Osmoregulation, ionoregulation and acid base regulation by the gastrointestinal tract after feeding in the elasmobranch (Squalus acanthias)

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1 1335 The Journl of Experimentl Biology 21, Pulished y The Compny of Biologists 27 doi:1.1242/je.2736 Osmoregultion, ionoregultion nd idse regultion y the gstrointestinl trt fter feeding in the elsmornh (Squlus nthis) Chris M. Wood 1,2,3, *, Mkiko Kjimur 1,3, Crol Buking 1,3 nd Ptrik J. Wlsh 2,3,4 1 Deprtment of Biology, MMster University, 128 Min St. West, Hmilton, Ontrio, L8S 4K1, Cnd, 2 Rosenstiel Shool of Mrine nd Atmospheri Sienes, University of Mimi, Mimi, FL 33149, USA, 3 Bmfield Mrine Sienes Centre, 1 Phen Drive, Bmfield, British Columi, Cnd nd 4 Deprtment of Biology, University of Ottw, 3 Mrie Curie, Ottw, Ontrio, K1N 6N5, Cnd *Author for orrespondene (e-mil: woodm@mmster.) Aepted 29 Jnury 27 In order to study the physiologil onsequenes of voluntry feeding in the gstrointestinl trt of ureoteli mrine elsmornh, dogfish (fsted for 96 h) were smpled t vrious times up to 36 h fter onsuming 56% rtion of teleost fish (hke) under nturl feeding onditions. Digestion nd sorption were ompleted etween 12 nd 36 h post-feeding. The tissue msses of different segments of the gstrointestinl trt inresed nd deresed mrkedly s the hyme moved through, minly euse of fluid engorgement rther thn hyperplsi. In fsted dogfish, the rdi nd pylori stomhs ontined only smll volumes of highly idi fluid (ph 1.77±1.12, 2.5±.8) similr in omposition to sewter. Feeding resulted in gstri phs of 3.2±.31 nd 3.95±.4 t 6 h, followed y slow delines through 6 h. An lkline tide in the lood lso ourred t 6 h. In the fe of lrge hnging msses of highly idi hyme in the stomhs, the ph (6.5±.1), ioni omposition nd volume of hyme in the intestine (spirl vlve) were preisely regulted from 6 to 6 h post-feeding t very different vlues from those in the stomhs, nd intestinl Summry HCO 3 remined low (5.12±.83 mmol l 1 ). The olon ws usully empty nd its ph onstnt t 7.2±.16 t ll times. Despite the ingestion of strongly hypo-osmoti teleost tissue, the osmollity of the hyme remined in equilirium with tht of the lood plsm in ll segments t ll times fter feeding. Muh of the osmoti equilirtion ws euse of the seretion of ure into the hyme, prtiulrly in the intestine. After feeding, gstri fluid onentrtions of N + nd Mg 2+ delined, K + nd C 2+ inresed, wheres Cl exhiited little hnge, inditing tht dditionl drinking of sewter ws miniml. N +, K +, wter nd espeilly Cl were sored in the intestine, wheres Mg 2+ nd C 2+ were lrgely exluded. Our results illustrte the omplex integrtion of digestive nd ionoregultory funtion in the elsmornh digestive trt, nd mrked differenes from the teleost pttern. Key words: gstri id seretion, hyme omposition, lkline tide, ure, osmollity, dogfish, shrk. Introdution The feeding eology of mrine shrks hs een widely investigted (Cortes, 1997; Cortes, 1999; Wetheree nd Cortes, 24), nd there is n undne of studies on the spiny dogfish Squlus nthis (Bonhm, 1954; Holden, 1966; Jones nd Geen, 1977; Tnsihuk et l., 1991; Hnhet, 1991; Lptikhovsky et l., 21; Alonso et l., 22). In generl, the dogfish ppers to e n opportunisti predtor with tholi tstes, feeding t irregulr intervls nd exploiting wide vriety of prey ording to their vilility. Mny dogfish re ught with empty stomhs, ut single mel my e mssive (up to 1% of ody mss). Averged on dily sis, onsumption ppers to e in the rnge of.52.5% (Jones nd Geen, 1977) or.84.1% of ody mss (Tnsihuk et l., 1991) in Squlus nthis, with sustntil yer-to-yer nd sesonl vrition. Gut pssge time is lulted to e more thn 5 dys in Squlus nthis (Jones nd Geen, 1977) nd 8 dys in Syliorhinus niul (Sims et l., 1996), with estimtes of 34 dys in other elsmornh speies t wrmer tempertures (Menon nd Kewlrmni, 1959; Wetheree et l., 1987; Wetheree nd Gruer, 199; Cortes nd Gruer, 199; Cortes, 1999). All uthors exept Menon nd Kewlrmni onur tht the digestive proess is muh slower in shrks thn in similrly sized teleost rnivores t omprle temperture (Menon nd Kewlrmni, 1959). In ontrst to this undne of trophi informtion, there re reltively few dt on the physiologil events ssoited with feeding (reviewed y Holmgren nd Nilsson, 1999), so

2 1336 C. M. Wood nd others reently, we hve egun to ddress this defiit with studies on Squlus nthis (Wood et l., 25; Kjimur et l., 26; Wlsh et l., 26). These investigtions hve reveled pronouned systemi disturnes inluding mrked lkline tide in the loodstrem peking 6 h fter feeding, presumly refletive of HCl seretion into the stomh (Wood et l., 25), nd slower tivtion of the ornithine ure yle in oth liver nd skeletl musle ssoited with rise in plsm ure nd osmollity levels (Kjimur et l., 26). In oth studies, postprndil mmoni-n nd ure-n exretion ws miniml, inditive of strong N-onservtion. There ws lso n tivtion of eroi enzymes, prtiulrly those of ketone ody metolism, with the most pronouned effets seen in the retl glnd, the orgn thought to del with ny exess NCl lod ompnying mel (Wlsh et l., 26). In the urrent investigtion, our fous hs turned to the events ourring in the gstrointestinl trt itself during the proessing of mel. Previous studies re sprse, nd hve onentrted on the ph of the stomh, with two ptterns desried in vrious speies. In one, the ph of the gstri fluids is lose to neutrlity when food is not present in the stomh, nd dereses gretly upon feeding (Sullivn, 195; Reolledo nd Vil, 1979; Cir nd Jolitz, 1989; Ppstmitiou nd Lowe, 25). In the other, the gstri ph is lwys low, ut inreses euse of the uffering tion of the food one mel is tken in (Bkin et l., 1935; Menon nd Kewlrmni, 1959; Ppstmitiou nd Lowe, 24). The only report on Squlus nthis (Sullivn, 195) suggested tht it follows the first pttern (i.e. id seretion ours only when food is present), lthough no tul ph dt were reorded. There is negligile informtion on ph levels in the reminder of the trt, or on ion onentrtions, osmollity or wter ontent of the hyme in ny prt of the gstrointestinl system during the proessing of mel. There is lso no informtion on whether the mss of the gstrointestinl trt itself hnges in response to feeding, s hs een doumented in nother intermittent eter, the python (Seor nd Dimond, 1995; Seor nd Dimond, 1998). With this kground in mind, we ddressed the following issues, using nturl feeding frenzy protool in whih dogfish were fed mel of the teleost hke (Merluius produtus), nd then terminlly smpled t vrious times up