Comparative Biochemistry and Physiology, Part A

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1 Comprtive iochemistry nd Physiology, Prt 162 (2012) Contents lists ville t SciVerse ScienceDirect Comprtive iochemistry nd Physiology, Prt journl homepge: rnchil nd extr-rnchil mmoni excretion in goldfish (Crssius urtus) following thermlly induced gill remodeling mnd. Smith, lex M. Zimmer, Chris M. Wood Deprtment of iology, McMster University, Hmilton, Ontrio, Cnd L8S 4K1 rticle info strct rticle history: Received 5 Octoer 2011 Received in revised form 17 Ferury 2012 ccepted 17 Ferury 2012 ville online 23 Ferury 2012 Keywords: mmoni excretion Gill remodeling Goldfish High externl mmoni (HE) Interlmellr cell mss (ILCM) Under cold cclimted conditions, goldfish (Crssius urtus) express n interlmellr cell mss (ILCM) which limits diffusive ion loss ut my lso impede rnchil mmoni excretion (J mm ). In the present study, goldfish were sujected to 2-week 5 or 25 C cclimtion in order to modulte the degree of ILCM gill coverge nd determine potentil effects on J mm. 25 C-fish displyed gill coverge which ws significntly lower thn the 5 C-fish, though the ILCM ws not completely sent in these fish. 5 C-fish demonstrted J mm vlues pproximtely 60% lower thn those of 25 C-fish. The mgnitude of nterior (rnchil) J mm strongly correlted with gill coverge (r 2 =0.83), suggesting tht the ILCM my impede rnchil J mm. Divided chmer experiments demonstrted tht reltive to the 25 C-fish, 5 C-fish relied more upon posterior routes of excretion. In response to high externl mmoni (HE; 1.5 mm NH 4 HCO 3 ) exposures, 25 C-fish displyed mmoni uptke while 5 C-fish mintined excretion ginst HE, suggesting tht the ILCM my ct s rrier preventing mmoni uptke. In summry, the ILCM ppers to impede rnchil J mm, such tht 5 C-rely more on extr-rnchil routes of excretion. We hypothesize tht gill remodeling in these fish my e intimtely tied to physiologicl djustments on the whole-ody scle Elsevier Inc. ll rights reserved. 1. Introduction The fish gill performs wide vriety of homeosttic control functions including ion regultion, osmoregultion, oxygen uptke, cron dioxide removl, nitrogen excretion, nd cid se regultion (Evns et l., 2005). Much ttention hs een devoted to nitrogenous wste removl, with focus on mmoni s the mjor end product of protein degrdtion. In mmoniotelic fishes, mmoni is pssed through the gill lipid ilyer nd into the externl environment down prtil pressure grdient vi fcilitted diffusion through Rhesus (Rh) glycoproteins (see Weihruch et l., 2009; Wright nd Wood, 2009 for reviews). The excretion or ccumultion of ure, less toxic nitrogenous wste product, is lso route for nitrogenous wste removl y freshwter fish wherey mmoni is converted into ure within the liver s mens to void the potentilly neurotoxic effects of ccumulted plsm mmoni (nderson, 2001; Ip et l., 2001). This is prticulrly pprent in zerfish (Dnio rerio) exposed to high externl mmoni (HE) where ure excretion (J ure ) ws seen to significntly increse following mrked decrese in mmoni excretion (J mm )(run et l., 2009) nd in mngrove revitions: HE, high externl mmoni; ILCM, interlmellr cell mss; J mm, mmoni excretion; J ure, ure excretion; T mm, totl mmoni. Corresponding uthor t: Deprtment of iology, McMster University, Hmilton, Ontrio, Cnd L8S 4K1. Tel.: x23237; fx: E-mil ddresses: smith_mnd_@hotmil.c (.. Smith), zimme@mcmster.c (.M. Zimmer), woodcm@mcmster.c (C.M. Wood). killifish (Rivulus mrmortus) where tissue ure levels incresed during ir exposure, lso likely in response to decresed J mm (Frick nd Wright, 2002). Moreover, numer of species which express fully functionl ornithine-ure cycle (OUC) often rely on ure excretion when mmoni excretion is inhiited (Mgdi tilpi, lcolpi grhm, Rndll et l., 1989; gulf todfish, Ospnus et, Sh nd Rth, 1990; wlking ctfish, Clris trchus, Wlsh et l., 1990; the Indin ir-rething teleost Heteropneustes fossilis, Sh et l., 2002). Under norml circumstnces, however, ure excretion ccounts for only 5 20% of nitrogenous wste removl in typicl freshwter fish (nderson, 2001). In the fish gill, high surfce re nd short diffusion distnces re eneficil for rpid nd efficient gs exchnge. However, s these nimls re hyper-osmotic compred to their environment, wter influx vi osmosis coupled to diffusive ion loss is prticulr prolem nd must e corrected y comintion of sustntil urine production, efficient renl tuule resorption, nd ctive ion uptke cross the gill. To void these energeticlly costly processes, some fish species hve developed the ility to remodel their gill structure in n ttempt to reduce diffusive ion loss cross the gills. (Sollid et l., 2003, 2005; Sollid nd Nilsson, 2006). Under normoxic conditions, the crucin crp (Crssius crssius) mintins cell mss enveloping the gill lmelle, deemed the interlmellr cell mss (ILCM), thought to prevent diffusive ion loss to the wter (Sollid et l., 2003; Sollid nd Nilsson, 2006). In response to hypoxi, fish grdully lose the ILCM through comintion of lowered rte of mitosis nd heightened rte of poptosis (Sollid nd Nilsson, 2006), therey incresing /$ see front mtter 2012 Elsevier Inc. ll rights reserved. doi: /j.cp

