Comparative Biochemistry and Physiology, Part B

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1 Comprtive Biochemistry nd Physiology, Prt B 164 (213) 53 6 Contents lists ville t SciVerse ScienceDirect Comprtive Biochemistry nd Physiology, Prt B journl homepge: Nturl vrition in enzyme ctivity of the Africn cichlid Pseudocrenilrus multicolor victorie Cindy D. Crocker, Luren J. Chpmn,c, Mery L. Mrtínez, Deprtment of Biology, Lurentin University, Sudury, Ontrio, Cnd P3E 2C6 Deprtment of Biology, McGill University, 125 Avenue Docteur Penfield, Montrél, PQ, Cnd 3HA 1B1 c Wildlife Conservtion Society, 23 Southern Boulevrd, Bronx, NY 146, USA rticle info strct Article history: Received 25 June 212 Received in revised form 25 Octoer 212 Accepted 28 Octoer 212 Aville online 1 Novemer 212 Keywords: Aeroic metolism Aneroic metolism Gluconeogenesis Hplochromine cichlid Hypoxi This study descries the metolic cpcities of the Africn cichlid Pseudocrenilrus multicolor victorie from four sites in Ugnd, Est Afric. Fish were cptured during the dry seson, from two qutic systems in different regions (Lke Nugo nd Mpng River). Within the Lke Nugo region, individuls were smpled from Lke Kynj (normoxic) nd Lwmund Swmp (hypoxic); within the Mpng River system, individuls were smpled from Bunog nd Khunge (chrcterized y sesonl vrition in dissolved oxygen (D.O.)). Enzyme ctivity levels of pyruvte kinse, lctte dehydrogense, citrte synthse, nd cytochrome C oxidse were mesured in four tissues: white skeletl muscle, hert, rin, nd liver. Two dditionl enzymes were mesured in the liver, mlte dehydrogense nd fructose 1,6-isphosphtse. l differences etween enzyme ctivities in most tissues were evident; however, little vrition ws oserved etween two sites within region despite differences in D.O. In generl, P. multicolor from the Mpng River system displyed greter neroic enzyme ctivity in white skeletl muscle, lower gluconeogenic enzyme ctivity in the liver, nd n overll higher enzyme ctivity in the hert nd rin tissues thn fish from the Nugo region. The ltter my reflect long-term dpttion to low-oxygen conditions t the metpopultion level in the Nugo region. 212 Elsevier Inc. All rights reserved. 1. Introduction Mny qutic hitts re chrcterized y low concentrtions of dissolved oxygen (D.O.) (Diz nd Rosenerg, 28; Diz nd Breiturg, 29; Richrds, 29), nd orgnisms tht persist in these hitts hve mechnisms tht confer tolernce to hypoxi (Hochchk, 198; Chpmn et l., 22; Chippri-Gomes et l., 25; Crmpton et l., 28; Mndic et l., 29). To mintin cellulr energy lnce during hypoxi, mny hypoxi-tolernt fishes reduce energeticlly expensive processes (e.g., protein turnover) nd increse oxygen-independent ATP production (Lnd et l., 1993; Hochchk et l., 1996; Ji nd Richrds, 28; Dvies et l., 211). When the severity of hypoxi increses, fish rely more hevily on the contriution of glycolysis to produce ATP, response tht is frequently ssocited with chnges in the ctivities of enzymes of glycolysis nd other pthwys of crohydrte metolism (Hochchk et l., 1996; DeKoning et l., 24; Pollock et l., 27). Support for this prdigm comes minly from lortory studies of fish held under different oxygen conditions (Chippri-Gomes et l., 25; Mrtínez et l., 26; Frwell et l., 27; Mrtínez et l., 29). Fewer studies hve exmined the metolic enzyme ctivity of fish cptured directly from their nturl environment, where individuls Corresponding uthor. Tel.: x2293; fx: E-mil ddress: mmrtinezgrci@lurentin.c (M.L. Mrtínez). re sujected to vrition in temperture, food vilility, nd predtion, long with vrition in D.O. (ut see Mrtínez et l., 211). In Est Afric, some freshwter systems experience drmtic reductions in D.O. during the dry seson, while others experience hypoxi during the riny seson s result of wter inflow from hypoxic swmps (Chpmn et l., 22; Crispo nd Chpmn, 28). In nturl popultions of the smll Africn hplochromine cichlid, Pseudocrenilrus multicolor victorie, fish from hypoxic hitts tend to e chrcterized y lrge gill surfce re (Chpmn et l., 2, 28), low criticl oxygen tension (Rerdon nd Chpmn, 21), high hemogloin nd hemtocrit levels (Chpmn et l., 22; Mrtínez et l., 29), nd smll rin size (Chpmn et l., 28) reltive to fish from well-oxygented sites. In lortory experiments where P. multicolor were rered under D.O. conditions similr to normoxic nd hypoxic field sites, there re lsting effects of popultion of origin, s well s pronounced influence of developmentl plsticity nd cclimtion, in determining these morphologicl nd physiologicl responses to hypoxi (Chpmn et l., 28; Mrtínez et l., 29; Crispo nd Chpmn, 21). In the context of metolic enzyme ctivities, Mrtínez et l. (29) compred F 1 offspring from low- nd high-oxygen popultion tht were rered under normoxi nd cclimted to low- nd high-d.o. conditions. They found tht lctte dehydrogense (LDH) ctivity in rin tissue ws influenced y popultion of origin, with lower ctivities found in fish of low-d.o. origin, wheres the D.O. levels during cclimtion /$ see front mtter 212 Elsevier Inc. All rights reserved.