to 15 dys. First, we wished to desrie the proessing of the food through the trt over time fter nturl feeding in quntittive fshion, nd to estlish whether there were ny trophi hnges in the mss of the gstrointestinl tissue. Seond, we sought to estlish tht the lkline tide (uild-up of plsm HCO 3 ), whih hd previously een seen in nimls fed y stomh tue (Wood et l., 25), lso ourred fter nturl feeding, euse the stomh tue feeding protool represented involuntry food intke. Third, we reorded ph nd HCO 3 levels in the digestive fluids nd hyme throughout the trt in strved nd post-feeding dogfish. Our gol here ws to vlidte the onlusions of Sullivn (Sullivn, 195), nd to understnd whether neutrliztion ours in the intestine (spirl vlve) in light of reent reports of high HCO 3 levels in this region in strved speimens of the moo shrk Chilosyllium plgiosum (Tylor nd Grosell, 26; Anderson et l., 26). A fourth gol ws to doument levels of osmollity, mjor eletrolytes nd wter ontent in the digestive fluids nd hyme during the proessing of the mel. We suspeted tht ingestion of osmoregulting teleost tissue y n osmoonforming elsmornh would rete lrge osmoti nd ioni grdients etween the hyme nd the lood plsm. The idse, ioni nd osmoti sttus of the gll ldder ile ws lso followed to ssess its ontriution to suh events. Finlly, in light of our reent finding tht the enzymes of the ornithine ure yle our in the intestine of Squlus nthis (Kjimur et l., 26), we determined ure levels in the vrious fluids to understnd whether this mjor osmolyte of the systemi fluids ws used to djust the osmollity of the hyme. Mterils nd methods Feeding Dogfish shrks (Squlus nthis,.93.7 kg) of oth sexes were olleted y trwl or y ngling in Brkley Sound, British Columi in July nd August 25. Prior to experimenttion, the fish were held s lrge group (~12 nimls) for severl weeks in 2 l irulr indoor tnk served with running sewter t the experimentl temperture (11±1 C), slinity (32±1 p.p.t.) nd ph (7.9±.15). Squlus nthis will not redily feed when held in smll tnks, ut in the lrge group tnk few fish strted feeding fter 1 week in ptivity, nd therefter the others quikly lerned to do so, s reported y Kjimur et l. (Kjimur et l., 26). During holding, the dogfish were fed every fourth dy (i.e. t pproximtely 96-h intervls) with freshly thwed whole hke (Merluius produtus) from whih the heds hd een removed. This is one of the most ommon nturl prey of dogfish in British Columi ostl wters (Jones nd Geen, 1977). A feeding frenzy ensued, nd ll the food ws onsumed within 3 min. The rtion supplied t eh feeding ws pproximtely 3% of ody mss, sed on the estimted mss of ll the dogfish in the group tnk. With prtie, it ws possile to disern whih dogfish hd fed sed on the ulging profile of the domen, nd t 1 h fter feeding, some of these nimls were ught y dip-net nd removed to isoltion enlosures. The enlosures were individul 4 l polyurethneoted wooden oxes (sewter flow=1 l min 1 ), s desried y Wood et l. (Wood et l., 1995; Wood et l., 25). In this mnner, group of nimls (N=68) ws set side for smpling t eh of the post-feeding times (6 h, 2 h, 3 h, 6 h, 12 h nd 36 h). Smpling At eh smple time, fish were terminlly nesthetized in their isoltion oxes y stopping the wter flow, lowering the wter level to 6 l nd dding n overdose of triine methnesulphonte (MS-222) (.2 g l 1 ). The fish were then quikly removed, weighed nd lood smpled y udl punture with #22 needle tthed to lithium-heprinized

3 Feeding in shrks ml syringe. Blood smples were entrifuged t 1 g for 2 min, the plsm ws removed, susmple ws divided into liquots for totl CO 2 nlysis, nd the reminder ws frozen in liquid N 2 for lter nlyses. The ody vity ws opened y long mid-ventrl inision. The ile dut ws ligted with silk suture, the gll ldder removed intt, nd then ut nd drined so s to ollet the entire ile volume into tred weighot. The ile ws weighed nd then dented into seled entrifuge tue, from whih liquots were removed for ph nd totl CO 2 nlysis, efore the reminder ws similrly frozen in liquid N 2. Doule ligtures were pled round the juntions of the esophgus with the rdi stomh, the rdi stomh with the pylori stomh, t the level of the pylori sphinter, t the posterior end of the intestine (spirl vlve), nd t the end of the olon (retum). This delineted four losed ss: rdi stomh (stomh-1), pylori stomh (stomh-2), intestine nd olon. The very short duodenum ws therey tken s prt of the intestinl s, whih ws dominted y the spirl vlve [see Holmgren nd Nilsson for ntomy (Holmgren nd Nilsson, 1999)]. Cuts were mde etween the doule ligtures, nd the ss removed to tred weighots. The entire proedure took pproximtely 1 min, fter whih dditionl tissues were hrvested for mesurements not reported in the urrent pper. Eh s ws weighed nd then ut open for the olletion of its ontents of digestive juies nd hyme into seprte weighot. The empty s ws reweighed, so s to yield the mss of the tissue nd the mss of the ontents. The ph of the fluid portion of the ontents ws then mesured, or if this ws not possile euse of insuffiient volume, ph mesurement ws tken from the muosl wll of the tissue. Tests demonstrted tht the mesurements from the fluid phse nd muosl wll were virtully identil. The fluid phse ws then mnully stirred nd smple (up to 1.5 ml) ws olleted. If the smple ws loudy, it ws entrifuged to yield ler superntnt, from whih susmple ws divided into liquots for totl CO 2 nlysis nd the reminder ws frozen in liquid N 2 for lter nlyses. The rest of the hyme nd the tissue of the empty s (exept the olon) were then dried to onstnt mss so s to yield their respetive wter ontents. Smples of the food nd the mient sewter were lso frozen for lter nlysis. Anlyses The ph of the ile nd the fluid phse of the hyme smples were mesured using Rdiometer GK241C (Copenhgen, Denmrk) glss omintion eletrode fitted into ustomuilt hmer thermosttted to the experimentl temperture of 11±1 C. The eletrode ws lirted with Rdiometer preision uffers nd its output displyed on Rdiometer phm 72 idse nlyzer. For very low-volume smples or when it ws neessry to tke mesurements from the muosl wll, n esophgel eletrode set (MiroEletrodes In., Bedford, NH, USA) lirted with the sme thermosttted uffers ws used. Totl CO 2 onentrtions of the vrious fluids were determined y the method of Cmeron (Cmeron, 1971). When oth totl CO 2 nd ph were mesured, HCO 3 onentrtions were lulted from rerrngement of the Henderson-Hssellh eqution using pproprite onstnts from Boutilier et l. (Boutilier et l., 1984). Smples