Accepted 5 March 2007

1935 The Journl of Experimentl Biology 21, 1935-1943 Pulished y The Compny of Biologists 27 doi:1.1242/je.5371 Responses to hypoxi nd recovery: repyment of oxygen det is not ssocited with compenstory protein synthesis in the Amzonin cichlid, Astronotus ocelltus J. M. Lewis 1, *, I. Cost 1, A. L. Vl 2, V. M. F. Almeid-Vl 2, A. K. Gmperl 1 nd W. R. Driedzic 1 1 Ocen Sciences Centre, Memoril University of Newfoundlnd, St. John s, NL, A1C 5S7, Cnd nd 2 Lortory of Ecophysiology nd Moleculr Evolution, Instituto Ncionl de Pesquiss d Amzôni, Almed Cosme Ferreir, 1756, 69.83-, Mnus, Amzons, Brzil *Author for correspondence (e-mil: jmlewis@mun.c) Accepted 5 Mrch 27 Oxygen consumption, s n indictor of routine metolic rte (RoMR), nd tissue-specific chnges in protein synthesis, s mesured y 3 H-lelled phenyllnine incorportion rtes, were determined in Astronotus ocelltus to investigte the cellulr mechnisms ehind hypoxi-induced metolic depression nd recovery. RoMR ws significntly depressed, y pproximtely 5%, when dissolved oxygen levels reched 1% sturtion (.67±.1 mg l 1 t 28±1 C). This depression in RoMR ws ccompnied y 5 6% decrese in liver, hert nd gill protein synthesis, ut only 3% decrese in rin protein synthesis. During recovery from hypoxi, n overshoot in RoMR to 27% of Summry the normoxic rte ws oserved, indicting the ccumultion of n oxygen det during hypoxi. This conclusion ws consistent with significnt increse in plsm lctte levels during the hypoxic exposure, nd the fct tht lctte levels rpidly returned to pre-hypoxic levels. In contrst, hyperctivtion of protein synthesis did not occur, suggesting the overshoot in oxygen consumption during recovery is ttriuted to n increse in cellulr processes other thn protein synthesis. Key words: hypoxi, recovery, routine metolic rte, protein synthesis, lctte production, Astronotus ocelltus. Introduction Dissolved oxygen is one of the most importnt environmentl fctors ffecting the survivl of nimls tht rely on qutic respirtion, nd nimls tht re exposed to periods of hypoxi show dpttions t the ehviourl, morphologicl nd/or physiologicl level. At the physiologicl level, nimls commonly resort to one of two strtegies: (1) mintennce of low levels of ctivity, which is fuelled y neroic metolism or (2) depression of metolism, ccomplished y decresing ATP producing nd consuming processes (Boutilier, 21; Lutz nd Nilsson, 1997). The ltter pproch llows survivl for longer periods of hypoxi/noxi ecuse of the conservtion of energy nd the limited ccumultion of toxic end products, such s lctte. However, there is trde-off to this pproch s deep metolic depression impirs the niml s ility to respond to externl stimuli nd leves the niml vulnerle to predtors. The mjority of successful oxyconformers re ectotherms tht survive short outs of hypoxi t wrm tempertures, ut require sesonl or ehviourlly regulted decreses in environmentl/ody temperture to survive extended noxi. Such nimls re the crucin crp (Crssius crssius), goldfish (Crssius urtus), common frog (Rn temporri) nd two species of freshwter turtle (Chrysemys pict ellii nd Trchemys script elegns) (Boutilier, 21). In these nimls depression of metolic rte y 7 95% occurs during hypoxi/noxi, sed on oxygen consumption rtes or clorimetry (Vn Wversveld et l., 1989; Jckson, 1968). This depression t the whole niml level is ccompnied y tissuespecific decreses in protein synthesis of 5% in crucin crp (Smith et l., 1996) nd 7 to >9% in freshwter turtles (Lnd et l., 1993; Biley nd Driedzic, 1996; Frser et l., 21). Protein synthesis is one of the mjor energy consuming processes, ccounting for 18 26% of cellulr energy expenditure (Hwkins, 1991). As such, the downregultion of protein turnover is one of the mjor contriuting fctors to the depression in ATP turnover nd metolic depression t the whole niml level (Guppy et l., 1994). Animls tht re exposed to prolonged period of oxygen deprivtion ccumulte n oxygen det tht is repid during recovery y sustntil increse in oxygen consumption. This oxygen det hs een shown to occur t oth the whole niml nd tissue levels in goldfish fter extended hypoxi exposure (Vn den Thillrt nd Vereek, 1991; Johnsson et