2 54 C.D. Crocker et l. / Comprtive Biochemistry nd Physiology, Prt B 164 (213) 53 6 were more importnt thn popultion of origin in determining the LDH ctivity in other tissues. The gol of this study ws to quntify vrition in metolic enzymtic ctivities in four field popultions of P. multicolor from sites tht differed in oxygen regimes to provide insights into nturl levels of metolic enzyme ctivities in field popultions. In these field popultions, dissolved oxygen is likely to interct with other vriles in determining the developmentl trjectory of tissue metolic enzyme ctivities. Fish were cptured from two qutic systems in Ugnd, Est Afric (Lke Nugo nd Mpng River), nd within ech region, two sites tht differed in oxygen regime were selected. Within the Lke Nugo region, fish were smpled from Lke Kynj (normoxic) nd Lwmund Swmp (chroniclly hypoxic); while within the Mpng region, fish were smpled from Bunog nd Khunge (oth chrcterized y fluctuting D.O. levels, ut Bunog hving higher levels thn Khunge cross sesons). Enzymes of eroic nd neroic crohydrte metolism were mesured in four tissues with distinct energy nd oxygen demnds. Under certin conditions (e.g., sence of llosteric nd covlent modifiction), mximl enzyme ctivity (Vmx) cn e used s mesure of enzyme concentrtion, nd therefore n indirect mesure of enzyme gene expression (Pierce nd Crwford, 1994, 1997). Thus, the results of these experiments llowed us to prtition vrition in metolic enzyme ctivities, nd potentilly enzyme gene expression, y geogrphic region, nd within region y the occurrence of low D.O. in nturl hitts. 2. Mterils nd methods 2.1. Study sites nd collection of fish In June 26, P. multicolor were collected from four sites in Western Ugnd, Afric. Two of the sites, Lke Kynj nd Lwmund Swmp re locted in the Nugo region close to Lke Victori, while the other two sites, Bunog nd Khunge re locted in the Mpng River of Western Ugnd (See mp in Crispo nd Chpmn, 28). Prior reserch hs shown tht the D.O. levels vry cross the four sites. Lke Kynj remins normoxic yer round (6.5 to 8.1 mg O 2 L 1 ; Rosenerger nd Chpmn, 2; Crispo nd Chpmn, 28). In contrst, the Lwmund Swmp is very hypoxic, with some sesonl vrition nd diel increse ssocited with periphyton mts in some res of the swmp. Rerdon nd Chpmn (29) reported men monthly D.O. vlues rnging from.37 to 1.61 mg O 2 L 1 in the morning nd 1.69 to 3.99 mg O 2 L 1 in the fternoon. The two sites in the Mpng River system re chrcterized y open, flowing wter ut fluctuting D.O. levels over the yer. At the Bunog site McNeil (212) reported rnge in monthly mens D.O. of 1.7 to 9. mg O 2 L 1, with lower levels ssocited with riny seson runoff. The Khunge site is situted immeditely downstrem of lrge ppyrus swmp tht contriutes to sesonl hypoxi in this site (rnge in monthly mens=1.1 to 5.7 mg O 2 L 1, Crispo nd Chpmn, 28). At the time of our fish smpling in 26, D.O. nd wter temperture were recorded twice (morning/fternoon) t ech site where the fish were cptured nd verged s follows: Lke Kynj, 7.43±.12 mg O 2 L 1 nd 24.±.5 C; Lwmund Swmp,.55±.3 mg O 2 L 1 nd 21.5±.4 C; Bunog, 7.53±.4 mg O 2 L 1 nd 2.8±.2 C; Khunge, 5.67±.7 mg O 2 L 1 ; 21.5±.3 C. Twenty individuls were cptured live from ech site using minnow trps ited with red. Fish were euthnized in situ with n overdose of uffered MS-222 (1 g L 1 MS-222 nd 4 g L 1 NHCO 3 ). Individuls were sexed nd mesured for mss (g±.1) nd totl length (cm±.1). Fish were then individully wrpped, leled, nd frozen in liquid nitrogen. Frozen smples were trnsported to McGill University in dry shipper, trnsferred to Lurentin University on dry ice, nd finlly stored t 8 C until ssyed. Permission to conduct reserch in Ugnd ws cquired from the Ugnd Ntionl Council for Science nd Technology, Mkerere University nd McGill University's Animl Cre committee (protocol no. 529) Preprtion of tissue homogentes Tissues (white skeletl muscle, hert, rin, nd liver) were quickly dissected on ice, immeditely weighed (g±.1), nd homogenized with Polytron homogenizer (PT 12) for three 2-s intervls in n ice cold uffer consisting of 1 mm of Hepes (ph 7.),.1 mm of dithiotheritol (DTT), 5 μg ml 1 soyen trypsin inhiitor, 5 mm β-mercptoethnol nd.2% Triton X-1. During nd etween homogeniztion, smples were kept on ice. Muscle nd rin were homogenized in 1 or 5 volumes of uffer, while liver nd hert were homogenized in 5 or 1 volumes depending on tissue mss. Homogentes were centrifuged using Thermo IEC Micromx RF Refrigerted Microcentrifuge t 24 g for 15 min t 4 C. Superntnts were kept on ice until enzyme ctivity ws ssyed Selection of enzymes Optiml concentrtions of sustrte, cofctors, nd llosteric modifiers were determined empiriclly for ech enzyme in ech tissue. Under these conditions, V mx my lso e n indictor of enzyme concentrtion, nd therefore n indirect mesure of enzyme