of the food were homogenized in lender, then digested in 5 volumes of 1N HNO 3 t 65 C prior to ssy. The reminder of the mesurements were mde on fluid smples, whih hd een frozen nd then lter thwed nd thoroughly mixed. N +, K +, C 2+ nd Mg 2+ onentrtions were determined y tomi sorption spetrosopy (SpetrAA- 22FS; Vrin) nd Cl y the olorimetri ssy of Zll et l. (Zll et l., 1956). Osmollity ws mesured y vpour pressure osmometry (Wesor 51C, Logn, UT, USA). Ure ws mesured y the dietyl monoxime method (Rhmtullh nd Boyde, 198). Wter ontents of hyme, gut tissues nd food smples were determined y drying to onstnt mss t 65 C for 7296 h. Dt hve een expressed s mens ± 1 s.e.m. (N). Msses of the gstrointestinl ontents nd the gstrointestinl tissue segments themselves were normlized to totl ody mss prior to nlysis. Dt were tested for normlity nd homogeneity of vrines, nd in some instnes dt were log-trnsformed prior to further nlysis to pss Brtlett s 2 test for homogeneity of vrines. Perentge dt were r-sin trnsformed. Dt were then nlyzed y one-wy nlysis of vrine (ANOVA), followed y Tukey s honestly signifint differene test to detet speifi differenes (Sttistix for Windows). In those few instnes in whih the dt still did not pss Brtlett s test, the non-prmetri KrusklWllis signedrnks test ws used in ple of the ANOVA nd Tukey s test. In the urrent study, dt were olleted from nimls tht hd een kept on 4-dy feeding yle nd then srified t vrious times fter their lst mel. Thus, in the figures dotted line t 96 h post-feeding indites the prole pre-feeding ondition (i.e. 96 h fsting), nd signifint differenes mong points t different times post-feeding re indited in the figure legends. Comprisons etween omprtments were mde using Student s pired t-test (two-tiled) with the Bonferroni orretion for multiple omprisons, nd pproprite log or rsin trnsformtions where neessry. A signifine level of P<.5 ws used throughout. Results Msses nd movements of hyme Bsed on the mss (fluid plus solid) of the ontents of stomh-1 plus stomh-2 t 6 h nd 2 h post-feeding (Tle 1), nd tking into ount wter ontent differenes, the verge mss of food onsumed in the single mel ws 56% of ody mss. This ws greter thn the estimted rtion (3%) fed to the entire group, ut n effort ws mde to selet those dogfish tht we knew hd eten, sed on ulging domens. The mss of ingested food ws signifintly greter in stomh-1 thn in stomh-2 t 6 h, ut this differene ws reversed y 2 h (Tle 1). At 3 h nd 6 h, this differene persisted, lthough the totl msses in eh setion were gretly

4 1338 C. M. Wood nd others Tle 1. Qulittive nd quntittive tultion of the time ourse of digestion from smpling of four setions of the gstrointestinl trt of the dogfish shrk t vrious times fter feeding Time fter mel (h) N Stomh-1 Stomh-2 Intestine Colon 6 7 Lrge mss of solid, some liquid Lrge mss of solid, some liquid Yellow-rown fluid Empty hyme hyme 36.6±5.2 (,A) 17.3±7. (,B) 6.2±1.1 (,C).±. (,D) 2 6 Smller mss of solid, more liquid Greter mss of solid, more liquid Similr mount of yellow-rown fluid, Empty hyme hyme ut pstier 22.±12.8 (,,A) 48.5±11.5 (,B) 7.3±1.3 (,A).±. (,C) 3 7 Smller mss of solid, smller Smller mss of solid, similr mount Similr mount of yellow-rown Empty or smll mount of drk pste mount of liquid hyme of liquid hyme fluid-pste 4.2±2.5 (,,A) 17.9±5.8 (,B) 6.2±2.2 (,A).2±.1 (,A) 6 6 Little solid (minly one), similr Similr mss of solid (minly one), Similr mount of yellow-rown Empty or smll mount of drk pste mount of hyme or ler fluid similr mount of liquid hyme fluid-pste, smll piees of one 5.5±2.9 (,A) 18.6±7.2 (,B) 5.6±1.3 (,A).1±.1 (,A) 12 8 Very smll volume of ler fluid Very smll volume of ler fluid only Very smll volume of wtery yellow- Empty only rown fluid, tiny piees of one.9±.4 (,A).4±.1 (,A).5±.1 (,A).±. (,A) 36 8 Very smll volume of ler fluid Very smll volume of ler fluid only Very smll volume of wtery yellow- Empty only rown fluid.4±.1 (,A).3±.1 (,A).2±.1 (,A).±. (,A) Vlues (mens ± 1 s.e.m.) re the reltive mss (g kg 1 ody weight) of the ontents in eh segment. Note: qulittive omprisons re with respet to onditions in the sme omprtment t the preeding smple time. Within the sme omprtment, vlues shring the sme lower-se letter re not signifintly different (P>.5). Within the sme time, vlues shring the sme upper-se letter re not signifintly different (P>.5). redued. The digestion of one lerly lgged ehind the digestion of soft tissue. By 12 h there ws no food or hyme remining in either stomh omprtment, only smll mount of ler fluid, nd this sitution persisted t 36 h. Right from 6 h through 6 h postfeeding there ws onstnt volume (pproximtely 6 ml kg 1 ) of yellowrown fluid-pste in the intestine, lthough its onsisteny hnged somewht over time. By 12 h this intestinl fluid ws redued pproximtely 1-fold, nd only tiny piees of ones remined. Similr smll mounts of intestinl fluid were present t 36 h. The olon ws empty t most times, nd only t 3 h nd 6 h postfeeding ws ny mteril found in this smll omprtment, nd only in few nimls, inditing tht fel mteril ws not held here for ny prolonged period. These oservtions indite tht digestion nd sorption were lrgely ompleted y 12 h post-feeding, nd finished efore 36 h post-feeding. Chnges in mss of the gstrointestinl trt tissues The reltive msses of the tissues of the gstrointestinl trt hnged during the proessing of the mel (Fig. 1). Thus, the mss of the tissue of stomh-1 ws signifintly lower t 3 h nd 6 h postfeeding thn t some other time points, differene of 1824% (Fig. 1A). By ontrst, the mss of stomh-2 ws gretest t these times, differene of pproximtely 3142% reltive to the 6 h vlue, nd 6883% reltive to the 36 h vlue, where stomh-2 mss ws t its lowest (Fig. 1B). The intestine underwent similr pttern of inrese followed y derese in mss fter feeding, with the lowest reltive mss gin ourring t 36 h. Reltive to the 6 h vlue, the pek t 2 h ws 28% rise, nd reltive to the 36 h vlue it ws 74% rise. To very lrge extent, these hnges were driven y ltertions in the wter ontents of the tissues, with only smll nd non-signifint hnges in the dry msses (Fig. 1A-C). For exmple, when the reltive mss of stomh-2 tissue inresed from 9.13±.53 (N=8) to 12.36±.62 (N=7) g kg 1 ody mss t