1936 J. M. Lewis nd others l., 1995). Johnsson et l. (Johnsson et l., 1995) predicted tht sustntil increse in protein turnover would ccompny the repyment of the oxygen det, ut consistent pttern in protein synthesis during recovery from hypoxi hs not een found. For exmple, n in vitro study on turtle heptocytes exposed to 12 h of noxi showed significnt overshoot in protein synthesis rtes to 16% of normoxic levels during recovery (Lnd et l., 1993). However, in vivo studies on crucin crp nd freshwter turtle species did not show hyperctivtion of protein synthesis during post-noxic recovery (Smith et l., 1996; Frser et l., 21). The Amzonin cichlid, the oscr or crá-çu Astronotus ocelltus, is n idel species to study hypoxi-induced metolic depression without the confounding vrile of decresed temperture. During periods of high wter, Amzon várzes ecome flooded nd the surfces of the lkes ecome densely covered with floting mrcrophytes (Vl nd Almeid-Vl, 1995). The dense surfce vegettion cuses extreme diurnl vrition in dissolved oxygen levels, with supersturtion occurring t middy when photosynthesis is t its mximum nd levels dropping close to zero during the night (McCormck et l., 23). A. ocelltus undergoes significnt decrese in routine metolic rte (RoMR; ~3%) when oxygen levels in the wter rech 2% sturtion nd only reverts to neroic metolism once oxygen levels drop elow 6% sturtion consumption, which is ccompnied y decrese in RoMR of pproximtely 6% (Muusze et l., 1998). These results suggest tht A. ocelltus, like the crucin crp nd freshwter turtles, is le to mintin eroic metolism in situtions of oxygen deprivtion y decresing the rte of ATP turnover until ner noxic conditions re reched. Until recently, little ws known out the cellulr mechnisms ehind the hypoxi-induced metolic depression in A. ocelltus nd of its response during recovery from severe hypoxi eyond the ehviourl nd physiologicl responses of A. ocelltus to hypoxi (Muusze et l., 1998; Slomn et l., 26). Recent studies hve egun investigtion into the vrious ATP-consuming processes tht contriute to the whole niml metolic depression. These studies hve shown significnt reduction in N +,K + -ATPse in gill nd kidney during hypoxi exposure (Richrds et l., 27) which is ccompnied y reduction of ion exchnge t the gills nd n overll reduction in metolic nitrogenous wste production (ure nd mmoni) (Wood et l., 27). The decrese in these ATP consuming processes re not ccompnied y chnges in concentrtion of ATP (Richrds et l., 27), indicting tht A. ocelltus is le to successfully tolerte extended hypoxi exposure ecuse of the reduction in key ATP-consuming processes. The ojectives of this study were to expnd our knowledge of the iochemicl processes ehind the hypoxi-induced metolic depression nd post hypoxi recovery in A. ocelltus through investigtion of the tissue-specific protein synthesis rtes in reltion to the whole niml metolic depression. In ddition, the present study is the first to otin mesurements of whole niml metolic rte nd protein synthesis under similr experimentl conditions on the sme popultion of fish. Mterils nd methods Experiments were conducted t the Lortory of Ecophysiology nd Moleculr Evolution, INPA, Brzil. A popultion of Astronotus ocelltus (Agssiz 1831) ws held in n outdoor holding tnk with erted well wter (O 2 sturtion of 8 1%) t 28 C nd fed commercil food once dily, until trnsferred to experimentl tnks (for protein synthesis studies) or respirometers (for mesurements of routine metolic rte, RoMR). In totl, eight fish were used for mesurement of RoMR (156 225 g, verge 186±1. g) nd 44 fish were used for the mesurement of protein synthesis (7 16 g, verge 95.8±3.8 g). All fish were held without feeding for 48 h prior to eginning mesurements for RoMR nd protein synthesis. In fish used for nlysis of RoMR nd protein synthesis, mesurements were tken under normoxic nd hypoxic conditions s well s during the recovery from cute hypoxi exposure. Normoxic conditions were identicl to those of the holding tnk (O 2 sturtion of 8 1%), nd the hypoxic chllenge consisted of step down decrese of the dissolved oxygen (DO) level. This ws ccomplished y uling nitrogen directly into the wter of the experimentl tnk, or into the reservoir tht supplied wter to the respirometer. Wter oxygen levels were stepped down from 1 to 7, 5, 3, 2 nd 1%, with O 2 levels mintined t ech step for 1 h, nd the fish held t 1% O 2 sturtion for 3 h prior to re-oxygention. Re-oxygention ws chieved y uling ir vigorously into the wter, nd wter O 2 levels returned to normoxic levels within 3 45 min. Mesurement of routine metolic rte Individul fish were trnsferred to specilly designed Plexigls respirometer (15 2 4 cm; 11.875 l) supplied with oxygen-sturted wter (8 1%) from 1 l reservoir nd llowed to cclimte for 48 h efore the eginning of the experiment. Wter from the reservoir ws continuously pumped through the respirometer using sumersile pump (model NK-1, Little Gint Co., Vernon Hills, IL, USA). Wter temperture nd oxygen concentrtion were monitored through circuit composed of tuing with extremely low gs permeility (Tygon Food & LFL, Cole Plmer, Inc., Oklhom City, OK, USA) using peristltic pump (Msterflex L/S model 772-12, Cole-Plmer) nd flowthrough chmers (D21, WTW, Weilheim, Germny) contining oxygen proes (model CellOx 325, WTW) positioned in the inflow nd outflow tuing of the respirometer. Mesurements of wter oxygen levels nd wter flow rte (rnge.8 1. l min 1 ) were tken t hourly intervls during the experiment (i.e. during normoxi, hypoxi nd recovery from hypoxi), s well s efore the plcement of the fish nd immeditely fter the removl of the fish from the respirometer