gene expression (Pierce nd Crwford, 1994, 1997). We mesured the following enzymes tht prticipte in the indicted metolic pthwys: pyruvte kinse (PK) glycolysis; lctte dehydrogense (LDH) glycolysis; citrte synthse (CS) citric cid cycle; mlte dehydrogense (MDH) citric cid cycle; cytochrome C oxidse (CCO) electron trnsport chin; nd fructose isphosphtse (FBPse) gluconeogenesis. Immeditely prior to enzyme ssys, some superntnts were further diluted to 1-fold reltive to the initil tissue mss using homogeniztion uffer. Rections were initited y the ddition of the sustrte specific for tht enzyme, or in some cses y the ddition of the tissue (underlined). The finl protocols for pyruvte kinse, mlte dehydrogense, nd fructose 1,6-isphosphtse were modified from Mrtínez et l., 26 s follows: Pyruvte kinse (PK; EC ): 1 mm Hepes uffer (ph 7.4); 1 mm KCl (hert, liver) 2 mm KCl (rin), or 4 mm KCl (muscle);.2mmnadh;2.5 mmadp(hert)or1 mmadp(muscle,liver rin);.25 UmL 1 L-LDH (muscle, rin),.5 UmL 1 L-LDH (hert) or 1 U ml 1 L-LDH (liver); 5 mm MgCl 2 (muscle, liver, rin) or 1 mm MgCl 2 (hert); 2mMPEP(muscle,liver,rin) or 4 mm PEP (hert). Mlte dehydrogense (MDH; EC ): 1 mm Hepes uffer (ph 7.4); 1 mm KCl,.2 mm NADH;.2 mm oxlocette. Fructose-1,6-isphosphtse (FBPse; EC ): 1 mm Hepes uffer (ph 7.4); 1 mm KCl;.4 mm NADP;.8 UmL 1 PGI;.72 UmL 1 Glucose-6-phosphte dehydrogense; 6 mm MgCl 2;.5 mm EDTA;.5 mm Fructose 1,6-isphosphte; tissue superntnt. The rection conditions for citrte synthse, cytochrome C oxidse, nd lctte dehydrogense were modified from stndrd protocols. Citrte synthse (CS; EC ): 5 mm Trizm Bse uffer (ph 8.);.1 mm 5, 5 -dithiois-2-nitroenzoic cid (DTNB);.1 mm Acetyl coenzyme A (muscle, hert) or.2 mm Acetyl coenzyme A (liver, rin);.75 mm oxlocette (muscle) or 1.5 mm oxlocette (liver, hert, rin) (Mrtínez et l., 1999). Cytochrome C oxidse (CCO; EC ): 5 mm K 2 HPO 4 nd 5 mm KH 2 PO 4 uffer (ph 7.);.7 mm reduced cytochrome C. Rections were run ginst.7 mm control of cytochrome C oxydized with.33% K-ferricynide; tissue superntnt (Zhou et l., 2).

3 C.D. Crocker et l. / Comprtive Biochemistry nd Physiology, Prt B 164 (213) Lctte dehydrogense (LDH; EC ): 1 mm Hepes uffer (ph 7.);.12 mm NADH; 1. mm pyruvte (Mrtínez et l., 29). Mximl enzyme ctivities were mesured in 96-well microplte reding spectrophotometer (Moleculr Devices, Spectrmx plus 384, Sunnyvle, CA, USA) t 25 C. Rtes from lnk rections (without sustrtes) were sutrcted for determintion of enzyme ctivities. For LDH, MDH, nd PK, rections were mesured y the disppernce of NADH t 34 nm. The reduction of NADP + t 34 nm, ws used to mesure the rection of FBPse. The CS rection ws mesured t 412 nm to detect the trnsfer of sulfhydryl groups from coenzyme A to 5,5 dithiois-2-nitroenzoic cid (DTNB), nd CCO ws mesured t 55 nm to oserve the oxidtion of reduced cytochrome C. The vlue of 3.89, 8.5, nd were used s the millimolr extinction coefficients for NAD(P)H, DTNB nd cytochrome C, respectively. All enzyme ctivities were mesured within 4 h of tissue homogeniztion, nd enzyme ctivity ws stle during this period of time. For the ssys, for ech fish nd for ech specific enzyme, four liquots per tissue were ssyed, rections were liner for 18 s. All chemicls nd iochemicls were purchsed from Sigm-Aldrich Co. (Okville, ON, Cnd), Roche Dignostics Corportion (Mississug, ON, Cnd) nd Thermo Scientific (Rockford, IL, U.S.A.) Protein nlysis Superntnts for protein nlysis were kept t 8 C until nlyzed. The icinchoninic cid (BCA) ssy ws used to determine the protein content in the superntnt frction of tissue homogentes (Smith et l., 1985). The protocol ws modified for use in 96-well microplte reding spectrophotometer (Brown et l., 1989). Smples were diluted using distilled wter to give finl dilution reltive to strting tissue mss of 1 for muscle, hert nd rin, nd 2 for liver. Ten μl of diluted smple ws dded to qudruplictes of 2 μl BCA working regent into individul wells of the 96-well microplte. Stndrds of 1 mgml 1 of ovine serum lumin (BSA) were included s stndrd curve with every plte. Since DTT concentrtion in the originl homogeniztion uffer ws less thn 1 mm, it did not hve ny effect on the solule protein levels mesured (Thermo Fisher Scientific Inc Technicl report TR68.3). Pltes were seled, nd incuted t 6 C for 3 min. After cooling for 1 min, pltes were red t 562 nm t room temperture Clcultions nd sttisticl nlysis All vriles were exmined for normlity using the Shpiro Wilks test, nd tested for homogeneity of vrince efore nlyses were completed. Enzyme specific ctivities re expressed in interntionl units (IUs; μmol sustrte trnsformed to product min 1 ) per milligrm of protein, nd were used for ll sttisticl nlyses. Muscle PK, CS, nd CCO ctivity, ll enzymes nlyzed in hert nd liver, nd rin PK nd CCO were log 1 trnsformed. Fish ody mss ws significnt