5 Feeding in shrks h, the perentge of wter inresed from 8.43±.62% (N=8) to 81.82±.44% (N=7), so the wter ontent inresed from 7.34±.42 (N=8) to 1.19±.51 (N=7) g kg 1 ody mss. Therefore, these hnges were lrgely ssoited with ltertions in the extent to whih the tissues were engorged with fluid. Indeed, we noted tht t the pek of the inreses, the exised tissue ontinued to serete fluid into the weighot for pproximtely 3 min fter the originl hyme hd een Tissue mss (g kg 1 ody mss) A B C,,,,,,, ,, Stomh-1 dry mtter Wter Stomh-2 dry mtter Wter Intestine dry mtter Wter Fig. 1. The influene of feeding on the reltive mss of the tissue (expressed s g kg 1 ody mss) of vrious segments of the gstrointestinl trt in the dogfish shrk (A) stomh-1 (rdi stomh), (B) stomh-2 (pylori stomh) nd (C) intestine. The inset rs indite the mss ontriuted y the tissue wter ontent, nd the differenes represent the mss ontriuted y the tissue dry-mtter ontent. Mens ± 1 s.e.m. (N=68 t eh time point). Mens not shring the sme letter re signifintly different (P<.5); the sme symols pply to the mss ontriuted y tissue wter ontent nd so hve not een repeted. removed nd the originl tissue mss tken. This did not hppen with tissues from nimls t 12 or 36 h post-feeding. Aidse hnges On the systemi side, totl CO 2 mesurements on lood plsm (Fig. 2) reveled ler evidene of postprndil lkline tide in the systemi loodstrem of these nturlly feeding shrks, with rise of pproximtely 4.5 mmol l 1 t 6 h post-feeding reltive to points t 3 h onwrds. As plsm ph ws not mesured, P CO 2 nd HCO 3 ould not e lulted, ut HCO 3 represented more thn 9% of this inrese, sed on ny resonle estimte of P CO 2. On the luminl side, feeding used mrked hnges in the idse sttus of the gstri fluids (Fig. 3). In fsted fish (i.e. 12 h), phs in the fluids of oth stomh-1 nd stomh-2 were low, verging 1.77 nd 2.5, respetively. However, these vlues rehed 3.2 nd 3.95 t 6 h post-feeding, inditing sustntil redution of H + onentrtion y the uffering tion of the food, despite the prole inrese in H + seretion rte t this time. Therefter, ph grdully fell in oth prts of the stomh s digestion proeeded until minim were rehed t 12 h. At ll times, ph ws lower in stomh-1 thn in stomh-2. At no time ws there ny detetle HCO 3 in the gstri fluids. In ontrst to these sustntil hnges in the stomh omprtments, phs in the intestine (~6.5) nd olon (~7.2) remined high nd remrkly stle, with no signifint hnges t ny times throughout the regime (Fig. 3). Similrly, the ph of gll ldder ile remined unhnged (dt not shown), with n overll men of 6.42±.8 (N=42). HCO 3 onentrtions in the intestinl fluid were rther vrile, rnging from to 19.6 mmol l 1 in individul nimls, ut there were no signifint differenes over time (dt not shown). The overll men ws 5.12±.83 (N=36) mmol l 1. HCO 3 onentrtions in gll ldder ile were low (4.21 mmol l 1 ) with n overll men of.61±.12 (N=41) mmol l 1. There ws [CO 2 ] (mmol l 1 ) Plsm totl CO 2, (Pre-feeding) 36 Fig. 2. The influene of feeding on plsm totl CO 2 onentrtion in lood smples drwn y udl punture in the dogfish shrk. Mens ± 1 s.e.m. (N=68 t eh time point). Mens not shring the sme letter re signifintly different (P<.5).

6 134 C. M. Wood nd others ph ph,,,,,, (Pre-feeding) Fig. 3. The influene of feeding on the ph of the fluid phse of the gstrointestinl ontents in stomh-1 (rdi stomh), stomh-2 (pylori stomh), intestine nd olon in the dogfish shrk. Mens ± 1 s.e.m. (N=68 t eh time point). Within omprtment, mens not shring the sme letter re signifintly different (P<.5). never suffiient fluid in the olon to otin mesurement of HCO 3 or of ny other ions or osmollity. Ioni nd osmoti hnges In the first smple tken fter eting (6 h), the perentge of wter in the ontents of stomh-1 (78.%, Tle 2) ws pproximtely the sme s in the originl food (8.2%, Tle 3), nd this remined more or less unhnged when the olus pssed into stomh-2 t 2 h (81.9%, Tle 2). This suggests tht very little sewter ws ingested with the originl mel. However, s digestion progressed, the perentge of wter in the hyme inresed in stomh-1, stomh-2 nd the intestine (Tle 2). When expressed on perentge sis s in Tle 2, the hnges in wter ontent re deeptively smll. Fig. 4,, illustrtes the lrge hnges in totl wter volumes in the hyme s it pssed down the trt. Colon Wter volumes peked t pproximtely Intestine 3 ml kg 1 ody mss t 6 h in stomh-1 nd 38 ml kg 1 ody mss t 2 h in stomh-2, nd remined signifintly elevted through 6 h. Of ourse, muh of this wter ws ontined within the ingested food, nd t lest initilly remined within the solid phse. Wter volume in the intestinl hyme remined t pproximtely 56 ml kg 1 ody mss throughout the 6 h to 6 h Stomh-2 post-feeding period. Stomh-1 A omprison of the omposition of the sewter, the teleost food, the lood plsm nd the smll mount of fluid in stomh-1 [.86±.42 (N=8) ml kg 1 ody mss] in fsting fish (12 h post-feeding) illustrtes severl points (Tle 3). First, the osmollity of sewter, plsm nd stomh-1 fluid were sttistilly identil. Seond, in other spets, stomh-1 fluid ws muh loser to sewter thn to lood plsm. These similrities inlude higher nd identil K + nd C 2+ onentrtions, muh higher N +, Cl nd Mg 2+ levels, nd muh lower ure levels in oth stomh-1 fluid nd sewter thn in lood plsm. Finlly, the food ws very different in omposition from ny of the fluids, nd would lerly offer n initil dilution of the osmollity, N + nd Cl levels of the ody fluids (stomh-1 or plsm), unless it ws ingested together with onsiderle mounts of sewter. Similrly, ure onentrtions in food were only frtion of those in these ody fluids. At the sme time, however, K + nd C 2+ onentrtions were onsiderly higher in the food thn in either plsm or stomh-1 fluid, wheres Mg 2+ onentrtion ws intermedite. The intke of the teleost food hd profound impt on the omposition of the fluid phse in the gstrointestinl ontents, ut surprisingly, osmollity remined t pproximtely plsm nd sewter levels (~95 mosm kg 1 ) in ll omprtments t ll times (Fig. 5A). This ourred despite the ft tht the Tle 2. Chnges over time in the perentge of wter of the ontents in vrious omprtments of the gstrointestinl trt in the dogfish shrk Time fter mel (h) N Stomh-1 Stomh-2 Intestine ±.95 (,A) 84.71±3.5 (,A,B) 88.51±2. (,B) ±2.73 (,A) 81.95±3.1 (,B) 87.91±1.18 (,A,B) ±3.45 (,A) 86.9±2.96 (,A) 91.64±1.25 (,A) ±3.53 (,A) 81.22±.98 (,B) 9.88±1.32 (,A) ±.49 (,A) 96.81±1.71 (,A) 93.71±1.79 (,,A) ±.15 (,A) 97.77±.14 (,A) 96.13±.51 (,A) Gstrointestinl omprtments studied were stomh-1 (rdi stomh), stomh-2 (pylori stomh) nd the intestine. Vlues re the mens ± 1 s.e.m. Within the sme omprtment, vlues shring the sme lower-se letter re not signifintly different (P>.5). Within the sme time, vlues shring the sme upper-se letter re not signifintly different (P>.5).