Hypoxi nd recovery in Astronotus ocelltus 1937 in order to correct for cteril O 2 consumption. Bcteril O 2 consumption ws consistently less thn 2% of the RoMR of the fish nd ws therefore considered to e negligile. The RoMR of ech fish ws clculted t ech mesurement intervl s: RoMR = [(C O 2(i) C O 2(o)] Vw 6 / M where RoMR is in mg O 2 kg h 1 ;C O 2(i) is the O 2 concentrtion in inflowing wter (mg O 2 l 1 ); C O 2(o) is the O 2 concentrtion in outflowing wter (mg O 2 l 1 ); Vw is the wter flow rte through the respirometer (l min 1 ) nd M is the mss of fish (kg) [modified from Cech (Cech, 199)]. Protein synthesis Normoxi Twelve fish were removed from the holding tnk, weighed, tgged for individul recognition nd trnsferred to seprte experimentl tnk under identicl environmentl conditions. After 48 h, fish were injected intrperitonelly, without nesthetic, with 1. ml 1 g 1 of [2,3-3 H]phenyllnine (Amershm Interntionl) solution. This injection solution consisted of 135 mmol l 1 phenyllnine in solution contining 125 mmol l 1 NCl, 3 mmol l 1 KCl, 1 mmol l 1 MgSO 4.7H 2 O, mmol l 1 CCl 2, 5 mmol l 1 Hepes (sodium slt), 5 mmol l 1 glucose, 2 mmol l 1 N 2 HPO 4, ph 7.8 t 28 C, in ddition to sufficient [2,3-3 H]phenyllnine to ensure dosge of 1 Ci ml 1. Following injection, fish were returned immeditely to the experimentl tnk nd fter n incution time of 1, 2 or 3 h, groups of four fish were killed y low to the hed nd immedite severing of the spine. Brin, hert, liver, white muscle nd gill tissue were excised in tht order, nd frozen in liquid nitrogen. All smples were kept t 7 C until nlysis. Hypoxi In this tretment, 12 fish were weighed, tgged nd trnsferred to the experimentl tnk. After 48-h cclimtion period, fish were exposed to stepwise decrese in dissolved oxygen levels s descried previously. Fish were injected immeditely once wter oxygen sturtion reched 1%, nd four fish were smpled (s ove) t 1, 2 nd 3 h fter injection with DO levels mintined t 1% for the 3 h hypoxi exposure. Recovery To ssess chnges in protein synthesis rtes during recovery from hypoxi, 2 fish were exposed to n cute hypoxi chllenge, s descried ove. After holding fish t 1% DO for 3 h, ir ws uled into the experimentl tnk llowing the dissolved O 2 level to return to normoxic levels (8 1%). Groups of five fish were injected t hourly intervls, strting when O 2 sturtion levels returned to normoxic levels (group 1) nd ending 4 h fter O 2 returned to normoxic levels (group 4). Ech group of fish ws smpled 1 h post-injection llowing protein synthesis to e trcked over 4-h time period during the post-hypoxic recovery. Tissues were excised nd stored s previously descried. Blood smpling for lctte Blood smples were otined from s mny fish s possile during the protein synthesis experiment, resulting in N=4, normoxic; N=8, hypoxic; nd N=7, recovery. Blood ws drwn from the cudl vein with heprinized syringe prior to smpling the fish for protein synthesis nlysis. Blood smples were centrifuged nd plsm ws stored t 7 C for lctte nlysis. Smple preprtion nd scintilltion counting The protocol used for the nlysis of protein synthesis followed tht of Treerg et l. (Treerg et l., 25), with techniques modified from the originl pper tht presents the flooding dose pproch to mesure rtes of protein synthesis (Grlick et l., 198). Smples were homogenized with Polytron homogenizer (Brinkmnn Instruments, Westury, NY, USA) in nine volumes of 6% perchloric cid (PCA) except for liver, which ws homogenized in four volumes of PCA. Homogenized smples were left on ice for 1 15 min nd 1 ml liquot ws trnsferred to microcentrifuge tue. Excess homogente (liver, muscle nd in some cses rin smples), ws stored t 7 C for the nlysis of lctte. The 1 ml liquot of homogente for protein synthesis ws centrifuged for 5 min t 15,6 g, fter which the superntnt ws removed nd frozen t 2 C for nlysis of the totl free phenyllnine content nd its rdioctivity. The protein pellet