covrite only in the cse of rin CCO, hert LDH, nd liver CS. Therefore, it ws included in the finl model s covrite for these three tissue enzyme ctivities. For ech response vrile, n ANOVA or ANCOVA ws used to test for effects of site of origin i.e. differences mong the four sites (Tukey post hoc nlysis). Sites were not nested within regions, ecuse we did not hve repliction of high-d.o. nd low-d.o. sites within ech dringe; rther we selected the four sites to mximize vrition in D.O. regime. Dringe effects were inferred vi post hoc comprisons. Sttisticl outliers detected in the ANOVA protocol (individuls where the vlue of the mesured trit ws more thn two stndrd devitions from the men), were removed from nlyses. All sttisticl nlyses were completed using SPSS Sttistics 17. (SPSS Inc., Chicgo, Illinois, U.S.A.). All figures represented re shown using the rw untrnsformed dt. Differences were considered significnt when P Results 3.1. Body size nd condition Fish mss, length, nd Fulton's condition fctor did not differ mong the four smpling sites (Tle 1). However overll, fish mss, length nd Fulton's condition fctor differed etween sexes (F [1,78] =19.523, P.1; F [1,79] =28.138, P.1; F [1, 79] =5.21, P=.27, respectively). In generl, mles were hevier nd longer thn femles, while femles hd greter Fulton's condition fctor thn mles. More specificlly, Bunog mles were hevier nd longer thn Bunog femles (F [1,18] =6.382, P=.21; F [1,18] =7.887, P=.12 respectively), Lke Kynj mles were hevier nd longer thn Lke Kynj femles (F [1,19] =4.457, P.1; F [1,19] =51.441, P.1 respectively), nd Lwmund Swmp mles were longer thn Lwmund Swmp femles (F [1,18] =4.776, P=.42). Furthermore, femles from Khunge hd greter Fulton's condition fctor thn mles (F [1,18] =2.127, P=.3). Sex only ffected the levels of muscle CS ctivity (F [1,78] =6.128, P=.15), where mles showed greter ctivity levels thn femles cross ll sites Tissue protein nd enzyme levels White skeletl muscle In white skeletl muscle, differences were detected mong the four sites for PK (F [3,76] =4.725, P=.4), LDH (F [3,73] =3.366, P=.23), nd CS (F [3,76] =6.95, P=.1), ut not for CCO (F [3,74] = 1.496, P=.223). Interestingly, white skeletl muscle PK nd LDH were significntly greter in fish from one or oth Mpng sites compred to fish from one or oth Nugo sites. Tukey post-hoc nlyses, reveled no differences etween sites within regions; ll differences occurred cross the two regions (Fig. 1), suggesting no cler effect of D.O. regime. PK ctivity ws higher in fish from oth Khunge nd Bunog (fluctuting D.O., Mpng region) thn in fish from Lke Kynj (normoxic, Nugo region). LDH ctivity ws lso higher in fish from Bunog thn in fish from Lke Kynj. Levels of CS in white skeletl muscle in fish from the Mpng sites were similr to those mesured in Lke Kynj, ut lower thn in fish from Lwmund Swmp (Fig. 1) Hert In hert, ll four enzymes were significntly greter in fish from one or oth Mpng sites compred to fish from one or oth Nugo sites [PK (F [3,76] =6.61, P=.1); LDH (F [3,71] =7.86, P.1); CS (F [3,76] =9.867,P.1); CCO (F [3,72] =3.66, P=.17)]. This vrition ppered to e independent of D.O. ecuse hert enzyme ctivities did not differ etween sites within region, despite the lrge difference in D.O. vilility etween the two Nugo sites. Specificlly, we oserved tht PK ctivity ws higher in P. multicolor from the Tle 1 Morphologicl trits of the Africn cichlid Pseudocrenilrus multicolor victorie. The Mpng region includes the Bunog nd Khunge (river, fluctuting dissolved oxygen sites), nd the Nugo region includes Lke Kynj (normoxic site) nd the Lwmund Swmp (hypoxic site). Dt represent mens ± S.E. Morphologicl trits Mpng Nugo Bunog Khunge Lke Kynj Lwmund Swmp Body mss (g) 4.29± ± ± ±.43 Totl length (cm) 6.33± ± ± ±.23 Fulton condition fctor 1.61± ± ± ±.3 (mss length 3 T ) 1 N Note: vlues re mens±s.e.

4 56 C.D. Crocker et l. / Comprtive Biochemistry nd Physiology, Prt B 164 (213) PK 5 LDH Bunog Khunge Lke Kynj Lwmund Enzyme Activity (Units * mg protein -1 ) CS CCO Mpng Nugo Mpng Nugo Fig. 1. White skeletl muscle enzyme ctivity of the Africn cichlid Pseudocrenilrus multicolor victorie cross divergent environments. Smples were collected from two different regions: Mpng (dshed rs) nd Nugo (plin rs). Two sites were smpled within ech region: Bunog (river, fluctuting dissolved oxygen) Khunge (river, fluctuting dissolved oxygen), nd Lke Kynj (normoxic) Lwmund Swmp (hypoxic). Enzymes mesured were PK (pyruvte kinse), LDH (lctte dehydrogense), CS (citrte synthse), nd CCO (cytochrome C oxidse). Letters indicte significnt differences mong sites of origin. Brs with the sme letter do not differ (Tukey post-hoc comprisons, P>.5). Dt re shown s men±s.e. Note: n=2 21 per popultion. Khunge (fluctuting D.O.) site in the Mpng region thn in either Nugo site (Fig. 2). Hert LDH