7 Feeding in shrks 1341 Tle 3. A omprison of osmollity nd the onentrtions of mjor osmolytes nd wter in sewter, food (deheded hke, Merluius produtus), lood plsm nd the fluid of stomh-1 (rdi stomh) prior to feeding (12 h fsting) in the dogfish shrk Sewter Food Blood plsm Stomh-1 fluid (N=4) (N=5) (N=8) (N=8) Osmollity (mosmol kg 1 ) 967±3 () 4 973±6 () 949±25 () Ion onentrtions (mmol l 1 ) N + 452±3 () 55.6±2.6 () 254±3 () 4±9 (d) Cl 515±3 () 46.4±2.4 () 249±2 () 44±14 (d) K + 9.8±.5 () 112.9±2.3 () 3.5±.9 () 11.6±.6 () C ±.1 () 73.±3.2 () 4.3±.1 () 8.7±.4 () Mg ±.4 () 15.9±.2 () 1.4±.1 () 35.3±1.2 (d) Ure.±. () 4.2±1. () 438±22 () 145±15 (d) Wter (%) (96.8)* 8.18±.23 () 96.15±.39 () 97.25±.49 () Vlues re the mens ± 1 s.e.m. *Clulted from slinity. Units for food re on per kg rther thn per l sis. Estimted vlue (see Holmes nd Donldson, 1969). Mesured vlues shring the sme letter for prmeter re not signifintly different (P>.5). originl osmoti onentrtion of the food ws less thn 5% of tht of these fluids. The only onsistent differene etween omprtments ws slightly higher osmollity (y pproximtely 5 mosm kg 1 ) in the fluid of the intestine, whih ws signifint t some smple times. Plsm osmollity exhiited minor flututions. Ure levels in the fluids of stomh-1 nd stomh-2 were pproximtely 2 mmol l 1 lower thn in intestinl fluid, wheres intestinl fluid ure ws only pproximtely 1 mmol l 1 elow tht in plsm (Fig. 5B). These differenes were mintined t ll times. Thus, onsiderle ddition of ure must our when the hyme moves from stomh-2 to the intestine. The impt of the mel, whih ontined negligile mounts of ure (~4 mmol kg 1 ), ppered to further lower ure Wter volume (ml kg 1 ody mss) Wter in gut ontents Stomh-1 d Stomh-2 Intestine, (Pre-feeding) levels in stomh-1 nd stomh-2 up to 36 h post-feeding. Notly, plsm ure peked t 2 h post-feeding. In fsted fish (1236 h post-feeding), N + onentrtions in the fluid of stomh-1 (~4 mmol l 1 ) were higher thn in stomh-2 (~325 mmol l 1 ), lood plsm (~255 mmol l 1 ) or intestinl fluid (~14 mmol l 1 ), ut lower thn in sewter (~45 mmol l 1 ) (Fig. 6A). All these differenes were signifint. The impt of the mel, whih ontined muh lower onentrtion of N + (~55 mmol kg 1 ), ws to use lower N + levels in stomh-1 nd stomh-2 y 35% t ll times through 6 h. By ontrst, N + levels in the intestinl fluid tended to e greter t these times. Plsm N + level ws mintined onstnt t ll times. Cl onentrtions in the gstrointestinl fluids (Fig. 6B) were generlly higher thn those of N +, prtiulrly in the intestine (.f. Fig. 6A), nd underwent less mrked hnges. In fsted fish (1236 h post-feeding), Cl onentrtions were similr in the fluids of stomh-1 nd stomh-2 (~43 mmol l 1 ) nd muh higher thn in the intestine or lood plsm (oth ~24 mmol l 1 ), lthough lower thn in sewter (~515 mmol l 1 ). Despite the intke of mel tht ontined muh lower levels of Cl (~45 mmol kg 1 ), these sustntil differenes were mintined fter feeding, nd there were only smll dereses in the Cl onentrtions in the gstri fluid. As with N +, intestinl fluid Cl tended to e greter through 6 h post-feeding. Plsm Cl remined unhnged. Fig. 4. The influene of feeding on totl wter volumes (expressed s ml kg 1 ody mss) of the gstrointestinl ontents in stomh-1 (rdi stomh), stomh-2 (pylori stomh) nd intestine in the dogfish shrk. This mesurement inludes wter in oth the fluid nd solid phses. Mens ± 1 s.e.m. (N=68 t eh time point). Within omprtment, mens not shring the sme letter re signifintly different (P<.5).

