t ech time point. Excluding the first 4 h of experimenttion s trining period, the men swimming speed of killifish, regrdless of time or diet, ws 1.1±0.1 BL s 1. There were no diel or sex effects s exmined with t-tests. On verge, there were no significnt differences in the sttionry time over ech min intervl etween or mongst ll tretments (9±3 min, dt not shown; N ). Series 2.1 Sttisticl testing reveled tht not only ws the interction effect of diet nd slinity significnt (P 0.009) for plsm C 2+ concentrtion ut lso diet nd slinity were significnt individul fctors s well (P nd P 0.005, respectively). In contrst, when exmining plsm N + nd chyme C 2+ concentrtions, the interction effect of diet nd slinity ws not significnt (P nd P 0.695, respectively), nor ws the slinity fctor itself (P nd P 0.361, respectively), ut the min fctor of diet ws (P<0.001 for oth). For chyme N + concentrtion, the interction effects etween slinity nd diet were not significnt (P 0.443) nd neither were the effects of diet or slinity when exmined individully (P nd P 0.1, respectively). Hence, for unfed fish, grdul cclimtion to 22 p.p.t. from p.p.t. (over Men speed (BL s 1 ) Time (h) Fig. 2. The men swimming speed [ody lengths (BL) s 1 ] cross ll tretments over min time intervls. No significnt differences were found when the tretments were exmined individully, nd therefore tretments were comined (N ). There ws no significnt effect of time (P>0.05) on the men swimming speed of the killifish. Htched res represent the drk period nd feeding occurred t 24 h s shown y the verticl line. 24 h) followed y rpid increse in slinity (from 22 to p.p.t. in 2 h) hd no effect on the plsm concentrtion of N + (men for the two slinities, 147.4±4.2 mmol l 1, N 14; Tle 3) or C 2+ (men for oth slinities, 2.4±0.4 mmol l 1, N 14; Fig. 3). Consuming diet 1 while mintining the externl slinity t 22 p.p.t. hd no significnt effects on plsm ion constituents when compred with the sme tretment of unfed fish though there ws tendency for higher plsm C 2+ in these fed fish (C ±1.4 mmol l 1, N 7, Fig. 3). There ws lso no significnt effect of chnging the externl slinity to p.p.t. on plsm C 2+ concentrtion (5.1±1.3 mmol l 1, N 7; Fig. 3). The sme held for fish consuming diet 2 when exmining N + nd C 2+, with plsm levels eing mintined regrdless of externl slinity, with no differences reltive to fish fed diet 1 or unfed fish (men for the two slinities, N ±4.9 mmol l 1, N 14; Tle 3; C ±0.4 mmol l 1, N 14; Fig. 3). Plsm N + concentrtion ws unffected y consuming diet 3 or incresing the externl slinity, similr to wht ws oserved following the consumption of diets 1 nd 2 (Tle 3). However, consumption of diet 3 when fish were constrined to slinity of 22 p.p.t. resulted in significnt increse in plsm C 2+ to 6.2±2.1 mmol l 1, N 7, sustntilly ove unfed nd diet 2 levels. Notly, when the externl slinity ws incresed to p.p.t. this post-feeding plsm C 2+ concentrtion ws no longer sttisticlly different from unfed vlues (3.0±0.9 mmol l 1, N 7; Fig. 3). Diet 4 showed the sme trends s diet 3, with the post-feeding rise in plsm C 2+ eing llevited when the fish were trnsferred to p.p.t. (Fig. 3). Diet 5 showed no effect of diet or externl slinity on plsm N + s with most other tretments (Tle 3). When fish ingested diet 5 there ws very lrge nd significnt increse in plsm C 2+ concentrtion (to 13.4±1.0 mmol l 1, N 7; Fig. 3) ut gin, the trnsition to higher slinity helped to ttenute the effect (8.8±0.9 mmol l 1, N 7; Fig. 3). The concentrtion of N + in the chyme ws mintined irrespective Tle 3. Plsm N + concentrtion nd chyme N + nd C 2+ concentrtion in killifish fed vrious diets nd either constrined to 22 p.p.t. or moved to p.p.t. Plsm Chyme N + N + C 2+ Diet Slinity (mmol l 1 ) (mmol l 1 ) (mmol l 1 ) Unfed 22 p.p.t. 144±4 p.p.t. 0±4 Diet 1 22 p.p.t. 143±6 p.p.t. 0±3 Diet 2 22 p.p.t. 144±5 96±6 53± p.p.t. 3±6 79±4 58±7 Diet 3 22 p.p.t. 0±2 2±4 5±23* p.p.t. 146±8 98±3 481±32* Diet 4 22 p.p.t. 7±4 p.p.t. 172±5 Diet 5 22 p.p.t. 149±6 95±4 502±22* p.p.t. 143±3 6±8 503±31* N 7 for ech slinity nd diet. *Significnt effect of diet (P<0.05).