ctivity ws higher in oth Mpng sites thn t either Nugo site (Fig. 2). Regrding the eroic enzymes, CS ctivity ws higher in oth Mpng sites thn in oth Nugo sites Brin In rin, ll enzymes differed mong smpling sites: PK (F [3,77] = 3.499, P=.19); LDH (F [3,76] =6.724, P.1); CS (F [3,76] = 5.465, P=.2); nd CCO (F [3,71] =5.395, P=.2). Agin, post-hoc nlyses did not detect ny differences etween sites within region, only etween sites cross the two regions. PK ctivity ws higher in Khunge thn in Lke Kynj (Fig. 3). Brin LDH ctivity ws significntly lower in Lke Kynj thn in oth Mpng sites. Brin CS levels were higher in fish from Khunge compred to fish from Lke Kynj; while rin CCO ctivity ws higher in the Khunge site thn in the Lwmund Swmp (Fig. 3) Liver Differences were detected mong sites in liver PK ctivity (F [3,76] = 3.496, P=.2), LDH ctivity (F [3,73] =5.835, P=.1), MDH ctivity (F [3,75] =5.971, P=.1), nd FBPse ctivity (F [3,74] =4.972, P=.3). Interestingly, PK ctivity ws higher in fish from Khunge site thn the Bunog site in the Mpng dringe, even though oth sites re chrcterized y fluctuting D.O. (Fig. 4). LDH ctivity ws higher in fish from oth Nugo sites thn in fish from Bunog in the Mpng region. MDH ctivities were higher in the Khunge site thn in oth Nugo sites; while FBPse ctivity ws lower in oth sites in the Mpng dringe thn in fish from the Lwmund Swmp (Fig. 4). 4. Discussion The present study provides further insights into the nturl vrition of tissue metolic potentil of the Africn cichlid P. multicolor victorie. Prior work on this species hs suggested high degree of phenotypic vrition cross popultions from divergent D.O. regimes (Chpmn et l., 22, 28; Mrtínez et l., 29; Crispo nd Chpmn, 21). To chieve roder understnding of phenotypic vrition cross environments, six key enzymes were nlyzed in four tissues of fish collected t two sites within ech of the two regions of Ugnd. In this study, there ws lrge vrition in tissue metolic potentil etween regions, while fewer differences etween sites within regions were oserved, despite vrition in D.O. regimes. We nticipted greter differences etween the sites in the Nugo region, ecuse we smpled during the dry seson, when D.O. is very low in the Lwmund Swmp nd high in Lke Kynj; wheres in the Mpng, similr fluctuting levels of D.O. re oserved throughout the yer, primrily with lower levels of D.O. during the wet seson due to influx from n upstrem ppyrus swmp. However, there were no significnt differences in metolic cpcities etween fish collected from the hypoxic Lwmund Swmp nd those from normoxic Lke Kynj. In generl, P. multicolor from the Mpng River system (Bunog nd Khunge sites) displyed greter neroic enzyme ctivity in white skeletl muscle, nd lower gluconeogenic enzyme ctivity in the liver thn fish from the Nugo region. Previous studies hve suggested tht for terrestril nd qutic orgnisms, n increse in the use of neroic pthwy is criticl when oxygen ecomes scrce, llowing for greter neroic energy production within tissues (Greney et l., 198; Wester, 23). However, some exceptionlly tolernt species cn lso reduce oxygen demnds vi metolic depression to help mtch oxygen supply nd

5 C.D. Crocker et l. / Comprtive Biochemistry nd Physiology, Prt B 164 (213) PK 5 LDH Bunog.6.4 c 4 3 Khunge Lke Kynj Lwmund Enzyme Activity (Units * mg protein -1 ) CS c CCO.1.5 Mpng Nugo Mpng Nugo Fig. 2. Hert enzyme ctivity of the Africn cichlid Pseudocrenilrus multicolor victorie cross divergent environments. Smples were collected from two different regions: Mpng (dshed rs) nd Nugo (plin rs). Two sites were smpled within ech region: Bunog (river, fluctuting dissolved oxygen) Khunge (river, fluctuting dissolved oxygen) nd Lke Kynj (normoxic) Lwmund Swmp (hypoxic). Enzymes mesured were PK (pyruvte kinse), LDH (lctte dehydrogense), CS (citrte synthse), nd CCO (cytochrome C oxidse). Letters indicte significnt differences etween sites of origin; rs with the sme letter do not differ (Tukey post-hoc comprisons, P>.5). Dt re shown s men±s.e. Note: n=2 21 per popultion PK LDH Bunog Khunge Lke Kynj Lwmund.4.5 Enzyme Activity (Units * mg protein -1 ) CS CCO Mpng Nugo Mpng Nugo Fig. 3. Brin enzyme ctivity of the Africn cichlid Pseudocrenilrus multicolor victorie cross divergent environments. Smples were collected from two different regions: Mpng (dshed rs) nd Nugo (plin rs). Two sites were smpled within ech region: Bunog (river, fluctuting dissolved oxygen) Khunge (river, fluctuting dissolved oxygen) nd Lke Kynj (normoxic) Lwmund Swmp (hypoxic). Enzymes mesured were PK (pyruvte kinse), LDH (lctte dehydrogense), CS (citrte synthse) nd CCO (cytochrome C oxidse). Letters indicte significnt differences etween sites of origin; rs with the sme letter do not differ (Tukey post-hoc comprisons, P>.5). Dt re shown s men±s.e. Note: n=2 21 per popultion.