8 1342 C. M. Wood nd others In ontrst to N + nd Cl, K + onentrtion in the food (~115 mmol kg 1 ) ws more thn 1-fold higher thn in sewter, lood plsm or ny of the gstrointestinl fluids of fsted fish (1236 h post-feeding; Fig. 6C). Sewter, stomh-1 nd stomh-2 fluids were ll pproximtely 1 mmol l 1, wheres plsm nd intestinl fluid were sustntilly lower t 35 mmol l 1. The impt of the K + -rih mel ws lso to triple the K + onentrtion in the fluids of oth stomh-1 nd stomh-2, nd these elevtions remined Osmollity (mosm kg 1 ody mss) [Ure] (mmol l 1 ) A B Osmollity Ure,,, x,y x,y y, x,y SW Food (Pre-feeding),, x signifint through 3 h. K + levels in the intestinl fluid were lso tripled nd remined elevted through 6 h, lthough the solute hnges were smller thn in the gstri fluids, nd K + onentrtions were lower in the intestinl fluids thn in the gstri fluids t ll times. There were no signifint hnges in plsm K +. Like K +, C 2+ ws in sustntilly higher onentrtion in the food (~73 mmol kg 1 ) thn in ny of the fluids smpled (Fig. 7A). Plsm C 2+ levels were pproximtely 4 mmol l 1, sewter, stomh-1 nd stomh-2 fluids were ll pproximtely 1 mmol l 1, wheres intestinl fluids were signifintly higher t pproximtely 2 mmol l 1 in fsted fish Intestine (1236 h post-feeding). In response to the, Plsm Intestine, Plsm Stomh-1 Stomh-2 Stomh-1 Stomh-2 x,y Fig. 5. The influene of feeding on (A) the osmollity nd (B) the ure onentrtion of the fluid phses of the gstrointestinl ontents in stomh-1 (rdi stomh), stomh-2 (pylori stomh) nd intestine in the dogfish shrk. Simultneously mesured vlues in the lood plsm re lso shown. Mens ± 1 s.e.m. (N=68 t eh time point). As points of referene, the drk rs represent the mesured vlues for mient sewter (SW) (N=4), nd the light rs represent the estimted vlue for the ingested food [N=5; vlue for osmollity from Holmes nd Donldson (Holmes nd Donldson, 1969)]. Signifint differenes re not mrked in A so s to void lutter; the only signifint differenes (P<.5) within omprtments re in plsm osmollity, s indited y mens not shring the sme letter in the following series: =6 h, 3 h, 36 h; =2 h, 12 h, 36 h; =2 h, 6 h, 12 h. In B, within omprtment, mens not shring the sme letter re signifintly different (P<.5). For lrity, x,y refers to stomh-2 nd, to stomh-1. C 2+ -rih mel, onentrtions inresed signifintly y 152 mmol l 1 in ll three omprtments of the gstrointestinl trt from 6 h through 6 h. Notly, C 2+ levels remined higher in the intestinl fluid thn in the gstri fluids throughout the entire period. Plsm C 2+ did not hnge. ws in lower onentrtion in the food (~16 mmol kg 1 ) thn in sewter (~52 mmol l 1 ), ut oth of these vlues were fr greter thn the low levels ( mmol l 1 ) in the lood plsm (Fig. 7B). At 12 h post-feeding, onentrtions of Mg 2+ were ll very similr (~35 mmol l 1 ) in the fluids of stomh-1, stomh-2 nd the intestine. These vlues were signifintly lower thn in sewter ut higher thn in food. However, fter longer-term fsting (36 h), Mg 2+ intestinl fluid Mg 2+ onentrtions were threefold greter (~12 mmol l 1 ), lthough highly vrile, wheres gstri fluid vlues remined the sme. After ingestion of the mel, Mg 2+ onentrtions were lowered y pproximtely 1 mmol l 1 in the fluids of stomh-1 nd stomh-2 from 6 h through 3 h. By ontrst, Mg 2+ onentrtions in the intestinl fluid tended to rise fter the mel, with n initil douling t 6 h. Mg 2+ onentrtions in the plsm exhiited minor flututions ut remined less thn 1.5 mmol l 1 t ll times. Gll ldder ile The gll ldder lerly ontrted fter the mel. The volume of ile in the gll ldder ws only.38±.9 (N=7),.38±.12 (N=6) nd.45±.6 (N=7) ml kg 1 ody mss t 6 h, 2 h nd 3 h fter the mel, respetively, ut then rose to signifintly higher levels t 6 h [.62±.13 (N=6)], 12 h [.82±.6 (N=8)] nd 36 h [.71±.18 (N=8)] ml kg 1 ody mss.

9 Feeding in shrks 1343 Fig. 6. The influene of feeding on (A) sodium, (B) hloride nd (C) potssium onentrtions of the fluid phses of the gstrointestinl ontents in stomh-1 (rdi stomh), stomh-2 (pylori stomh) nd intestine in the dogfish shrk. Simultneously mesured vlues in the lood plsm re lso shown. Mens ± 1 s.e.m. (N=68 t eh time point). As points of referene, the drk rs represent the mesured vlues for mient sewter (SW) (N=4), nd the light rs represent the mesured vlues for the ingested food (N=5). Within omprtment, mens not shring the sme letter re signifintly different (P<.5). There were no signifint differenes in the plsm omprtment in AC. In C, for lrity, x,y,z refers to stomh-2 nd,, to stomh A N +,,, Stomh-1 Stomh-2 Plsm Intestine, Bile ws in osmoti equilirium with lood plsm (~95 mosm kg 1 ) nd exhiited similr N + onentrtions (~27 mmol l 1 ), ut other spets of its omposition were rther different (Fig. 8). Thus, K + (~7 mmol l 1 ), Mg 2+ (~12 mmol l 1 ) nd C 2+ (~3 mmol l 1 ) onentrtions were ll sustntilly higher thn in lood plsm, wheres ure levels (~31 mmol l 1 ) were lower (Fig. 8 versus Tle 3). Prtiulrly notle were the very low levels of Cl in ile (~6 mmol l 1 ), only pproximtely 25% of those in plsm, inditing tht muh of the negtive hrge ws rried y n unmesured nion, presumly ile id nions. There were only modest hnges in the omposition of gll ldder ile fter feeding (Fig. 8). These inluded slightly lower levels of osmollity nd C 2+ t 2 h nd 6 h, nd of Cl t 6 h, nd higher level of ure t 2 h. Biliry Mg 2+, N + nd C 2+ onentrtions were lso slightly higher fter 36 h of strvtion thn t other times. 12 Disussion The pttern of feeding nd digestion 1 Contrry to the erly oservtions of Sullivn (Sullivn, 195), the dogfish shrk lerly flls into tht group of elsmornhs tht mintin highly idi ph in the gstri fluids during fsting, nd exhiit rise in ph one the olus of food is ingested. The ph hnges in the stomh of Squlus nthis (from pproximtely 1.8 to 3.9 t 6 h fter the mel; Fig. 3) were very similr to those in the leoprd shrk Trikis semifsit (1.5 to 3.1) (Ppstmitiou nd Lowe, 24) ut muh more mrked thn in severl other speies exhiiting this sme pttern (3.5 to 5.) (Bkin et l., 1935; Menon nd Kewlrmni, 1959). Ppstmitiou nd Lowe [Ion] (mmol l 1 ) B C K + Cl z, z y,z,,,,,, x,y, SW Food (Pre-feeding) Stomh-2 Stomh-1 Intestine Plsm speulted tht mintining stomh ph ontinuously low my e dvntgeous for n opportunisti predtor (Ppstmitiou nd Lowe, 24), llowing it to e physiologilly prepred to digest mel soon fter feeding event. The digestion nd gut pssge time oserved in the urrent study (lrgely omplete,, x, Stomh-1 Stomh-2, Intestine Plsm x