2 186.. Smith et l. / Comprtive iochemistry nd Physiology, Prt 162 (2012) surfce re y up to 7.5-fold nd effectively incresing the re ville for gs exchnge (Sollid et l., 2003). In ddition, temperture chnges hve lso een shown to elicit similr effects (Sollid et l., 2005; Rissnen et l., 2006). s fish re ectothermic, n increse in temperture increses metolic rte which, in turn, cretes greter demnd for O 2 consumption. t high tempertures, cclimted crucin crp (25 C) lose the ILCM, while 5-dy cclimtion t low temperture (10 15 C) promotes the regenertion of the cell mss (Sollid et l., 2005). Similrly, the goldfish (Crssius urtus) lso demonstrtes temperture-dependent ILCM coverge (Sollid et l., 2005; Mitrovic nd Perry, 2009; Perry et l., 2010). Though reduction of gill surfce re in colder tempertures, where oxygen demnd is lower, is eneficil in preventing diffusive ion loss, this process my consequently result in reduction of rnchil mmoni excretion if the ILCM cts s rrier towrds mmoni movement. Perry et l. (2010) demonstrted tht goldfish cclimted to high temperture (25 C) were le to cler n intrperitonelly injected mmoni lod (either NH 4 HCO 3 or NH 4 Cl) t greter rte thn cold-cclimted (7 C) fish, suggesting tht the ILCM my in fct provide physicl rrier to rnchil mmoni excretion. J mm under norml conditions, however, ws not significntly ffected y temperture cclimtion (Perry et l., 2010). This result is curious s fish t higher temperture would e expected to hve n incresed metolic rte nd, hence, incresed J mm (see Wood, 2001). One potentilly importnt fctor tht ws not tken into ccount ws the possile role of shunting mmoni excretion to extr-rnchil routes of excretion. It is generlly well-ccepted tht the gills ccount for the mjority of mmoni excretion nd tht posterior routes (i.e., gstrointestinl, renl, nd/or cutneous routes) ccount for significntly smller proportion under norml conditions (Smith, 1929). If the ILCM impedes the rnchil movement of mmoni, fish my compenste with n increse in extr-rnchil J mm ; this hs een seen in response to certin environmentl conditions in other species (see Wood, 1993). Indeed, the skin my ply such role during mmoni chllenges s indicted y n increse of the mrn expression of Rh proteins in the skin of some species under mmoni-loded conditions (Hung et l., 2007; Nwt et l., 2007; Nwt nd Wood, 2009). The gol of this study ws to first investigte the potentil of 5 C-induced ILCM cting s rrier to mmoni movement in goldfish. We hypothesized tht 5 C-fish, in comprison to 25 C-fish, would disply reduction in J mm under norml conditions, even though Perry et l. (2010) did not show the sme in their fish which were much lrger in size. We further hypothesized tht these fish would utilize two physiologicl mechnisms to del with this ILCM-induced reduction in rnchil J mm : redistriution of J mm to posterior routes of excretion nd/or shift to rely more upon ure-n production nd excretion to void mmoni toxicity. lterntively, we hypothesized tht ILCM coverge my result in n ccumultion of plsm mmoni to which goldfish my e prticulrly resistnt s studies hve demonstrted these fish tolerting totl plsm mmoni levels up to 14 mmol/l (Sinh et l., 2012). Finlly, we predicted tht during high environmentl mmoni exposure, 25 C-fish, with lesser degree of ILCM coverge, would show greter mmoni uptke thn 5 C-fish, further suggesting tht the ILCM cts s rrier towrds mmoni movement. 2. Mterils nd methods 2.1. nimls Goldfish (Crssius urtus) (mss=4.6±1.3 g) were purchsed from ig l's Pet Store, Hmilton, Ontrio, Cnd. Fish were held in plstic tnks contining erted, dechlorinted Hmilton tp wter (modertely hrd: [N + ]=0.6 mequiv/l, [Cl ]=0.8 mequiv/l, [C 2+ =0.8 equiv/l, [Mg 2+ =0.3 mequiv/l, [K + ] =5 mequiv/l; titrtion lklinity 2.1 mequiv/l; ph ~8.0; hrdness ~140 mg/l s CCO3 equivlents) t 5 nd 25 C. Fish were cclimted to these two tempertures for 2 weeks prior to ny experimenttion. Fish were fed 1% tnk ody mss with ig l's rnd stple fish flke. 25 C-fish were fed dily, nd 5 C-fish were fed once every 2 dys s their nutritionl demnd ws noticely lower t this temperture. ll fish were fsted for 48 h prior to experimenttion Whole ody mmoni/ure flux (J mm /J ure ) Fish from ech cclimtion temperture were chosen rndomly nd plced into continers contining 350 ml erted dechlorinted, Hmilton tpwter thermosttted to the given cclimtion temperture. oth groups were plced in the continers for 2 h, nd 3-mL wter smples were drwn from ech continer every 0.5 h for mmoni nd ure-n nlyses. ph ws mesured following smpling times, nd ws mintined t 7.6 y the ddition of 0.1 M KOH or HCl Divided chmer pprtus Fish were fsted for 48 h prior to experimenttion. Rndomly selected fish were initilly nesthetized using 125 ppm of 1:10 clove oil in ethnol solution. Ltex dms were plced directly ehind the opercul, seprting the gills nd hed from the rest of the ody. The dm ws then secured into plce using elstic nds over Flcon tue filled with 50 ml dechlorinted tp wter of the respective cclimtion temperture. This pprtus ws then plced within the continers descried ove. dilute clove oil nesthetic concentrtion (15 ppm) ws present in the wter in these experiments to mildly sedte the fish in order to prevent escpe from the ltex dm. The effects of this nesthetic were ssessed in seprte series of experiments (see elow). Continers were plced in wter th t the