6 58 C.D. Crocker et l. / Comprtive Biochemistry nd Physiology, Prt B 164 (213) PK.2.15 LDH Bunog Khunge Lke Kynj.4.1 Lwmund.2.5 Enzyme Activity (Units * mg protein -1 ) CS CCO 3 MDH.12 FBPse 2 c c Mpng Nugo Mpng Nugo Fig. 4. Liver enzyme ctivity of the Africn cichlid Pseudocrenilrus multicolor victorie cross divergent environments. Smples were collected from two different regions: Mpng (dshed rs) nd Nugo (plin rs). Two sites were smpled within ech region: Bunog (river, fluctuting dissolved oxygen) Khunge (river, fluctuting dissolved oxygen) nd Lke Kynj (normoxic) Lwmund Swmp (hypoxic). Enzymes mesured were PK (pyruvte kinse), LDH (lctte dehydrogense), CS (citrte synthse), CCO (cytochrome C oxidse), MDH (mlte dehydrogense) nd FBPse (fructose 1,6-isphosphtse). Letters indicte significnt differences etween sites of origin; rs with the sme letter do not differ (Tukey post-hoc comprisons, P>.5). Dt re shown s men±s.e. Note: n=2 21 per popultion. demnd (Ji nd Richrds, 28; Richrds, 29). The suggested generl increse in the levels of neroic enzymes ws not oserved in P. multicolor. Rther, this species displyed gret del of vrition mong tissues nd enzymes, similr to findings oserved in other hypoxi tolernt species. For exmple, in the Gulf killifish Fundulus grndis hypoxi exposure ltered the glycolytic ctivity levels of some enzymes however, the enzymes ffected nd the direction of response ws tissue specific(mrtínez et l., 26). Furthermore, in the common crp Cyprinus crpio, the eroic enzymes, CS nd CCO displyed differences in the direction under hypoxi exposure (Zhou et l., 2). It hs een oserved in the mummichog Fundulus heteroclitus, tht differences in gene expression cuse specific chnges within metolic pthwys, improving the cpcity of individuls to survive under vrying environmentl conditions (Oleksik et l., 25; Grnt et l., 27). As such, one cn infer tht prolonged or recurring hypoxi exposure, my work s strong dptive force to trigger protein gene expression chnges, llowing individuls to conserve sufficient levels of ATP s they do not hve to uniformly ctivte or dectivte genes controlling ll of the enzymes of specific metolic pthwy. This my llow individuls living in such fluctuting environments to etter respond to the vrious environmentl chnges oserved with sesons such s D.O. nd food vilility. The pprent lower enzyme ctivity, regrdless of oxygen regime in the Nugo region compred to the Mpng region my reflect long-term dpttion to specific conditions within the metpopultion. Interestingly, this ws more evident in the hert nd rin tissues, which re more metoliclly ctive t rest nd re very importnt in the mintennce of the orgnisms homeostsis (Soengs nd Aldegunde, 22; Nilsson nd Lutz, 24), thn muscle for instnce. Although sesonl vrition in D.O. is chrcteristic of the Lwmund Swmp, levels remin well elow sturtion yer round. The two sites in the Mpng River dringe, Bunog nd Khunge, experience hypoxi in the wet seson, ut rrely rech vlues s low s the miniml vlues in the Lwmund Swmp. Hence, fish from Bunog nd Khunge pper to undergo lrger vrition in the levels of dissolved oxygen through the yer thn fish from the Lwmund Swmp, soliciting therefore, greter level of chnge. On the other hnd, similrities in the enzyme ctivities etween Lwmund Swmp nd Lke Kynj fish,

7 C.D. Crocker et l. / Comprtive Biochemistry nd Physiology, Prt B 164 (213) despite of the gret differences in dissolved oxygen levels, could suggest tht Lwmund Swmp fish re well cclimted to low D.O. levels, prompting over time, very different response to the sme stressor. Hence, fish from Lwmund Swmp my no longer perceive hypoxi s stressor, nd they do not need to respond in the sme roust mnner to this stressor s if they were fcing it for the first time, s oserved in our lortory cclimtion study (Mrtínez et l., 29). The reltively low degree of chnge in D.O. levels occurring in the Nugo region compred to those in the Mpng region, might hve selected for n overll reduction on the metolic cpcity in P. multicolor in order to mtch their metolic needs to their specific environmentl surroundings. In doing so, long term dpttion to such conditions should promote regultion of specific genes for enzymes from different metolic pthwys (Chuukov et l., 212) mximizing survivl nd therefore fitness. This venue could e of interest to pursue y using microrry studies to look more closely t the dptive chnges in gene expression s well s the use of popultion-genomics (Oleksik, 21) to oserve DNA vrition etween individuls nd etween popultions over sesons. Dt from prior study in P. multicolor F 1 offspring from the sme two popultions in the Nugo (Mrtínez et l., 29) suggested tht tissue LDH specific ctivity depends on the popultion from which prentl stock ws smpled. In their study, F 1 offspring were rered under normoxi, nd then the fish were cclimted to hypoxi or normoxi. They found evidence for popultion effect with high levels of rin LDH in fish of hypoxic swmp origin. Although similr trend ws oserved in the present study, the difference ws not sttisticlly significnt. Rther, in the present study, vrition in tissue enzyme ctivity is more clustered y region thn y dissolved oxygen regime (within region). The difference etween the studies highlights the vlue of smpling oth field popultions nd fish rered under controlled lortory conditions. In the field popultions, fish hve developed under divergent D.O. environments, ut these hitts lso differ in environmentl fetures tht my ffect tissue enzyme ctivity. The results of our field study do mirror previous results from our reserch group, where genetic structure of P. multicolor ws clustered