7 Diet influences slinity preference 1971 Plsm C 2+ concentrtion (mmol l 1 ) 5 0,c,c p.p.t. Unfed Diet 1 Diet 2 Diet 3 Diet 4 Diet 5 Fig. 3. Plsm C 2+ concentrtion of fish fed vrious diets nd constrined to slinity of 22 p.p.t. or moved to p.p.t. Brs tht shre letters re not significntly different (P>0.05). N 7 for ech tretment. of diet (men concentrtion cross ll diets nd slinities; 95.9±4.1 mmol l 1, N 42; Tle 3). C 2+ concentrtion ( mol g 1 ) in the chyme of fish fed diet 2 (men cross the two slinities, 55.4±8.2 mmol l 1, N 14) ws significntly lower thn tht seen in the chyme of fish fed diet 3 (men cross the two slinities, 495.0±27.7 mmol l 1, N 14) nd diet 5 (men cross the two slinities, 502.4±26.5 mmol l 1, N 14; Tle 3). Series 2.2 Overll, when the proportion of 45 C 2+ found in the ody nd wter ws exmined sttisticlly, there ws significnt effect of diet (P 0.009, P 0.0), slinity (P 0.021, P 0.027) nd significnt interction etween the two fctors (P 0.038, P 0.041). In contrst there were no significnt effects found in the gut (interction P 0.376, diet P 0.064, slinity P 0.978; Fig. 4A). Hence, there ws no effect of incresing the externl slinity on the distriution of 45 C 2+ mong the externl wter, the whole-ody comprtment nd the intestine (including chyme) when fish were fed diet 2 (Fig. 4A). At this time (6 h post-feeding), ech comprtment ccounted for roughly equl proportions of the ingested 45 C 2+, pproximtely 29 41% ech. Fish tht consumed diet 3 showed similr pttern in postprndil 45 C 2+ distriution, regrdless of externl slinity (Fig. 4A). While there ws no significnt difference etween the distriutions seen with diet 2 nd diet 3, fish tht were fed diet 5 nd constrined to 22 p.p.t. retined the mjority of 45 C 2+ within the ody comprtment, nd relesed only very smll proportion to the externl wter (2.1±1.4%, N 7). However, when these fish were exposed to n increse in externl slinity to p.p.t., there ws significnt increse in the proportion relesed to the wter (.8±6.1%, N 7) nd corresponding significnt decrese in the proportion retined in the ody comprtment compred with fish t 22 p.p.t. (42±12.4%, N 7 t p.p.t. vs 81.6±12%, N 7 t 22 p.p.t.; Fig. 4A). There ws then no difference when these proportions were compred with the other two diet tretments (Fig. 4A). There were no significnt differences in the proportion of 45 C 2+ tht ws found in the intestine, either within diet 5 etween the slinities, or etween the other two diets (Fig. 4A). There ws significnt interction of diet nd slinity (P<0.001) s well s significnt effects of oth diet (P<0.001) nd slinity (P<0.001) on the solute mount of C 2+ found in the wter, ody nd gut (Fig. 4B). Hence, the corresponding totl mount of dietry,c,,c d c Proportion of 45 C (% of ingested) Asolute mount of C 2+ (µmol) A B Wter Body Gut p.p.t. Diet 2 Diet 3 Diet p.p.t. Diet 2 Diet 3 Diet 5 C 2+ [sed on the specific ctivity of the food (Tle 1) nd the rdioctivity found in ech comprtment (Fig. 4A)] shows the very lrge ody C 2+ urden generted y diets 3 nd 5 (note the difference in scle, right-hnd xis, for the dt for diets 3 nd 5). This ws prticulrly pronounced when the slinity ws restricted to 22 p.p.t. (Fig. 4B). The clculted lod of dietry C 2+ in the ody t 22 p.p.t. is 1.7-fold higher thn when the niml is moved to p.p.t. (Fig. 4B). DISCUSSION The unique system employed in the current study ws first used to test the slinity preference of the grey snpper, Lutjnus griseus (Serrno et l., ). Interestingly, the lortory slinity preference of L. griseus contrdicted field oservtions of L. griseus occurrence, stressing the importnce of externl iotic nd iotic fctors (Serrno et l., ). In the present study, we used this sme system to discover the importnce of comined iotic/iotic fctor, the slt content of mel, in ltering the slinity preference of the killifish. In our study, the men preferred slinity of unfed killifish in the lortory ws pproximtely 22 p.p.t. (Fig. 1A,B) which is similr to previous study on this suspecies [ p.p.t. (Fritz nd Grside, 