given cclimtion temperture. Smll mgnetic stir rs were plced within the weighted Flcon tue to fcilitte full mixing of interior wters. Five microliters of 0.1 μci/μl 22 N ws injected into the wter within the Flcon tue, nd 200 μl wter smple ws withdrwn from the externl comprtment every 0.5 h to ssess the integrity of the sel creted y the ltex dm. mximum of 10% isotope loss from the Flcon tue to the exterior comprtment, s mesured y gmm counting (Perkin Elmer Wizrd uto Gmm Counter), ws ccepted s successful dm. Wter smples (3 ml) were withdrwn from the Flcon tue nd exterior chmer every 30 min nd ph ws mintined in oth chmers in the sme mnner s descried ove. To ssess the potentil effects of the 15 ppm clove oil nesthetic on mmoni nd ure-n excretion, whole-ody nesthetic controls were performed for ech cclimtion temperture. These controls consisted of nimls tht were initilly nesthetized in 125 ppm clove oil solution nd then plced into individul continers descried erlier which contined 15 ppm nesthetic solution. The dm ws not used; ll other experimentl procedures were the sme s for the whole-ody excretion testing High externl mmoni (HE) mmoni flux (J mm ) Rndomly selected fish were plced within continers of 350 ml erted 1.5 mm NH 4 HCO 3 in dechlorinted tp wter neutrlized to ph 7.6 with 0.1 M HCl. ll solutions were prepred t respective cclimtion temperture, nd chmers were housed within wter ths of the given cclimtion temperture. oth groups were plced in the continers for 2 h nd 1.5-mL wter smples withdrwn from ech continer every 0.5 h for mmoni nlysis. ph ws mintined using methods descried ove.

3 .. Smith et l. / Comprtive iochemistry nd Physiology, Prt 162 (2012) Coverge Index 1.2 C lood testing t the end of every experiment, lood smples were tken y lind cudl puncture from experimentl fish using lithiumheprinized Hmilton syringe fter scrificing the fish with neutrlized 0.1 g/l MS-222 solution. It ws not possile to otin smples from every fish. Smples were then centrifuged nd the decnted plsm ws stored t 80 C Dissections Fig. 1. Coverge indices following 2 week cclimtion period in 25 C nd 5 C-fish (see text for scoring system). : Whole-ody control series (n=8). : Whole-ody nesthetic series (n=5 8). C: Divided chmer series (n=6 8). Mens shring the sme letter re not significntly different within temperture cclimtion (25 C, upper cse letters; 5 C, lower cse letters). sterisks represent significnt differences etween 25 C nd 5 C-fish within ech experimentl series. Following lood smpling, entire gill skets were removed nd plced on ice. Dissected gill rches were viewed under dissecting microscope to determine the degree of coverge of the secondry lmelle y the ILCM. totl of 40 rndomly selected filments were counted on four gill rches of fish, resulting in 160 filments counted per fish t oth cclimtion tempertures. The degree of coverge ws scored under the following vlues: (0) no coverge (ll lmelle of the exmined filment re exposed); (1) prtil coverge (the lmelle of the exmined filment re only prtilly covered); nd (2) full coverge (none of the lmelle of the exmined filment re exposed). These scores were verged out of the numer of filments counted to ttin coverge index Environmentl scnning electron microscopy In order to vlidte the light microscopy method of ssessing gill coverge descried ove, gill coverge in seprte set of fish ws viewed using environmentl scnning electron microscopy (ESEM). seprte set of fish ws cclimted to 25 nd 5 C using the sme protocol descried ove nd fish were treted in n identicl mnner throughout the cclimtion period. Following 2 weeks of cclimtion, three fish from ech temperture cclimtion were chosen rndomly, scrificed using neutrlized MS-222, nd two rches (one from ech side) were removed nd plced in Krnovsky's fixtive (2% glutrldehyde, 1% prformldehyde) overnight. Gill rches were then dehydrted in scending concentrtions of ethnol strting from 30% nd ending with 100% efore eing viewed using n environmentl scnning electron microscope (Philips ElectroScn 2020 ESEM, ElectroScn Corportion, Wilmington, M, US). Photos of filments per fish were cptured nd were scored lindly using the sme scoring system descried ove nlyticl techniques Wter levels of mmoni were mesured using the slicyltehypochlorite colorimetric ssy (Verdouw et l., 1978). mmoni flux rtes (μmol/g/h) were clculted using the following eqution: J mm ¼ ½mmŠ i ½mm Š f ðv=t MÞ where [mm] i nd [mm] f re initil nd finl mmoni concentrtions (μmol/l) etween 0.5-h time (t) intervls, V is volume (L) of wter within individul continers, nd M is mss (g). Plsm totl mmoni concentrtions (T mm ) were tested using n enzymtic ssy kit (Richem, Mumi, Indi). Plsm clernce of mmoni (ml/g/h) ws clculted using the following eqution: Plsm clernce rte ¼ J mm =plsm T mm where J mm is mmoni excretion (μmol/g/h) over the 2-hour flux nd plsm T mm is the terminl plsm concentrtion of mmoni (μmol/l). Ure-N levels were mesured using colorimetric ssy (Rhmtullh nd oyd, 1980) modified for low ure concentrtions. Fig. 2. Two exmple ESEM photos from 25 C-fish demonstrting fully covered filments (single sterisks), prtilly covered filments (doule sterisks), nd fully uncovered filments (triple sterisks). Twenty-three photos were scored for totl of filments per fish.