ws wshed y mnully re-suspending the pellet in 1. ml of 6% PCA, vortexing, centrifuging s descried ove, nd then discrding the superntnt. This wsh step ws repeted until the rdioctivity in the discrded superntnt ws t ckground levels to ensure only protein ound [ 3 H]phenyllnine ws eing mesured in the protein pellet. After sufficient wshing, 1. ml of.3 mol l 1 NOH ws dded to the tue contining the protein pellet. The protein pellet ws incuted in wter th held t 37 C until fully dissolved. The dissolved protein ws stored t 2 C until nlysis for protein content nd protein-ound rdioctivity. Aliquots of the originl superntnt from the homogenized tissue nd the dissolved protein were dded to 1 ml of Ultim Gold TM liquid scintilltion cocktil nd counted on Beckmn Coulter LS65 liquid scintilltion counter to otin the [2,3-3 H]phenyllnine content of the free nd protein-ound phenyllnine pools of the tissues, respectively. Phenyllnine specific ctivity ws considered to e c.p.m./phenyllnine content (nmol). This ssumes negligile conversion of rdiolelled phenyllnine to other rdiolelled components, which hs een proved with HPLC nlysis in species of mollusc (Pky et l., 22). This ssumption is considered to e cceptle for the current study given the elevted nd constnt c.p.m./phenyllnine rtio in ll tissues over the time course of the study nd the liner rte of incorportion of rdiolel into the protein pool for ll tissues. Biochemicl ssys Free pool phenyllnine content ws mesured from the PCA extrction superntnt nd phenyllnine stndrds in 6% PCA

1938 J. M. Lewis nd others using fluorometric ssy following the protocol descried in McCmn nd Roins (McCmn nd Roins, 1962). Protein content of the tissue ws determined from the NOH-soluilized protein pellet y using the BioRd D c kit (Bio-Rd Lortories, Hercules, CA, USA) using stndrds mde from ovine serum lumin. Lctte ws mesured in stndrds in 6% PCA, plsm, liver, white muscle nd rin tissue vi the reduction of NAD + to NADH t 34 nm using Sigm dignostics kit. Sttisticl nlyses Comprison of oxygen consumption dt ws crried out y using repeted mesures ANOVA followed y Dunnett s post-hoc test, to compre ll vlues with the normoxic (control) vlue. Lctte concentrtions for normoxi, hypoxi nd recovery tretments were compred using one-wy ANOVA, with Tukey s post-hoc test for multiple comprisons. In the protein synthesis experiment, men tissue phenyllnine content nd specific ctivity over the incution time were compred using one-wy ANOVA with Tukey s post-hoc test for multiple comprisons, nd the incorportion of rdioctivity into protein ws exmined y liner regression. Once dt were confirmed to fit the vlidtion criteri, phenyllnine incorportion rtes for ech tissue were compred using onewy ANOVA followed y Tukey s post-hoc test. In ll cses P<.5 ws considered significnt. Results Routine metolic rte The RoMR of A. ocelltus under normoxic conditions ws 1 138.7±16.5 mg kg 1 h (Fig. 1). Despite decresing oxygen sturtion of the wter, there ws no significnt decrese in RoMR until wter O 2 levels reched 1% sturtion (.67±.5 mg l 1 t 28±1 C). At this level, O 2 consumption ws decresed to 65.1±2. mg kg 1 h 1, vlue pproximtely 5% of the normoxic rte. Although RoMR t 1 h post-hypoxi RoMR (mg O 2 kg 1 h 1 ) 4 3 2 1 M O2 [O 2 ] in wter * 1 2 3 4 5 6 7 8 9 1 11 Time (h) Fig. 1. Routine metolic rte (RoMR) in A. ocelltus in reltion to chnging levels of O 2 sturtion in the wter. RoMR (M O 2) mesurements re shown s mens ± s.e.m., N=8 fish. *Significnt differences in RoMR from time (normoxi) vlues (P<.5). * * * 1 9 8 7 6 5 4 3 2 1 DO 2 in wter (%) significntly incresed to 35.5±29.7 mg kg 1 h 1, 27% of rtes otined under normoxi, RoMR returned to pre-hypoxic levels (174.±3.1 mg kg 1 h 1 ) y 2 h post-hypoxi nd remined t similr levels for the reminder of the experiment. Lctte concentrtion Lctte concentrtions under normoxic conditions in the vrious tissues were.4±.3 mol ml 1 for plsm (N=4) nd.25±.6,.84±.11 nd 2.63±.3 mol g 1 tissue for liver (N=11), rin (N=12) nd white muscle (N=6), respectively (Fig. 2). As there were no significnt differences in lctte concentrtion in ny of the tissues during the 3-h hypoxi nd 4-h recovery periods, results were pooled within ech tretment to give men vlue for hypoxic nd recovery smples. During the 3-h hypoxic exposure, only plsm exhiited significnt increse in lctte concentrtion (1.13±.27 mol ml 1, N=8). During the post-hypoxic recovery period, plsm lctte returned to levels (.42±.3 mol ml 1, N=7) tht were not significntly different from pre-hypoxic vlues. Finlly, significnt decreses in lctte concentrtions in liver (.2±.1 mol g 1 tissue, N=2) nd rin.55±.6 mol g 1 tissue, N=2) occurred during the recovery period, wheres lctte concentrtion in white muscle ws mintined t similr concentrtions over ll three tretments. Vlidtion of protein synthesis methodology In order to ccurtely interpret protein synthesis rtes otined vi the flooding dose methodology severl vlidtion criteri must e met: (1) the injection dose must e shown to e sufficient to elevte the free phenyllnine pool of the vrious tissues; (2) the specific ctivity of the free [Lctte] ( mol g 1 ) 3.5 3. 