y geogrphicl region etween Mpng River sites nd Nugo sites, thn ccording to dissolved oxygen regime (Crispo nd Chpmn, 28). In fct, in three out of the four tissues exmined, regionl difference ws detected. Levels of PK ctivity in the muscle, nd levels of PK, LDH, nd CS ctivity in the hert were higher in fish from the Mpng region compred to those inhiting the Nugo region. The opposite trend ws oserved in liver FBPse ctivity, with greter FBPse ctivity occurring in P. multicolor from the Nugo region. With the exception of liver PK ctivity, we did not detect differences etween enzyme ctivity levels etween sites within region, regrdless of the oxygen regime. As stted ove, other environmentl fctors etween regions tht we did not consider during this study, such s food vilility, food qulity, wter flow, nd predtion pressure my hve gret impct on the tissue metolic potentil. For exmple, other studies hve shown tht reduction of food intke or diverse type of energy source (crohydrtes, lipid, nd proteins) cn hve direct effect on orgnisms' metolism (Pilkis nd Grnner, 1992; Méton et l., 1999; Enes et l., 29). Although we did not directly mesure levels of glucose or glycogen, chnges in key enzymes cn e used to infer vrition in feeding regimes s well s to indicte fish nutritionl sttus nd condition. Due to the fct tht smpling occurred during the dry seson, it is most likely tht the lower levels of PK enzyme ctivity oserved in fish from the Bunog site re due to reduced food vilility or qulity s hs een oserved in other fish (Cowey et l., 1977; Mrtínez et l., 22; Kirchner et l., 23). Lower levels of FBPse from oth sites in the Mpng my indicte lower gluconeogenic cpcity s oserved in strved fish (Enes et l., 29). On the other hnd, wter flow my lso e plying role in determining tissue metolic potentil. The sites in the Mpng region were oth flowing wter sites, wheres the two sites in the Nugo region re chrcterized y low or no flow. The slightly greter tissue glycolytic ctivity oserved in the white skeletl muscle of individuls from the Mpng River sites my suggest greter urst swimming cpcity during prey cpture or to escpe from predtors s oserved in yellow perch, lke trout nd cod (Sherwood et l., 22; Mrtínez et l., 24; Rennie et l., 25). Previous studies of P. multicolor hve demonstrted high level of developmentl plsticity in morpho-physiologicl trits such s gill size nd rin size in response to divergent oxygen regimes in the l, where lrger gill size is chrcteristic of field popultions from low-oxygen sites in oth the Mpng nd Nugo regions (Chpmn et l., 2, 28; L. Chpmn unpulished dt). Therefore, it is possile tht the lrge gill size long with other morphophysiologicl trits, oserved in low-oxygen sites my serve s primry mechnism to extrct oxygen in the hypoxic environment, reducing the need to compenste metoliclly, s hs een oserved in different cclimtion or cclimtiztion experiments (Pnepucci et l., 2; Mrtínez et l., 29, 211). In conclusion, when we comine our results of vrition in tissue enzyme ctivities nd the genetic structure of P. multicolor, there is some indiction tht specific regionl chrcteristics nd geogrphic distnce my e fcilitting locl dpttion to environments on regionl scle, ut t the sme time, gene flow my limit locl dpttion of tissue metolic potentil etween divergent hitts on locl scle s seen in other species (Grnt et l., 27; Crispo nd Chpmn, 21; Somero, 21). Acknowledgments This work ws supported y the Ntionl Science nd Engineering Reserch Council of Cnd (discovery grnt to MLM nd LJC), Lurentin University nd McGill University Reserch Funds (MLM nd LJC); the Ntionl Science Foundtion (IBN to LJC), the Cnd Foundtion for Innovtion, the Wildlife Conservtion Society nd the Cnd Reserch Chir progrm (LJC). We thnk B.B. Rees for feedck on erlier versions of this mnuscript. References Brown, R.E., Jrvis, K.L., Hylnd, K.J., Protein mesurement using icinchoninic cid: elimintion of interfering sustnces. Anl. Biochem. 18, Chpmn, L.J., Glis, F., Shinn, J., 2. Phenotypic plsticity nd the possile role of genetic ssimiltion: hypoxi induced trde-offs in the morphologicl trits of n Africn cichlid. Ecol. Lett. 3, Chpmn, L.J., Nordlie, F.G., Siefert, A., 22. Respirtory oxygen consumption mong groups of Pseudocrenilrus multicolor victorie sujected to different oxygen concentrtions during development. J. Fish Biol. 61, Chpmn, L.J., Alert, J., Glis, F., 28. Developmentl plsticity, genetic differentition, nd hypoxi-induced trde-offs in n Africn cichlid fish. Open Evol. J. 2, Chippri-Gomes, A.R., Gomes, L.C., Lopes, N., Vl, A.L., Almeid-Vl, V.M.F., 25. Metolic djustments in two Amzonin cichlids exposed to hypoxi nd noxi. Comp. Biochem. Physiol. B 141, Chuukov, V., Zulet, I., Li, H., 212. Regultory rchitecture determines optiml regultion of gene expression in metolic pthwys. Proc. Ntl. Acd. Sci. 19, Cowey, C.B., Knox, D., Wlton, M.J., Adron, J.W., Regultion of gluconeogenesis y diet nd insulin in rinow-trout (Slmo girdneri). Br. J. Nutr. 38, Crmpton, W.G.R., Chpmn, L.J., Bell, J., 28. Interspecific vrition in gill size is correlted to mient dissolved oxygen in the Amzonin electric fish Brchyhpopomus (Gymnotiformes: Hypopomide). Environ. Biol. Fish. 83, Crispo, E., Chpmn, L.J., 28. Popultion genetic structure cross dissolved oxygen regimes in n Africn cichlid fish. Mol. Ecol. 17, Crispo, E., Chpmn, L.J., 21. Geogrphic vrition in phenotypic plsticity in response to dissolved oxygen in n Africn cichlid fish. J. Evol. Biol. 23, Dvies, R., Moyes, C.D., Wng, Y.S., 211. Inr- nd inter-specific vrition in metolic gene expression in reltionship to environmentl hypoxi. Comp. Biochem. Physiol. A159, DeKoning, A.B.L., Picrd, D.J., Bond, S.R., Schulte, P.M., 24. Stress nd inter-popultion vrition in glycolytic enzyme ctivity nd expression in teleost fish, Fundulus heteroclitus. Physiol. Biochem. Zool. 77, Diz, R.J., Rosenerg, R., 28. Spreding ded zones nd consequences for mrine ecosystems. Science 321, Diz, R.J., Breiturg, D.L., 29. The Hypoxic Environment. In: Richrds, J.G., Frrell, A.P., Bruner, C.J. (Eds.), Hypoxi in fishes. Elsevier Inc., Sn Diego, pp