4 188.. Smith et l. / Comprtive iochemistry nd Physiology, Prt 162 (2012) Ure-N flux (μmol N/g/h) ws determined using the following eqution: J ure ¼ ½Ure NŠ i ½Ure N Š f ðv=t MÞ where [Ure-N] I nd [Ure-N] f re initil nd finl ure-n concentrtions (μmol N/L) etween hlf hour time (t) intervls, V is volume (L) of wter within individul continers, nd M is mss (g) Sttistics Dt re expressed s mens±1 SEM (n=numer of fish tested) nd significnce ws ccepted t P5. Over every 2-h flux conducted in this study, one-wy NOV test ws used (n NOV on rnks in the cse of filed equl vrince test) to determine if there were significnt differences etween ech 0.5-h time intervl within flux mesurements. In fct, no differences occurred over ny flux experiment for mmoni or ure excretion nd s such dt reported re verge excretion rtes over ech 2-h flux. Significnce etween cclimtion groups within given tretment ws determined y Student's t-test (Mnn Whitney rnk sum test in the cse of filed equl vrince or normlity). Significnce within temperture cclimtions etween individul experimentl series, where more thn two tretments re compred, ws determined using one-wy NOV with Holm Sidk post-hoc test. Significnce within temperture cclimtions etween individul experimentl series, where only two tretments re compred, ws determined y Student's t-test (Mnn Whitney rnk sum test in the cse of filed equl vrince or normlity). ll vlues represented s percentges were normlized using n rcsine trnsformtion prior to performing sttisticl nlyses. 3. Results 3.1. Effects of the ILCM on mmoni nd ure hndling Degree of coverge of the gills y the ILCM, s ssessed y light microscopy, ws significntly greter in the 5 C-fish of ll 3 experimentl series (Fig. 1). Mesurements of coverge index using ESEM (see Fig. 2 for exmple photos) confirmed this difference. The solute vlues of coverge index y ESEM, compred to light microscopy, were significntly lower in 5 C-fish nd slightly lower in 25 C-fish (P=56) (Tle 1). The reltive difference in coverge etween ech cclimtion temperture, however, ws similr using light microscopy (1.3-fold difference) nd ESEM (1.5-fold difference). No significnt differences occurred etween experimentl series within given cclimtion temperture. Men J mm of 25 C-fish in the whole-ody control series ws significntly greter thn tht of the 5 C-fish (Fig. 3). Fsting 25 Cfish for 5 dys did not result in significntly different men J mm ( 1.17±0.12 μmol/g/h; dt not shown). J mm ws lso greter in the 25 C-fish of the totl nd divided chmer series thn the 5 C-fishofthesmeseries(Fig. 3 nd C). 25 C-fish did not show Tle 1 Men coverge indices for control, non-nesthetized fish s scored using light microscopy or environmentl scnning electron microscopy. Light microscopy Environmentl scnning electron microscopy Temperture ( C) ± ± ± ±.068, Crosses represent significnt differences etween scoring methods t given cclimtion temperture. sterisks represent significnt differences etween temperture cclimtions using the sme scoring method. ll comprisons were mde using unpired Student's t-tests (or Mnn Whitney rnk sum test in the cse of filed equl vrince or normlity). J mm (µmol/g/h) C Fig. 3. Men mmoni excretion (J mm ) over 2-h flux period in 25 C nd 5 C-fish. : Whole-ody control series (n=8). : Whole-ody nesthetic series (n=5 8). C: Totl (nterior nd posterior) divided chmer series (n=8 10). Mens shring the sme letter re not significntly different within temperture cclimtion (25 C, upper cse letters; 5 C, lower cse letters). sterisks represent significnt differences etween 25 C nd 5 C-fish within ech experimentl series. significnt differences in men J mm etween experimentl series; however, the J mm vlues of 5 C-fish were sustntilly lower in the whole-ody nesthetic nd divided chmer series, respectively, in comprison to the control series. J ure within the whole-ody control series ws lso significntly greter in the 25 C-fish thn in the 5 C-fish (Fig. 4) nd this sme oservtion ws mde within the divided chmer series (Fig. 4). There were no significnt differences in J ure within temperture cclimtion etween the two experimentl series. In the whole-ody control series, the % totl N-excretion s ure-n ws greter in 25 C-fish (29%) thn in 5 C-fish (13%) ut this difference ws not sttisticlly significnt (dt not shown; P=52). The sme result ws oserved in the totl divided chmer series (24% nd 12% in 25 C-fish nd 5 C-fish, respectively. P=0.31; dt not shown). Terminl plsm T mm ws not significntly different etween ny temperture cclimtion or experimentl series (Tle 2). In the whole-ody control series, plsm mmoni clernce rte ws out 2-fold higher in 25 C-fish thn in 5 C-fish, difference which ws not significnt (P=66) (Fig. 5). In contrst, plsm clernce rtes were 33-fold greter in 25 C-fish thn in the 5 Cfish in the whole-ody nesthetic series (Fig. 5). 25 C-fish lso displyed n pproximtely 10-fold greter plsm clernce rte thn J ure (µmol N/g/h) Fig. 4. Men ure-n excretion (J ure ) over 2-h flux period in 25 C nd 5 C-fish. : Whole-ody control series (n=8). : Totl (nterior nd posterior) divided chmer series (n=6 8). Mens shring the sme letter re not significntly different within temperture cclimtion (25 C, upper cse letters; 5 C, lower cse letters). sterisks represent significnt differences etween 25 C nd 5 C-fish within ech experimentl series.