2. 1..5 Normoxi Hypoxi Recovery, Plsm Brin Muscle Liver Fig. 2. Lctte concentrtion in plsm, rin, white muscle nd liver tissue of A. ocelltus during normoxi, hypoxi nd recovery from hypoxi. Vlues re mens ± s.e.m.; numers of smples/fish were: plsm normoxic (N=4), hypoxic (N=8), recovery (N=7); rin normoxic (N=6), hypoxic (N=1), recovery (N=2); muscle normoxic (N=12), hypoxic (N=12), recovery (N=2); liver normoxic (N=11), hypoxic (N=1), recovery (N=9). Significnt differences within ech tissue re indicted y different letters, P<.5.,

Hypoxi nd recovery in Astronotus ocelltus 1939 phenyllnine must increse rpidly post-injection nd remin stle throughout the time protein synthesis is mesured; nd (3) the rte of phenyllnine incorportion must e liner nd egin immeditely fter injection. As shown in the following sections, ll three criteri were fulfilled during this experiment. Elevtion of free phenyllnine pool content The concentrtion of free pool phenyllnine in the vrious tissues ws not significntly different etween the three tretments or etween smple times within tretments. Therefore, results for normoxic, hypoxic nd post-hypoxic fish were pooled nd referred to s injected fish (N=44). Injected fish hd free phenyllnine levels of.55±.4,.16±.1,.8±.1,.2±.1 nd.19±.1 mmol phe g 1 fresh tissue (N=44) for liver, white muscle, rin, hert nd gill, respectively. When compred to levels of free phenyllnine in un-injected fish (.12,.8,.3,.5,.9 mmol phenyllnine g 1 fresh tissue; N=1), levels were twofold higher in rin, white muscle nd gill, nd fivefold higher in liver nd hert tissue. The seline levels of free phenyllnine from un-injected oscrs nd the increse chieved vi the flooding dose of phenyllnine re comprle to levels mesured in crucin crp (Smith et l., 1999). Intrcellulr free pool phenyllnine specific ctivity Intrcellulr specific ctivity of the free phenyllnine pool for oth normoxic nd hypoxic fish ws elevted 1 h postinjection, nd remined constnt over the 3 h tht protein synthesis ws mesured (Fig. 3). On verge, the specific ctivity for normoxic fish ws 139±79, 641±27, 711±37, 788±61 nd 656±22 c.p.m. nmol 1 phenyllnine for liver, white muscle, rin, hert nd gill, respectively (N=12). For hypoxi-exposed fish, the specific ctivity for the sme tissues ws 78±44, 642±17, 696±26, 622±34 nd 62±14 c.p.m. nmol 1 phenyllnine (N=12). As there ws no significnt difference in the specific ctivity etween posthypoxi recovery smpling times, results were pooled for ech tissue to give men vlues of 83±23, 641±12, 72±7., 665±1 nd 672±17 c.p.m. nmol 1 phenyllnine for liver, white muscle, rin, hert nd gill, respectively (N=2). Phenyllnine incorportion into tissue protein Protein synthesis rtes were expressed s nmol phenyllnine incorported per mg protein. Regression equtions clculted over the 3-h smpling time demonstrted significnt nd liner incorportion of phenyllnine into liver, rin, hert nd gill tissue of oth normoxic nd hypoxic fish (Fig. 4). As the intercepts of the regression lines were not significntly different from zero (i.e. time ), it cn e ssumed tht the incorportion of phenyllnine into liver, rin, hert nd gill tissues egn immeditely following injection. Rtes of protein synthesis for white muscle in oth normoxic nd hypoxic tretments were elow detectle levels. As liner incorportion rtes were chieved for oth normoxic nd hypoxic fish over the three time points nd phenyllnine incorportion egn immeditely post-injection, rtes of protein synthesis during recovery were determined only t 1 h post-injection. Tissue-specific rtes of phenyllnine incorportion Rtes of phenyllnine incorportion under normoxic conditions were.92±.13,.8±.5,.9±.16 nd c.p.m. nmol 1 phenyllnine A 16 Liver 12 8 4 1 8 6 4 2 12 8 4 8 6 4 2 8 6 4 2 B Brin C Hert D Gill E Muscle 1 2 3 4 Time (h) Fig. 3. Post-injection chnges in the specific ctivity of the intrcellulr free phenyllnine pool in (A) liver, (B) rin, (C) hert, (D) gill nd (E) white muscle of A. ocelltus during normoxi (closed circles) nd hypoxi (open circles). Vlues re mens ± s.e.m., N=4.