1974)] ut in mrked contrst to our initil hypothesis. A slinity of 22 p.p.t. is ove the iso-osmotic point, where most ions would d c c Fig. 4. (A). The proportion of 45 C 2+ (% of ingested) in the wter, the ody nd the intestine of killifish fed vrious diets nd either constrined to slinity of 22 p.p.t. or moved to p.p.t. N 7 for ech tretment. Brs tht shre letters re not significntly different (P>0.05). (B) The solute mount of dietry C 2+ excreted into the wter, sored into the ody nd remining in the intestine for ech killifish fed vrious diets nd either constrined to slinity of 22 p.p.t. or moved to p.p.t. The left y-xis refers to diet 2; the right y-xis refers to diets 3 nd 5. Brs tht shre letters re not significntly different (P>0.05). 50 Asolute mount of C 2+ (µmol)

8 1972 C. Bucking, C. M. Wood nd M. Grosell neither move into nor out of the ody. In fct, t 22 p.p.t., the externl N +, Cl nd C 2+ levels re pproximtely 2-fold higher thn plsm vlues. In comprison, n environment of 12 p.p.t. hs N +, Cl nd C 2+ concentrtions tht re reltively similr etween the wter nd the lood (e.g. Dene nd Woo, 04; Edwrds et l., 05). Energeticlly speking, n iso-osmotic environment would eliminte the need for ctive regultion of ion nd wter homeostsis. Aove or elow this slinity, fish will e forced to ionoregulte nd osmoregulte, n energeticlly expensive endevour (reviewed y Boeuf nd Pyn, 01) s it relies hevily on the N + /K + -ATPse to crete ion grdients nd to drive trnsport. Fritz nd Grside lso showed tht prior cclimtion to either freshwter or sewter efore experimenttion did not ffect overll slinity preference (Fritz nd Grside, 1974). In contrst, prior cclimtion of killifish to freshwter ppered to reduce the dptive responses of trnsepithelil potentils to vrying slinities (Wood nd Grosell, 08). These results suggest tht the preferred hitts of euryhline fish my e distinct from those expected sed on individul physiologicl systems. Slinity is one of the most importnt environmentl fctors to influence the geogrphic distriution of killifish, nother proly eing temperture (e.g. Fngue et l., 06). Although considered euryhline, their distriutions in Atlntic Cnd nd the Estern USA re such tht killifish occur lmost exclusively in esturies nd slt mrshes. Additionlly, the Fundulus genus hs more thn species, mny of which occur in symptry. Sptil seprtion of these species is proly not due to physiologicl rriers in slinity tolernce, s ll species cn tolerte overlpping rnges of slinity (Weiserg, 1986; Griffith, 1974). However, slinity preference differences [e.g. F. diphnus slinity preference of 8 p.p.t. vs F. heteroclitus preference of ~ p.p.t. (Fritz nd Grside, 1974)] my e fctor in prtitioning ech species within the hitt. Additionlly, the suspecies F. heteroclitus mcrolepidotus used in the current study overlps geogrphiclly with the other suspecies F. heteroclitus heteroclitus with whom they interreed nd form hyrids. The heteroclitus suspecies tolertes lower slinity less well thn the mcrolepidotus suspecies (Scott et l., 04). In future, it would e of interest to test whether there re corresponding differences in slinity preference etween the two suspecies. Other physiologicl differences etween heteroclitus nd mcrolepidotus include temperture preference differences (Fngue et l., 09), mitochondril differences (Fngue et l., 09) nd differentil het shock responses (Fngue et l., 06; Hely et l., ), suggesting tht the two suspecies my hve slightly different physiology. Finlly, the preference of fish exposed to less frequent slinity chnges (e.g. migrtory species such s slmonids or eels) or less predictle slinity chnges (e.g. tide pool inhitnts such s tide pool sculpins) is uncler. We hypothesize tht nimls exposed to these types of slinity chllenges during their life history would not utilize this strtegy of ltering their surroundings y locl movements to compenste for physiologicl imlnce, proly ecuse of lck of opportunity to do so. The men swimming speed (nd conversely the lengths of sttionry periods) of