5 .. Smith et l. / Comprtive iochemistry nd Physiology, Prt 162 (2012) Tle 2 Plsm totl mmoni (T mm ; μmol/l) in 25 C nd 5 C-fish in whole-ody control, nesthetic control, divided chmer, nd HE experimentl series. Experimentl series Whole-ody control Whole-ody nesthetic control Divided chmer HE Temperture ( C) ± ± ± ± ± ± ± ±285.0 No significnt differences existed etween ny of the non-he groups or etween whole-ody control nd HE series (n=6 8). 5 C-fish in the divided chmer series (Fig. 5C). Clernce rtes in 25 C-fish did not vry significntly cross experimentl series; however, t 5 C, clernce rtes in the whole-ody series did vry significntly from oth the nesthetic nd the totl divided chmer series (Fig. 5). When nterior J mm ws compred to coverge index in oth temperture groups within the totl divided chmer series, liner trend emerged (r 2 =0.83; P001) where fish with higher degree of coverge (5 C-fish) showed mrkedly lower J mm thn those with lower degree of coverge (25 C-fish) (Fig. 6). This liner trend ws not seen in plsm mmoni concentrtions s no difference in plsm T mm existed etween temperture cclimtion groups (Tle 2). Interestingly, plsm mmoni clernce rtes lso correlted with ILCM coverge (r 2 =0.92; P001; Fig. 6), though this reltionship did not fit the liner curve seen in Fig. 6. Insted, 5 C-fish ll displyed low clernce rtes, irrespective of the degree of ILCM coverge, wheres in the 25 C-fish, plsm clernce of mmoni correlted linerly with ILCM coverge (r 2 =0.64; P=109) (Fig. 6). J ure did not show correltion with coverge index (r 2 =0.17, P=793; dt not shown) The effect of the ILCM on mmoni excretion prtitioning etween temperture cclimtion groups, verging 95% nterior nd 5% posterior (dt not shown) Effect of the ILCM on mmoni excretion in response to HE J mm ws reversed in 25 C-fish exposed to HE (Fig. 9), representing mmoni uptke. 5 C-fish, however, mintined excretion during HE, though t pproximtely hlf the rte seen under control conditions (Fig. 9). Plsm T mm in HE exposed fish ws greter thn control vlues for oth 25 C nd 5 C-fish; however, this difference ws not significnt in either temperture group (P=72 nd P=81 for 25 C nd 5 C-fish, respectively) (Tle 2). When compred ginst degree of ILCM coverge, J mm decresed linerly with incresing coverge index (r 2 =0.525; P=37), where 25 C-fish, with the lest degree of coverge showed the highest uptke, nd 5 C-fish, with higher degrees of coverge, demonstrted either very smll mounts of uptke or excretion ginst the imposed T mm grdient (Fig. 10). 0.5 nterior J mm in 25 C-fish ws pproximtely 6-fold greter thn in 5 C-fish (Fig. 7). Posterior J mm ws lso greter in 25 C-fish though only y pproximtely 2-fold (Fig. 7). nterior J mm ws 11-fold nd 4-fold greter thn posterior J mm in 25 C-fish nd 5 C-fish, respectively (Fig. 7). When tken s percentge of totl J mm, nterior excretion ccounted for 90% in 25 C-fish with posterior excretion ccounting for the remining 10% (Fig. 8). 5 C-fish depended significntly more upon posterior routes of excretion, with 74% of J mm from nterior routes, nd 26% excretion from posterior routes (Fig. 8). nterior to posterior prtitioning of J ure ws not significntly different Plsm Clernce (ml/g/h) nterior J mm (µmol/g/h) Plsm Clernce (ml/g/h) C Fig. 5. Men plsm mmoni clernce rte (ml/g/h) over 2-h flux period in 25 C nd 5 C-fish. : Whole-ody control series (n=8). : Whole-ody nesthetic series (n=5 8). C: Totl (nterior nd posterior) divided chmer series (n=6 8). Mens shring the sme letter re not significntly different within temperture cclimtion (25 C, upper cse letters; 5 C, lower cse letters). sterisks represent significnt differences etween 25 C nd 5 C-fish within ech experimentl series Coverge Index Fig. 6. Reltionships of () nterior mmoni excretion (J mm )(r 2 =0.83; P001) vs. coverge index nd () totl (nterior nd posterior) plsm clernce rte (r 2 =0.92; P001) vs. coverge index in 25 C nd 5 C-fish within the divided chmer series (n=6 8).