194 J. M. Lewis nd others 2. 1..5 A Liver RoMR (mg O 2 kg 1 h 1 ) 4 3 2 1 A c Normoxic Hypoxic Recovery [Phenyllnine] (nmol mg 1 protein) 3. 2. 1..5 3.5 3. 2. 1..5 8. 7. 6. 5. 4. 3. 2. 1. B Brin C Hert D Gill 1 2 3 4 Time (h) Fig. 4. Post-injection time course for the incorportion of rdiolelled phenyllnine into protein in A. ocelltus during normoxi (closed circles) nd severe hypoxi (open circles). (A) Liver (y=.47x+.62, r 2 =.6, P r =.5, P y =.22; y=.44x.22, r 2 =.8, P r =.3, P y =.39); (B) rin (y=.82x.5, r 2 =.92, P r =2.35 1 5, P y =.84; y=.57x+.7, r 2 =.95, P r =2.97 1 6, P y =.61); (C) hert (y=.94x+.2, r 2 =.8, P r =.5, P y =.71; y=.55x.16, r 2 =.84, P r =6.78 1 4, P y =.53); (D) gill (y=2.34.24, r 2 =.93, P r =6 1 5, P y =.72; y=x+.21, r 2 =.68, P r =.15, P y =.79). For ech tissue regression equtions refer to normoxic nd hypoxic fish, respectively. All r 2 vlues re significnt (P r <.5), ll y- intercept vlues re not significnt from zero (P y >.5). Vlues re mens ± s.e.m., N=4, except for hypoxi-exposed liver 2 h smple, where N=3. White muscle not shown ecuse rtes of incorportion were not detectle. [Phenyllnine] (nmol mg 1 protein h 1 ) 1.4 1.2 1..8.6.4.2 1..8.6.4.2 1.2 1..8.6.4.2 2. 1..5 B C D E,,,,,,c,,, Norm Hyp 1 2 3 4, Recovery time (h) Fig. 5. (A) Routine metolic rte RoMR nd (B E) tissue phenyllnine incorportion in liver (A), rin (B), hert (C) nd (D) gill, in A. ocelltus exposed to normoxi, severe hypoxi, nd during recovery from hypoxi. Vlues re mens ± s.e.m., N=8 for routine metolic rte mesurements. For phenyllnine incorportion mesurements, N=12 for normoxi nd hypoxi exposure, nd N=4 for ech time point during recovery. Different lowercse letters indicte significnt difference; P<.5.

Hypoxi nd recovery in Astronotus ocelltus 1941 2.2±.11 nmol phenyllnine mg 1 protein h 1 for liver, rin, hert nd gill, respectively. During exposure to hypoxi, rtes of protein synthesis decresed to.41±.12,.62±.3,.36±.6, nd 1.14±.15 nmol phe mg 1 protein h 1, depression of 56% for liver, 27% for rin, 6% for hert nd 5% for gill (Fig. 5). During recovery from cute hypoxi, no hyperctivtion of protein synthesis occurred, nd two different ptterns in post-hypoxic phenyllnine incorportion were oserved. In liver nd gill, rtes of phenyllnine incorportion (.74±.8 nd 1.46±.27 nmol phenyllnine mg 1 protein h 1, respectively) were not significntly different from normoxic levels y 1 h post-hypoxi, nd remined t similr levels for the durtion of the recovery period. By contrst, phenyllnine incorportion took longer to return to normoxic levels in rin (3 h) nd hert (2 h), nd in oth these tissues phenyllnine incorportion ws significntly less thn normoxic vlues for the reminder of the recovery period. This ltter result suggesting tht full recovery of protein synthesis in rin nd hert tissues tkes longer thn 4 h. Discussion Hypoxi-induced metolic depression Routine metolic rte In the present study, routine metolic rte (RoMR) ws mintined t normoxic rtes until dissolved oxygen levels in the wter decresed to 1%. At this time, RoMR underwent 5% depression, which ws mintined for the full 3 h of hypoxi exposure. This response to hypoxi ws similr to tht otined previously for oscrs, in which depression in RoMR of pproximtely 5% ws oserved once wter O 2 levels reched 1% oxygen sturtion, nd reduction of 6% ws mesured when wter O 2 levels pproched those of noxic conditions (Muusze et l., 1998). At the lowest level of hypoxi tested in the present study (1% DO), lctte levels were only significntly incresed in plsm. However, this increse only rought lctte levels to one-fifth of levels otined in A. ocelltus t 6% DO (Muusze et l., 1998). These results, in comintion with the sence of lctte ccumultion in white muscle, indicte neroic metolism is only eginning to e employed to supplement energy demnds t this level of oxygen deprivtion, nd metolic depression is n effective wy of conserving ATP until A. ocelltus is fced with lmost noxic conditions. In other studies with comprle lengths of hypoxi exposure, levels of lctte incresed to greter extent in plsm [4.9-fold (Richrds et l., 27); 11 16-fold (Wood et l., 27)] nd white muscle [2 3-fold (Richrds et l., 27; Wood et l., 27)] thn in the current study. This discrepncy in lctte ccumultion during hypoxic exposure is most likely result of the quick entry into hypoxi (~1 h) for these two studies s compred to the grdul trnsition into hypoxi of our study (~5 h). The level of metolic depression chieved y A. ocelltus