F. heteroclitus ws unffected y feeding regrdless of the diet, diel ptterns or sex (Fig. 2), suggesting tht the slinity preference oserved ws not influenced y ctivity levels. In contrst, Edeline nd collegues showed tht slinity preferences in Anguill nguill were linked to locomotor ctivity, with more ctive eels preferring lower slinity (Edeline et l., 05). They hypothesized tht the more ctive eels were in the process of migrting to freshwter, which suggests tht slinity preference cn e trnsient nd depend on life history stges. Additionlly, Serrno nd collegues showed tht ctivity levels of unfed L. griseus were correlted with externl slinity, with spontneous swimming decresing t slinity extremes (i.e. oth low nd high slinities), phenomenon thought to relte to energy conservtion for osmoregultory needs (Serrno et l., ). This prticulr difference my e prtilly ccounted for y the urst swimming nture of killifish vs the continul swimming nture of the grey snpper [current study vs tht of Serrno nd collegues (Serrno et l., )]. After consuming either live worm prey (diet 1) or worm pellets (diet 2), the slinity preference of killifish ws either unchnged or slightly decresed (Tle 2). These worms presented the fish GI trct with miniml ion lods. In fct, the ion levels in the live worm diet were much less thn those found in the surrounding wter (Tle 1). This suggests tht physiologicl chnges ssocited with the process of digestion re not responsile for driving the ehviourl externl slinity selection seen with ion-enriched diets. Addition of C 2+ to the worm pellets (diet 3) cused mrked increse in the slinity preference of killifish (Tle 2). This phenomenon ws exggerted when fish consumed not only high levels of C 2+ ut lso high levels of NCl (diet 5; Tle 2). Interestingly, dietry C 2+ ws shown to e ssimilted in sewter trout despite high environmentl C 2+ levels (Bucking et l., 11). This ssimiltion suggests tht dietry C 2+ my e importnt for ion regultion in sewter teleosts despite the fct tht soring C 2+ my e unfvourle in sewter fish s they re fced with n inwrdly directed C 2+ concentrtion grdient. Additionlly, ion regultion imlnces ssocited with feeding hve een suspected of intercting with C 2+ -sensing receptors (CSRs) in fish through ltertions in plsm ion concentrtions. Fellner nd Prker suggest tht increses in plsm N + concentrtion following feeding reduce the signlling ctivity of the CSR found in the rectl glnd of elsmornchs, nd consequently increse rectl glnd lood flow nd slt secretion (Fellner nd Prker, 02; Fellner nd Prker, 04). A controversil role for piscine CSRs in sensing the externl slinity hs een theorized. It hs een suggested tht the CSRs, locted minly in ionoregulting nd osmoregulting tissues such s the gill, kidney nd intestine in ddition to the rin, re ctivted y increses in ions ssocited with increses in externl slinity (Nering et l., 02). This is either through increses in plsm ion concentrtions creted y elevted environmentl levels or through direct contct with the externl environment (Nering et l., 02). It is thought tht the CSRs would hence provide mechnism of notifying orgns of chnges in externl slinity. However, how this theory fits in with previous evidence tht Cl is the stimulus for slinity sensing nd control of drinking rtes (Ando nd Ngshim, 1996; Hirno, 1974) is unknown. The reson for the move to higher slinity ppers to e plsm C 2+ regultion, s when the fish were confined to 22 p.p.t., the plsm C 2+ levels rose to impressive levels (Fig. 3), pprently cused y compromised ility to unlod C 2+ to the wter (Fig. 4A). Generlly, C 2+ is hndled y three orgns: the intestine, the kidney nd the gills. Dietry C 2+ ppers to e tken up in sustntil mounts in oth freshwter (Bucking nd Wood, 07) nd sewter fish (Bucking et l., 11). Intestinl uptke of C 2+ is thought to e N + dependent (Flik et l., 1993; Flik et l., 1990) nd is likely to involve N + /C 2+ exchnger. The results of the current study suggest tht C 2+ uptke from the diet is not influenced y chnges in slinity (Tle 3, Fig. 4) nd proly does not explin the pprent inility to regulte plsm C 2+. Renl excretion of divlent ions is thought to e importnt in oth freshwter nd sewter fish, despite significnt reduction in urine production t