6 190.. Smith et l. / Comprtive iochemistry nd Physiology, Prt 162 (2012) J mm (µmol/g/h) -0.8 J mm (µmol/g/h) Fig. 7. Prtitioning of mmoni excretion (J mm ) in 25 C nd 5 C-fish. : nterior J mm. : Posterior J mm. Mens shring the sme letter re not significntly different within temperture cclimtion (25 C, upper cse letters; 5 C, lower cse letters). sterisks represent significnt differences etween 25 C nd 5 C-fish within ech experimentl series (n=8 10). 4. Discussion 4.1. Overview In the current study, fish were nesthetized in order to ssess the reltive distriution etween nterior nd posterior J mm. Our control tests demonstrted tht nesthesi lone cn pprently reduce J mm, ut this oservtion ws only true of 5 C-fish (Fig. 3). The reson for this effect occurring only for the lower temperture cclimtion group is unknown. Wht is of significnce, however, is tht no differences occurred etween the nesthetic control group nd whole-ody divided chmer groups (Fig. 3). We interpret this result to men tht the nesthetic tretment effectively reduced ny stress which my hve een ssocited with this experimentl procedure. In the proceeding text, we rgue tht the difference in J mm etween cclimtion tempertures ppers to e relted to the degree of ILCM coverge, regrdless of ny confounding nesthetic or temperture effects. To ssess gill coverge, novel method of scoring gill coverge sed on oservtions mde using light microscopy ws used. In order to vlidte this technique, seprte group of fish ws cclimted to 25 C nd 5 C using the sme cclimtion protocol s ll other fish used in the study nd gill rches were exmined using ESEM (Fig. 2). Though solute vlues of coverge index were lower using ESEM thn using light microscopy, the reltive difference in coverge etween temperture cclimtion groups remined the Fig. 9. Men mmoni excretion (J mm ) over 2-h flux period in 25 C nd 5 C-fish. : Whole-ody control series (n=8). : Whole-ody HE series (n=6 8). Mens shring the sme letter re not significntly different within temperture cclimtion (25 C, upper cse letters; 5 C, lower cse letters). sterisks represent significnt differences etween 25 C nd 5 C-fish within ech experimentl series. sme. Thus, it ppers tht our method of ssessing gill coverge using light microscopy, though not s sensitive s ESEM, ppers to e vlid technique for determining reltive coverge indices etween 25 C-fish nd 5 C-fish. To our knowledge, this is the first time tht ESEM hs een used for this purpose. n dvntge of this new pproch, reltive to trditionl SEM, is its ese the gill tissue needs only simple fixtion prior to visuliztion. There is no need to sputter-cot the smples, so it is fster, nd there is less chnce of loss of the intr-lmellr cell mss during processing. Previous studies on the plstic remodeling of goldfish gills hve reported tht complete loss of the ILCM occurs t 25 C in response to hypoxi or n increse in temperture (Sollid et l., 2005; Perry et l., 2010), wheres we were only le to chieve prtil loss of ILCM coverge. However, it is importnt to note the differences in cclimtion regimes etween these studies nd the study presented here. Sollid et l. (2005) cclimted goldfish for n entire month t 25 C prior to experimenttion. Perry et l. (2010) cclimted their nimls to 18 C for t lest week, susequently rised the tnk temperture y 1 C per dy until 25 C ws reched, nd then kept the nimls t this temperture for further 2 weeks prior to experimenttion. In oth studies, the cclimtion regime resulted in complete loss of the ILCM t 25 C. In the present study, however, the ILCM ws not entirely shed during the 2-week 25 C cclimtion period within ny experimentl series (Fig. 1). This likely reflects the different % nterior Excretion % Posterior Excretion % Totl J mm J mm (µmol/g/h) Coverge Index Fig. 8. Reltive nterior nd posterior mmoni excretion (J mm ) in divided chmer series. : 25 C-fish (n=8). : 5 C-fish (n=6). Fig. 10. The reltionship of mmoni excretion (J mm )(r 2 =0.525; P=37) vs. coverge index in 25 C nd 5 C-fish exposed to HE (n=6 8).

7 .. Smith et l. / Comprtive iochemistry nd Physiology, Prt 162 (2012) protocols employed. Time-dependence of ILCM loss hs een demonstrted in the goldfish, where exposure to hypoxi t 7 C for 1 dy reduced the ILCM to pproximtely 69% of originl interlmellr re coverge, nd n dditionl 6 dys of hypoxi only reduced this vlue to 39% (Mitrovic et l., 2009). Thus, it is possile tht with different cclimtion protocol, totl ILCM loss could hve een chieved. Nevertheless, higher degree of coverge ws seen in 5 C-fish (Fig. 1), suggesting tht these fish my hve een suject to n impednce of rnchil mmoni excretion, if the ILCM cts s diffusive rrier for mmoni movement Effects of the ILCM on net mmoni nd ure excretion In ll experimentl series, J mm ws greter in 25 C-fish fish thn in 5 C-fish (Fig. 3). Perry et l. (2010), using similr temperture cclimtion (7 C nd 25 C), though considerly lrger fish (25 g), did not show differences in J mm etween cclimtion tempertures under control, unfed conditions. These differences my reflect llometric effects where the smller fish used in this study, hving greter surfce re to volume rtio, would e impcted y temperture chnges to greter degree thn lrger fish. This group lso demonstrted tht feeding history hd profound effect upon control J mm in these fish (Perry et l., 2010). Thus, it ws initilly hypothesized tht perhps the differences in J mm etween cclimtion tempertures in our study my hve een due to considerle difference in feeding rtes etween the two temperture groups, s the nutritionl demnd of 25 C-fish ws much greter thn tht of 5 C-fish (see Mterils nd methods). Fsting 25 C-fish for n dditionl 72 