is similr to tht of goldfish nd crucin crp, oth of which decrese metolic rte y pproximtely 7% under noxi (Vn Wversveld et l., 1989), ut not s gret s demonstrted y freshwter turtles (9 95% reduction) (Jckson, 1968). Metolic depression is less in teleosts s result of mintennce of ion exchnge with the environment nd low levels of predtor voidnce. For exmple, in their nturl environment A. ocelltus re susceptile to predtion from ir-rething fish nd eril predtors, nd lortory experiments show tht they split their time eqully etween unprotected normoxic environments nd sheltered hypoxic environments (Slomn et l., 26). Turtles, however, re essentilly closed system s they retret into their protective shell nd enter comtose-like stte during periods of oxygen deprivtion. Protein synthesis The use of the flooding dose methodology to mesure in vivo protein synthesis requires tht severl vlidtion criteri e fulfilled. The results from this study show tht the injection dosge used successfully flooded the free phenyllnine pool during oth normoxi nd 3 h of hypoxic exposure, cusing two- to fivefold increse in phenyllnine concentrtion in the vrious tissues. As well, the specific ctivity of the free phenyllnine pool ws elevted 1 h post-injection nd remined stle t this level for the 3 h over which protein synthesis ws mesured. The finl vlidtion criterion requires the incorportion of rdiolelled phenyllnine into the tissues to e liner post-injection. This ws shown for ll tissues in oth normoxi- nd hypoxi-exposed fish, except for white muscle (Fig. 4). The rdioctivity of protein-ound phenyllnine in white muscle ws elow detectle levels, indicting rtes of protein synthesis in this tissue to e miniml. Given tht rtes of protein synthesis in fish white muscle re extremely low s compred to mmmls (Fuconneu et l., 1995), nd A. ocelltus hs much lower mss specific oxygen uptke thn other teleosts, including tropicl species (Almeid- Vl et l., 26), it is not surprising tht protein synthesis ws undetectle in the white muscle of A. ocelltus. The role of protein synthesis in hypoxi-induced metolic depression in ectothermic nimls hs een previously descried in freshwter turtles (specificlly Trchemys script elegns nd Chrysemys pict ellii) nd in the crucin crp (Crssius crssius), nd these studies show tht the extent to which protein synthesis is depressed is positively linked with the degree to which ctivity is curtiled. For exmple, rtes of protein synthesis were suppressed y pproximtely 7% in the hert of T. script elegns (Biley nd Driedzic, 1996) nd y >95% in vrious tissues in C. pict ellii; oth these species enter comtose-like stte during noxi (Lnd et l., 1993; Frser et l., 21). By contrst, the crucin crp, which mintins low levels of ctivity during hypoxi/noxi exposure, exhiits depression in protein synthesis of pproximtely 5% in hert nd white muscle, 95% in liver tissue, ut no significnt depression in the rin (Smith et l., 1996). Similr to the crucin crp, A. ocelltus exhiited tissuespecific depression in protein synthesis when exposed to cute hypoxi exposure. Rtes of protein synthesis in liver, hert nd

1942 J. M. Lewis nd others gill were depressed y 5 6%, wheres rtes of protein synthesis in the rin were only depressed y 27%. Thus, our results reinforce the ide tht fish need to mintin protein synthesis in the rin to prevent dmge to neurl tissue, nd to sustin pproprite rin functions so tht predtors cn e effectively voided. The depression of protein synthesis in gills is ccompnied y simultneous decrese in N + pumping nd lek rtes in the gills, s shown y mesurement of N +,K + - ATPse ctivity nd N + flux (Richrds et l., 27; Wood et l., 27). One of the suggested mechnisms controlling the depression of protein synthesis, nd therefore depression of metolic rte, is decrese in ph (Hochchk nd Somero, 22; Richrds et l., 27). The reduction of protein synthesis hs een linked to n increse in recominnt elongtion fctor 2 kinse (EF2K) cused y exposure to low ph (Dorovkov et l., 22). The significnt reduction of protein synthesis in liver, rin, hert nd gill tissues in hypoxi-exposed A. ocelltus, oserved in the present study, comined with the decreses in extrcellulr nd intrcellulr ph in A. ocelltus exposed to comprle hypoxic conditions (Richrds et l., 27) strengthens the rgument of Richrds et l., tht ph my hve direct effect on protein synthesis nd therefore, metolic rte in A. ocelltus. Recovery from cute hypoxi exposure A significnt overshoot in oxygen consumption