10 1344 C. M. Wood nd others [Ion] (mmol l 1 ) A B C 2+ Mg 2+,,,,, x y x,y x x,y y 5 dys, nd finished y 15 dys; Tle 1) ws omprle to tht reported y Jones nd Geen for the sme speies forefed omprle food items (Jones nd Geen, 1977). Our reent mesurements of N-udget in nturlly feeding Squlus nthis (Kjimur et l., 26) indite tht the nimls must hve to feed every 56 dys just to mintin enough N for uresed osmoregultion, let lone grow. In ord with these ides, stomh-1 did not pper to inrese its mss fter initil ingestion of the mel (Fig. 1A), suggesting tht it is mintined in stte of rediness during strvtion so s to e le to ept mel t ny time. The fll in stomh-1 mss t 3 h nd 6 h post-feeding ws SW Food (Pre-feeding) Intestine Stomh-1 Stomh-2 Plsm, y Intestine Stomh-1 Stomh-2 Plsm Fig. 7. The influene of feeding on (A) C nd (B) Mg onentrtions of the fluid phses of the gstrointestinl ontents in stomh-1 (rdi stomh), stomh-2 (pylori stomh) nd intestine in the dogfish shrk. Simultneously mesured vlues in the lood plsm re lso shown. Mens ± 1 s.e.m. (N=68 t eh time point). As points of referene, the drk rs represent the mesured vlues for mient sewter (SW) (N=4), nd the light rs represent the mesured vlues for the ingested food (N=5). Within omprtment, mens not shring the sme letter re signifintly different (P<.5). There were no signifint differenes in the plsm omprtment in A. In B, for lrity, x,y refers to stomh-2 nd, to stomh-1. ssoited with fll in tissue wter ontent of unknown etiology. We speulte tht ontrtile tivity t these times resulted in the mehnil displement of fluid from the musle tissue. By ontrst, stomh-2 did inrese its mss sustntilly, s did the intestine, inditing tht these segments n e tivted firly quikly when required (Fig. 1B,C). Although these hnges were reltively lrge, they were minly euse of fluid engorgement rther thn tul prolifertion of the tissue. We ttriute these hnges to inresed lood flow nd seretory tivity for digestion nd sorption. By 36 h fter the lst mel, these were proly gretly redued, resulting in the oserved flls in tissue msses t this time. By wy of omprison, in the 3-dy strved Burmese python, whih is perhps the hmpion in terms of upregultion of the digestive trt (Seor nd Dimond, 1995), stomh mss inresed y 26%, minly euse of inresed hydrtion only t 6 h postfeeding, wheres intestinl mss inresed more slowly, rehing 3% y 3 dys, ut the reltive roles of hydrtion versus prolifertion were not seprted in tht study. However, more reently, Holmerg et l. hve reported tht dry s well s wet intestinl mss inreses in the python t 48 h fter feeding, lthough the perentge of wter dereses (Holmerg et l., 23). By nlogy to teleosts (e.g. Axelson et l., 1989; Axelson nd Fritshe, 1991), overll inreses in regionl lood flow re expeted fter feeding, ut these hve not yet een mesured in elsmornhs (Holmgren nd Nilsson, 1999). The volume of hyme in the intestine (pproximtely 6 ml kg 1 ody mss; Tle 1) nd its ioni nd osmoti hrteristis (Figs 5-7) remined pproximtely onstnt from 6 h through 6 h post-feeding, despite lrge hnges in these prmeters in stomh-1 nd stomh-2. The omposition of the hyme ws lso very different from tht in the stomhs, with muh higher ph (Fig. 3) nd ure levels (Fig. 5B), higher C 2+ (Fig. 7A) nd Mg 2+ (Fig. 7B) onentrtions, nd muh lower N + (Fig. 6A), Cl (Fig. 6B) nd K + (Fig. 6C) onentrtions. This suggests tht the pylori sphinter ts s n effiient meter, letting through only s muh mteril from stomh-2 s the neutrliztion, trnsport nd sorptive proesses in the intestine n del with t ny one time. As in higher vertertes, this pylori trnsit is proly under omplex neurl nd hormonl ontrol (Holmgren nd Nilsson, 1999). In most vertertes, the finl setion of the trt, the olon or retum, serves for wter sorption, therey drying the fees (Holmgren nd Nilsson, 1999). In the dogfish, there ws

11 Feeding in shrks 1345 rrely muh mteril in the olon, nd when present, it ws thik pste (Tle 1), in ord with this ide. Our impression is tht overll sorptive effiieny is very high in Squlus nthis, nd tht the mount of fel mteril dishrged is smll. Aidse responses ssoited with feeding nd digestion The inrese in plsm HCO 3 onentrtion t 6 h fter the nturl mel (56% rtion; Tle 1) ws more thn 4 mmol l 1 (Fig. 2), sustntilly greter thn the mmol l 1 seen erlier in dogfish fed 2% rtion y stomh tue (Wood et l., 25). Thus, the originl oservtion of postprndil lkline tide ws not n rtift of involuntry forefeeding, nd these dt suggest tht the mgnitude of the response is proportionl to the rtion. Squlus nthis hs remrkle pity to resist lklosis during experimentl HCO 3 loding, omplished y exretion of se (HCO 3 equivlents) t high rte through the gills (Wood et l., 1995). This ppers to our vi mehnism driven y solterl V-H + -ATPse in speilized rnhil ells (Tresguerres et l., 25; Tresguerres et l., 26). Therefore, the true extent of metoli se genertion during digestion my tully hve een muh greter thn indited y the oserved lklosis in the systemi loodstrem. Mesurements of possile se flux to the wter fter feeding will e required to ddress this possiility. The use of the lkline tide is the seretion of idi equivlents y the gstri muos to digest the food olus: solterl HCO 3 efflux into the extrellulr omprtment mthes the rte of pil H + seretion. In higher vertertes (reviewed y Shs et l., 1995; Hersey nd Shs, 1995; Niv nd Frser, 22; Wng et l., 21), K + -stimulted, H + - trnsloting ATPse is responsile for the pil H + seretion, nd Cl /HCO 3 exhnger for the solterl HCO 3 export nd Cl entry. Cl is elieved to exit pilly vi Cl hnnel, suh tht there is net seretion of HCl into the stomh. At present, it is unertin s to whether the mehnism is the sme in elsmornhs where the HCl nd pepsinogen seretion funtions pper to e omined into single gstri glnd ell type, the oxyntiopepti ell (Hogen, 1967; Reolledo nd Vil, 1979). Certinly, K + -stimulted, H + -ATPse very similr to tht of mmmls hs een lolized in gstri glnd ells of the elsmornh Dsytis sin (Smolk et l., 1994), ut HCl seretion ours with the genertion of negligile trnsepithelil potentil, in ontrst to higher vertertes (Hogen, 1959; Hogen, 1967; Rehm, 1962; Kidder, 1976; Kidder, 1991). The hyme entering the intestine from stomh-2 during the Bile omponents (mmol l 1 ) 1 Bile,,, Osmollity 8 (mosml kg 1 ) ,,,,,,,,,,, (Pre-feeding) Ure N + Cl C 2+ Mg 2+ K + Fig. 8. The influene of feeding on the omposition of gll ldder ile in the dogfish shrk. Mens ± 1 s.e.m. (N=68 t eh time point). For eh prmeter, mens not shring the sme letter re signifintly different (P<.5). There were no signifint differenes for K +. period from 6 h to 6 h post-feeding hd ph in the rnge , yet ph in the intestinl hyme ws preisely regulted t 6.5 (Fig. 3). This indites very urte nd effetive neutrliztion mehnism in the intestine. Although the volume of the gll ldder ws depressed t 63 h, gll ldder ile is unlikely to hve plyed muh of role in neutrliztion euse it ontined only very low HCO 3 onentrtions (men=.61 mmol l 1 ). However, in strved Squlus nthis, Boyer et l. reported HCO 3 onentrtions in hepti ile to e 1-fold higher (5.8 mmol l 1 ), with sustntil flow rte (74 l kg 1 h1 ) (Boyer et l., 