h (5 d totl), however, did not result in men J mm significntly different from tht of the 48 h-fsted control group, demonstrting tht differences in feeding history likely did not contriute to the oserved differences in J mm. The differences in J mm oserved etween temperture cclimtion groups my hve lso een induced y temperture itself, rther thn the ILCM. The connection etween temperture increses nd resulting increses in mmoni excretion hs een seen in vriety of fish species (goldfish: Metz, 1972; trout: Pyn nd Mtty, 1975; wlleye: Ci nd Summerfelt, 1992; sturgeon: Gershnovich nd Pototskij, 1995). This temperture-dependent increse in mmoni excretion my e t lest prtly relted to n increse in rnchil perfusion nd rnchil mmoni diffusion tht cn occur during periods of incresed temperture (Pyn nd Mtty, 1975). Metz (1972) lso determined tht J mm in non-fed fish, eing derived solely from metolic production, displyed Q 10 vlue of 4, suggesting high dependency upon temperture. The interprettion of this result, common to vriety of teleost species, is tht t higher tempertures, nonfed fish exhiit incresed eroic metolism fueled y the oxidtion of proteins (see Wood, 2001 for review). Nevertheless, Metz (1972) lso demonstrted tht the diffusive portion of mmoni excretion (tht ove metolic excretion), using NH 4 Cl plsm loding, hd Q 10 of 1.9. This lower Q 10 implies tht portion of the temperture sensitivity in goldfish, specificlly, is independent of metolic production nd occurs presumly s result of chnges in rnchil permeility. In the present study, strong liner correltion (r 2 =0.83; P001) of rnchil (nterior) J mm with the degree of ILCM coverge ws seen within the totl divided chmer control series (Fig. 6). Thus, it ppers tht the oserved differences in J mm etween temperture cclimtion groups re not likely due entirely to temperture-driven chnges in respirtion or metolism. This is consistent with the study y Perry et l. (2010) where clering of intrperitonel injections of NH 4 Cl or NH 4 HCO 3 ws greter in 25 C-cclimted thn in 7 C-cclimted goldfish. Moreover, in our 25 C-fish, plsm mmoni clernce rtes lso correlted with the degree of ILCM coverge, implying tht ILCM presence increses the time it would tke the fish to completely cler mmoni from given volume of plsm. This gin suggests tht the ILCM my somehow impede the effectiveness of the gills in eliminting mmoni from the ody. In 5 C-fish, however, clernce rtes were independent of ILCM coverge (Fig. 6). Perhps t this cclimtion temperture, clernce rtes re more dependent upon possile temperture-induced chnges in rnchil permeility nd diffusion, rther thn upon the degree of ILCM coverge. Plsm T mm vlues did not fit with our initil hypothesis of the ILCM cting s rrier to mmoni excretion (Tle 2). We then speculted tht perhps 5 C-fish were le to convert mmoni to ure in order to void toxicity s hs een demonstrted in zerfish exposed to HE (run et l., 2009). However, J ure ws lso much lower in 5 C-fish (Fig. 4) nd did not correlte with ILCM coverge. This difference ws likely due to the forementioned temperture-driven physiologicl effects on respirtion nd protein ctolism. Moreover, % totl N-excretion s ure-n ws not significntly different etween temperture cclimtion groups which my imply tht 5 C-fish do not selectively excrete one nitrogenous wste product or nother in response to incresed ILCM presence. Unfortuntely, in light of the smll volumes of lood plsm ville, it ws not possile to mesure plsm ure concentrtion. Therefore, the ccumultion of ure within the plsm or tissue to void toxicity my still e possile mechnism to void potentil mmoni toxicity The effect of the ILCM on mmoni prtitioning Homer Smith (1929), using crp nd goldfish, ws the first to demonstrte tht the mjority of mmoni excretion (94%) occurred rnchilly. This nterior posterior prtitioning is in greement with the 25 C-fish of this study (Figs. 7 nd 8). 5 C-fish, however, relied more on posterior routes of excretion on reltive sis (Fig. 8), suggesting tht ILCM presence, impeding rnchil excretion, forces these fish to shunt mmoni excretion to these extr-rnchil routes. In this study, we were unle to determine which posterior routes would e most utilized y these fish. Interestingly, recent studies on the cutneous expression of Rh genes in rinow trout (Oncorhynchus mykiss), in response to HE exposure, suggest tht the skin my serve s potentil extr-rnchil route of mmoni excretion (Nwt et l., 2007; Nwt nd Wood, 2009). Note tht this trend of 5 C-fish relying more upon posterior routes of excretion ws not oserved for ure excretion The effects of the ILCM on mmoni excretion in response to HE The reversl nd susequent re-estlishment of J mm is consistent response to HE exposure in mny freshwter fish (Wilson et l., 1994; Nwt et l., 2007; run et l., 2009; Perry et l., 2010; Zimmer et l., 2010). In this study, 25 C-fish exhiited the chrcteristic reversl of excretion (Fig. 9) over the 2-h flux period, in ccord with these previous studies. However, the ility of 5 C-fish to mintin excretion, leit to lesser degree thn under control conditions, ws notle (Fig. 9). This is consistent with the notion tht the ILCM my ct s diffusive rrier for mmoni, in this cse preventing in mmoni uptke in 5 C-fish. The reltively lower degree of ILCM coverge in 25 C-fish llowed for the uptke of mmoni during HE exposure. The notion of the ILCM preventing mmoni ccumultion during HE could not, however, e confirmed y plsm T mm vlues. oth cclimtion groups demonstrted tendency for incresed plsm T mm during HE exposure ut this ws not significnt (Tle 2). Note, however, tht since only very smll volumes of plsm were otined, we hd to dilute our smples sustntilly to e le to mesure T mm, so these dt should e interpreted with cution. Perhps with longer exposure, more pronounced differences in these vlues would hve een oserved, though 2 h ws sufficient to elevte plsm T mm in juvenile rinow trout exposed to the sme concentrtion of mmoni (Zimmer et l., 2010). Finlly, J mm in this