to 27% of normoxic rtes ws oserved during the first hour of recovery, indicting tht the 3-h hypoxic exposure ws sustntil enough to cuse the fish to ccumulte n oxygen det. Crucin crp hve lso een shown to ccumulte sustntil oxygen det during periods of hypoxi (Vn den Thillrt nd Vereek, 1991), nd it hs een suggested the hyperctivtion of metolic rte during noxic/severe hypoxic recovery is ssocited with the restortion of phosphocretine, the conversion of lctte into glycogen, nd possily n increse in protein synthesis (Johnsson et l., 1995). Although there ws no ccumultion of lctte in the white muscle in the present study, Richrds et l. (Richrds et l., 27) hve shown 3% decrese in cretine phosphte in white muscle fter 4 h exposure to hypoxi, which returned to pre-hypoxi exposure levels during recovery. As such, the sustntil increse in oxygen consumption seen in our study during the first hour of recovery my e linked to the restortion of phosphocretine. An in vitro study on turtle heptocytes exposed to 12 h of noxi hs shown significnt increse in protein synthesis (to 16% of normoxic rtes) during the first hour of recovery (Lnd et l., 1993). However, the present study, which mesured in vivo protein synthesis rtes, did not show ny hyperctivtion of protein synthesis in the vrious tissues during the recovery period. These results gree with other in vivo studies showing hyperctivtion of protein synthesis does not occur in noxic exposed turtles (Frser et l., 21) or crucin crp (Smith et l., 1996). There were two distinct ptterns oserved in post-hypoxic phenyllnine incorportion in A. ocelltus. In tissues tht re min source for protein synthesis, liver nd gill, phenyllnine incorportion returned to pre-hypoxic rtes y 1 h posthypoxi. By contrst, protein synthesis in rin nd hert tke longer thn 4 h post-hypoxi to fully recover. The slow recovery in rin tissue is prticulrly interesting s its hypoxi-induced reduction in protein synthesis is hlf of tht shown y the other tissues. The resons for this remin elusive; however, it my e linked to the removl of dietry source of mino cids (due to the cesstion of feeding), requiring A. ocelltus to rely on the recycling of existing protein (i.e. protein turnover) to replenish diminished supplies due to the decrese of protein synthesis during metolic depression. Conclusions The present study ws successful in furthering the insight into the iochemicl dpttions of A. ocelltus to conditions of extreme low oxygen to include description of the role protein synthesis plys in contriuting to the whole niml metolic depression. The response of A. ocelltus to cute hypoxi nd susequent recovery, t oth the physiologicl nd iochemicl level, ws similr to tht of the well studied noxi-tolernt teleost, the crucin crp. However, there re tissue-specific differences in the mgnitude of the hypoxiinduced depression of protein synthesis (rin 2%, other tissues 5 6%), which suggest tht rin function is mintined during hypoxi to fcilitte ctive predtor voidnce. As well, this study demonstrted tht n cute (3 h) exposure to severe hypoxi is sustntil enough to cuse A. ocelltus to ccumulte n oxygen det, ut the repyment of this oxygen det is not ccompnied y compenstory hyperctivtion in protein synthesis. This ltter finding indictes the high metolic rte A. ocelltus during the first hour of recovery is ttriuted to cellulr processes other thn the ssimiltion of protein. Comining the results from the current work with recent discoveries from comprle studies on the ehviourl, physiologicl nd iochemicl dpttions of A. ocelltus (Muusze et l., 1998; Almeid-Vl et l., 2; Slomn et l., 26; Richrds et l., 27; Wood et l., 27) it cn now e concluded the metolic depression oserved ehviourlly is chieved through decrese in physicl ctivity, ctivtion of neroic metolism nd the reduction of energy consuming processes (nitrogenous wste production, ion exchnge nd protein synthesis). These iochemicl dpttions enle Astronotus ocelltus to mintin stle ATP levels, nd therefore extend survivl time when fced with conditions of extreme low oxygen. The uthors would like to thnk Nzré Pul-Silv for her technicl support throughout the project, Dr Adrin Chippri- Gomes for her ssistnce with otining the rdioisotope permit, nd Fio Soller d Silv for his help with the smpling of fish. This project ws funded though NSERC grnts wrded to W.R.D. nd A.K.G., nd funds from CNPq nd FAPEAM wrded to A.L.V. nd V.M.F.A.-V.

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