1976). Potentilly, these vlues ould e even higher in fed nimls, so HCO 3 originting from the ile dut epitheli ould ply signifint role, s in higher vertertes. The other, more importnt soure of HCO 3 in higher vertertes is the inr dut epitheli of the pnres, nd the flow rte nd HCO 3 onentrtion of pnreti juie inrese fter mel (Guyton nd Hll, 26). The dogfish shrk hs lrge disrete pnres (Holmgren nd Nilsson, 1999), so the role of pnreti seretion in neutrliztion ould e sustntil. However, we re not wre of ny reports on this topi in elsmornhs. A third possile soure is HCO 3 seretion y the intestinl epithelium itself. In mrine teleost fish, this is known to our t very high rte in exhnge for Cl uptke, nd to e involved in the preipittion of divlent tions (priniplly C 2+ ) nd CO 3 in the intestine, therey llowing the sorption of more free wter from the imied sewter (Wilson et l., 1996; Wilson et l., 22; Grosell et l., 25). Reent work hs reported very high HCO 3 onentrtions (4575 mmol l 1 ) in the intestinl fluid of strved speimens of the moo shrk,

12 1346 C. M. Wood nd others Chilosyllium plgiosum, experiening hypertoni shok nd resulting in sustntil drinking (Tylor nd Grosell, 26; Anderson et l., 26). These HCO 3 onentrtions were omprle to those in mrine teleosts tht drink regulrly. However, HCO 3 seretion rtes were very low in isolted intestinl s preprtions from these hypertonilly exposed elsmornhs, s well s from ontrol speimens of Chilosyllium plgiosum (Anderson et l., 26), so the soure of tht HCO 3 in the shrk is unertin. In the urrent study on Squlus nthis, HCO 3 onentrtions in intestinl fluid were muh lower (men=5.1 mmol l 1 ) thn in Chilosyllium plgiosum nd did not vry etween strved nd fed nimls. We onlude tht the soure of the se used for preise ph ontrol of the intestinl hyme is unler nd deserves future investigtion. Iono- nd osmoregultory responses ssoited with feeding nd digestion Contrry to our initil hypothesis, ingestion of lrge mel of teleost tissue (pproximtely 6 mosm kg 1 elow shrk plsm vlues) never resulted in osmoti grdients etween the gstrointestinl ontents nd the ody fluids (Fig. 5A). Within 6 h, the gstrointestinl fluids were rought into osmoti equilirium (Fig. 5A), lthough lrge ioni grdients persisted throughout the digestive proess (Figs 6, 7). One possile explntion would e lrge osmoti wter flux ross the stomh wll. Bsed on 5.5% rtion, n 8% wter ontent in the food (Tle 3) nd the 6 mosm kg 1 osmoti differene, this would neessitte flux of pproximtely 17.6 ml of wter per kg of dogfish mss ross the stomh wll from food to plsm, reduing the wter ontent of the ingested mteril to pproximtely 61.5%. However, wter ontent of the intestinl ontents styed more or less unhnged initilly nd lter inresed (Tle 2), so it is unlikely tht this lrge sorptive flux of wter ourred. Another possile explntion for the rpid djustment of hyme osmollity would e the ingestion of ppreile mounts of sewter with the food. The whole question of drinking in elsmornhs hs een ontroversil sine the originl onlusion of Smith tht these nimls do not need to drink s prt of their osmoregultory strtegy (reviewed y Anderson et l., 26; Smith, 1936). However, erlier we doumented drinking t very low rte (~.16 ml kg 1 h1 ) in strved speimens of Squlus nthis (We nd Wood, 2), nd the omposition of the smll mount of fluid in stomh-1 in the urrent study suggests tht it ws derived from sewter (Tle 3). The prtiulrly high, lthough vrile Mg 2+ onentrtion in intestinl fluid t 36 h (Fig. 7B) ould reflet greter drinking rte in longer-term strvtion i.e. intestinl fluid sorption with Mg 2+ exlusion, s disussed susequently. To our knowledge, drinking hs never een mesured in elsmornhs during or soon fter feeding. However, the fts tht the perentge of wter of stomh-1 ontents t 6 h fter the mel (Tle 2) ws virtully identil to tht of the food (Tle 3), nd tht the N + nd, to lesser extent, Mg 2+ onentrtions in the fluid phse oth deresed t this time through 36 h (Fig. 6A,C) suggest tht drinking remined low following feeding. Osmollity ppered to e sustined y the ddition of ions, ure nd unmesured sustnes to the gstrointestinl fluids. Although some of these unmesured sustnes were undoutedly the orgni nions normlly present in the prey, dditionl unmesured osmolytes my hve een reted y the susequent digestion of proteins to polypeptides nd mino ids, nd of triglyerides to free ftty ids, et. The degree to whih they ontriuted to hyme osmollity would depend on the lne etween the rte t whih they were reted y digestion versus the rte t whih they were removed y sorption. By omprison of onentrtions of mesured osmolytes, the ure must hve originted from the dogfish systemi fluids rther thn the food (Fig. 5B); indeed, MIntosh first doumented ure seretion in the stomhs of two ry speies (MIntosh, 1936). However, N + (Fig. 6A) nd Cl (Fig. 6B) must hve originted from the systemi fluids nd/or imied sewter rther thn the food, wheres K + (Fig. 6C) nd C 2+ (Fig. 7A) lerly me from the food. Mg 2+ proly originted minly from sewter (Fig. 7B). Fig. 9 ompres totl mesured osmolyte onentrtions with mesured osmollity in the fluid phses of the gstrointestinl trt nd the lood plsm. No orretion hs een mde for osmoti tivity oeffiients. These oeffiients for inorgni ions nd ure re in the rnge of.9.96 in dogfish plsm (Roertson, 1975; Roertson, 1989), ut re unknown for inorgni nd orgni osmolytes in dogfish hyme. It is likely tht there is some degree of overestimtion in the summted osmolyte onentrtions, nd therefore underestimtion of unmesured osmolytes (minly orgni osmolytes) in this nlysis, ut it serves to illustrte importnt trends. In strved dogfish, mesured osmolytes stisftorily ounted for totl osmollity in ll omprtments exept the intestinl fluid (Fig. 9A). In fed nimls (2 h), there were disrepnies of 23 mosm kg 1 in ll three gstrointestinl omprtments (Fig. 9B), proly euse of orgni osmolytes from the digested food, with perhps smll ontriutions from ile ids (Boyer et l., 1976) nd SO 4 (Tylor nd Grosell, 26; Anderson et l., 26). In the fluids of stomh-1 nd stomh-2 in oth strved nd fed dogfish, the lrgest mesured omponent ws Cl, nd the other mjor omponents were N + nd ure. The ft tht Cl onentrtion ws well-mintined (Fig. 6B) reltive to N + (Fig. 6A), ure (Fig. 5B) nd Mg 2+ (Fig. 7B) in the fluids of stomh-1 nd stomh-2 fter feeding ws inditive of HCl seretion y the gstri muos. When hyme moved from stomh-2 into the intestine, C 2+ nd Mg 2+ levels oth inresed, despite strong onentrtion grdients fvouring their uptke into the plsm fter feeding (Fig. 7A,B). This suggests tht these potentilly toxi divlents were poorly tken up or perhps even sereted into the lumen in this prt of the trt. In quntittive terms, muh more importnt ws two- to threefold inrese in its ure onentrtion, whih ws prtiulrly mrked in the fed