8 192.. Smith et l. / Comprtive iochemistry nd Physiology, Prt 162 (2012) series lso correlted with ILCM coverge (Fig. 10), though in the opposite direction thn under non-he conditions (Fig. 6), providing further evidence tht ILCM my impede mmoni movement nd tht, furthermore, this impednce ppers to e idirectionl. In conclusion, there is n pprent direct effect of the ILCM on rnchil (nterior) J mm. We hve demonstrted tht J mm in control nd HE conditions cn e correlted to the degree of ILCM coverge. This suggests tht the ILCM my, in fct, ct s rrier for mmoni trnsport nd tht fish my undergo physiologicl chnges in order to compenste for reduction in rnchil J mm. Though 5 C-fish in the present study did not demonstrte shift to rely more upon ure excretion to excrete nitrogenous wste, we cnnot rule out the potentil of these fish storing ure in the plsm or tissues to void mmoni toxicity. Furthermore, 5 C-fish demonstrted greter reltive proportion of posterior J mm thn did 25 C-fish. Thus, our two initil hypotheses (reduced whole-ody J mm nd incresed relince on extrrnchil excretion t 5 C) re supported. The present reserch sheds light on the physiologicl strtegies of this unique fish species in response to rnchil compenstions which re mde to djust oxygen uptke nd ion loss rtes. We suggest need for future studies to investigte the effects of hypoxi-induced loss of the ILCM on J mm nd if this would lso led to chnges in nitrogen hndling (i.e., ure excretion/storge) or chnges in extr-rnchil J mm. Finlly, in light of recent reserch, it will e interesting to determine whether or not these fish express Rh proteins in the skin nd if cutneous surfces re utilized for mmoni excretion during periods of reduced rnchil excretion. cknowledgements Supported y n NSERC Discovery grnt to CMW. Z is supported y n Ontrio Grdute Scholrship nd CMW y the Cnd Reserch Chir progrm. References nderson, P.M., Ure nd glutmine synthesis: environmentl influences on nitrogen excretion. Fish Physiol. 20, run, M.H., Steele, S.L., Perry, S.F., The responses of zerfish (Dnio rerio) to high externl mmoni nd ure trnsporter inhiition: nitrogen excretion nd expression of rhesus glycoproteins nd ure trnsport proteins. J. Exp. iol. 212, Ci, Y., Summerfelt, R.C., Effects of temperture nd size on oxygen consumption nd mmoni excretion y wlleye. quculture 104, Evns, D.H., Piermrini, P.M., Choe, K.P., The multifunctionl fish gill: dominnt site of gs exchnge, osmoregultion, cid se regultion, nd excretion of nitrogenous wste. Physiol. Rev. 85, Frick, N.T., Wright, P.., Nitrogen metolism nd excretion in the mngrove killifish Rivulus mrmortus II. Significnt mmoni voltiliztion in teleost during ir-exposure. J. Exp. iol. 205, Gershnovich,.D., Pototskij, I.V., The peculirities of non-fecl nitrogen excretion in sturgeons (Pisces: cipenseride) 2. Effects of wter temperture, slinity nd ph. Comp. iochem. Physiol. 111, Hung, C.Y.C., Tsui, K.N.T., Wilson, J.M., Nwt, C.M., Wood, C.M., Wright, P.., Rhesus glycoprotein gene expression in the mngrove killifish Kryptoleis mrmortus exposed to elevted environmentl mmoni levels nd ir. J. Exp. iol. 210, Ip, Y.K., Chew, S.F., Rndll, D.J., mmoni toxicity, tolernce nd excretion. Fish Physiol. 20, Metz, J., rnchil sodium exchnge nd mmoni excretion in the goldfish Crssius urtus. Effects of mmoni loding nd temperture chnges. J. Exp. iol. 56, Mitrovic, D., Perry, S.F., The effects of thermlly induced gill remodeling on ionocyte distriution nd rnchil chloride fluxes in goldfish (Crssius urtus). J. Exp. iol. 212, Mitrovic, D., Dymovsk,., Nilsson, G., Perry, S., Physiologicl consequences of gill remodeling in goldfish (Crssius urtus) during exposure to long-term hypoxi. m. J. Physiol. 297, R224 R234. Nwt, C.M., Wood, C.M., mrn expression nlysis of the physiologicl responses to mmoni infusion in rinow trout. J. Comp. Physiol. 179, Nwt, M.C., Hung, C.C.Y., Tsui, T.K.N., Wilson, J.M., Wright, P.., Wood, C.M., mmoni excretion in rinow trout (Oncorhynchus mykiss): evidence for Rh glycoprotein nd H + -TPse involvement. Physiol. Genomics 31, Pyn, P., Mtty, J., The chrcteristics of mmoni excretion y perfused isolted hed of trout (Slmo girdneri): Effect of temperture nd CO 2 -free ringer. J. Comp. Physiol. 96, Perry, S.F., Schwiger, T., Kumi, Y., Tznev, V., run, M.H., The consequences of reversile gill remodeling on mmoni excretion in goldfish (Crssius urtus). J. Exp. iol. 213, Rhmtullh, M., oyd, T.R.C., Improvements in the determintion of ure using dicetyl monoxime; methods with nd without deproteinistion. Clin. Chem. ct 107, 3 9. Rndll, D.J., Wood, C.M., Perry, S.F., ergmn, H.L., Mloiy, G.M.O., Mommsen, T.P., Wright, P.., Ure excretion s strtegy for survivl in fish living in very lkline environment. 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(Ed.), The physiology of fishes, Vol. 1. CRC Press, oc Rton, pp Wood, C.M., Influence of feeding, exercise, nd temperture on nitrogen metolism nd excretion. Fish Physiol. 20, Wright, P.., Wood, C.M., new prdigm for mmoni excretion in qutic nimls: the role of Rhesus (Rh) glycoproteins. J. Exp. iol. 212, Zimmer,.M., Nwt, C.M., Wood, C.M., Physiologicl nd moleculr nlysis of the interctive effects of feeding nd high externl mmoni on rnchil mmoni excretion nd N + uptke in freshwter rinow trout. J. Comp. Physiol. 180,