9 Diet influences slinity preference 1973 higher slinities. The kidney ntomy of the killifish is consistent with tht of other mrine stenohline teleosts; the nephron lcks the distl tulule responsile for resorption of N + nd Cl (Edwrds nd Schnitter, 1933), suggesting tht they my e using this route for C 2+ excretion. In fct, renl clcium resorption is significntly reduced in sewter-dpted fish (Schmidt-Nielsen nd Renfro, 1975; Bjornsson nd Nilsson, 1985; Elger et l., 1987), which ultimtely results in higher excretion to the urine. Finlly, C 2+ is ctively tken up y the gills of fish for growth nd homeostsis (Flik et l., 1985; Perry nd Flik, 1988; Flik nd Verost, 1994); however, gill C 2+ trnsporter ctivity is not reduced with incresing slinity (reviewed y Flik et l., 1996). Complex electrochemicl conditions my offer n explntion, t lest in prt. The incresed plsm C 2+ levels my trigger move to higher slinity. As this would result in n increse in the trnsepithelil electricl potentil (TEP; lood-side positive) of the gills (Wood nd Grosell, 08), the niml my ccount for the increse in the equilirium potentil (clculted ccording to the Nernst eqution) of C 2+ cused y the incresed plsm concentrtions, nd mintin net outwrd driving force for pssive C 2+ loss. Indeed, it would e of interest to record the TEP in these C 2+ -loded fish. Elevted dietry NCl levels my lso result in elevted plsm N + concentrtions, incresing the ctivity of the rnchil N + /C 2+ exchnger, which would drive internl plsm C 2+ concentrtions even higher, resulting in the need to move to wter of even higher slinity to mintin the equilirium potentil of C 2+ elow tht of the TEP. However, Prodocimo nd collegues found C 2+ influx to increse with incresing slinity in killifish, with no chnge in C 2+ efflux (Prodocimo et l., 07), which contrdicts our hypotheses, s does our finding of minly nonsignificnt chnges in plsm N + concentrtion. Prdoxiclly, Prodocimo nd collegues lso found tht t higher slinities, killifish hd decresed whole-ody clcium concentrtions (Prodocimo et l., 07). Additionlly, the influx rtes mesured t full-strength sewter were more thn 2-fold greter thn those previously mesured (Myer-Gostn et l., 1983). Clerly, this is complicted physiologicl response nd much more experimenttion is needed to verify tht n increse in plsm C 2+ is more esily relieved t higher slinities (either through the kidney or the gill) nd tht this is the reson for the oserved slinity preference in the current study. Overll, this study confirms the slinity preference of F. heteroclitus to e out 22 p.p.t., clerly greter thn iso-osmotic vlues. However, this is influenced y the ionic composition of the diet, with elevted C 2+ driving the preferred slinity higher, n effect tht is exggerted y elevted dietry NCl. The dptive significnce ehind the chnge in slinity preference my e relted to ion regultion, with n inility to excrete the dietry C 2+ lod t lower slinities. This is not to sy tht the C 2+ -loded killifish were unle to survive t lower slinities, ut given the opportunity they sought out n environment tht ugmented their ionoregultion, showing dptive ehviourl regultion. Bsed on the current study, killifish ctively seek out slinities tht cn rectify internl C 2+ imlnces creted y feeding on C 2+ -rich diets. Ultimtely, this could extend to multitude of esturine fish species. As pointed out in the Introduction, environmentl slinity plys significnt role in determining the physiology of fish. However, this study illustrtes tht the physiology of orgnisms conversely plys n importnt role in determining their preferred environment. The implictions of this on the overll niche occuption of esturine fish could e lrge, ffecting the wy we interpret the ecology of these fish species. 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