8 6 C.D. Crocker et l. / Comprtive Biochemistry nd Physiology, Prt B 164 (213) 53 6 Enes, P., Pnsert, S., Kushik, S., Oliv-Teles, A., 29. Nutritionl regultion of heptic glucose metolism in fish. Fish Physiol. Biochem. 35, Frwell, M., Fox, M.G., Moyes, C.D., Burness, G., 27. Cn hypoxi tolernce explin differences in distriution of two co-occurring north temperte sunfishes? Environ. Biol. Fish. 78, Grnt, D., Forde, S.E., Hendry, A.P., 27. The multifrious effects of dispersl nd gene flow on contemporry dpttion. Funct. Ecol. 21, Greney, G.S., Plce, A.R., Cshon, R.E., Smith, G., Powers, D.A., 198. Time course of chnges in enzyme ctivities nd lood respirtory properties of killifish during long-term cclimtion to hypoxi. Physiol. Zool. 53, Hochchk, P., 198. Living Without Oxygen: Closed nd Open Systems in Hypoxi Tolernce. Hrvrd University Press, Cmridge. Hochchk, P., Buck, L.T., Doll, C.J., Lnd, S.C., Unifying theory of hypoxi tolernce: moleculr/metolic defence nd rescue mechnisms for surviving oxygen lck. Proc. Ntl. Acd. Sci. 93, Ji, L.A., Richrds, J.G., 28. AMP-ctivted protein kinse ctivity during metolic rte depression in the hypoxic goldfish, Crssius urtus. J. Exp. Biol. 211, Kirchner, S., Kushik, S., Pnsert, S., 23. Low protein intke is ssocited with reduced heptic gluconeogenic enzyme expression in rinow trout (Oncorhynchus mykiss). J. Nutr. 133, Lnd, S.C., Buck, L.T., Hochchk, P.W., Response of protein-synthesis to noxi nd recovery in noxi-tolernt heptocytes. Am. J. Physiol. 265, R41 R48. Mndic, M., Todghm, A., Richrds, J.G., 29. Mechnisms nd evolution of hypoxi tolernce in fish. Proc. R. Soc. B 276, Mrtínez, M., Couture, P., Guderley, H., Temporl chnges in tissue metolic cpcities of wild Atlntic cod Gdus morhu (L.), from Newfoundlnd. Fish Physiol. Biochem. 2, Mrtínez, M., Guderley, H., Nelson, J.A., Weer, D., Dutil, J.D., 22. Once fst cod, lwys fst cod: mintennce of performnce hierrchies through chnging food vilility in cod (Gdus morhu). Physiol. Biochem. Zool. 75, 9 1. Mrtínez, M., Bédrd, M., Dutil, J.D., Guderley, H., 24. Does condition of Atlntic cod (Gdus morhu) hve greter impct upon swimming performnce t U crit or sprint speeds? J. Exp. Biol. 27, Mrtínez, M.L., Lndry, C., Boehm, R., Mnning, S., Cheek, A.O., Rees, B.B., 26. Effects of long-term hypoxi on enzymes of crohydrte metolism in the Gulf killifish, Fundulus grndis. J. Exp. Biol. 29, Mrtínez, M.L., Chpmn, L.J., Rees, B.B., 29. Popultion vrition in hypoxic responses of the cichlid Pseudocrenilrus multicolor victorie. Cn. J. Zool. 87, Mrtínez, M.L., Rynrd, E., Rees, B.B., Chpmn, L.J., 211. Oxygen limittion nd tissue metolic potentil of the Africn fish Brus neumyeri: Roles of ntive hitt nd cclimtiztion. BMC Ecol. 11, 2. McNeil, G.V., 212. Hypoxi, crotenoids, nd colour expression in the widespred Africn cichlid fish Pseudocrenilrus multicolor victorie. M.Sc. thesis, McGill University, Montrel, Cnd. Méton, I., Medivill, D., Csers, A., Cntó, E., Fernández, F., Bnnte, I.V., Effects of diet composition nd rtio size on key enzymes ctivities of glycolysis gluconeogenesis, the pentose phosphte pthwy nd mino cid metolism in liver of gilthed se rem (Sprus urt). Br. J. Nutr. 82, Nilsson, G.E., Lutz, P.L., 24. Anoxi tolernt rins. J. Cere. Blood Flow Met. 24, Oleksik, M.F., 21. Genomic pproches with nturl fish popultions. J. Fish Biol. 76, Oleksik, M.F., Roch, J.L., Crwford, D.L., 25. Nturl vrition in crdic metolism nd gene expression in Fundulus heteroclitus. Nt. Genet. 37, Pnepucci, L., Fernndes, M.N., Snchez, J.R., Rntin, F.T., 2. Chnges in lctte dehydrogense nd mlte dehydrogense ctivities during hypoxi nd fter temperture cclimtion in the rmored fish, Rhinelepis strigos (Siluriformes, Loricriide). Rev. Brs. Biol. 6, Pierce, V., Crwford, D., Rpid enzyme ssys investigting the vrition in the glycolytic pthwy in field-cught popultions of Fundulus heteroclitus. Biochem. Genet. 32, Pierce, V.A., Crwford, D.L., Phylogenetic nlysis of therml cclimtion of the glycolytic enzymes in the genus Fundulus. Physiol. Zool. 7, Pilkis, S.J., Grnner, D.K., Moleculr physiology of the regultion of heptic gluconeogenesis nd glycolysis. Annu. Rev. Physiol. 54, Pollock, M.S., Clrke, L.M.J., Dué, M.G., 27. The effects of hypoxi on fishes: from ecologicl relevnce to physiologicl effects. Environ. Rev. 15, Rerdon, E.E., Chpmn, L.J., 29. Hypoxi nd life-history trits in eurytopic Africn cichlid. J. Fish Biol. 75, Rerdon, E.E., Chpmn, L.J., 21. Energetics of hypoxi in mouth-rooding cichlid: evidence for interdemic nd developmentl effects. Physiol. Biochem. Zool. 83, Rennie, M.D., Collins, N.C., Shuter, B.J., Rjotte, J.W., Couture, P., 25. A comprison of methods for estimting ctivity costs of wild fish popultions: more ctive fish oserved to grow slower. Cn. J. Fish. Aqut. Sci. 62, Richrds, J.G., 29. Metolic nd Moleculr Responses of Fish to Hypoxi. In: Richrds, J.G., Frrell, A.P., Bruner, C.J. (Eds.), Hypoxi in fishes. Elsevier Inc., Sn Diego, pp Rosenerger, A.E., Chpmn, L.J., 2. Respirtory chrcters of three species of hplochromine cichlids: implictions for use of wetlnd refugi. J. Fish Biol. 57, Sherwood, G.D., Pzzi, I., Moeser, A., Hontel, A., Rsmussen, J.B., 22. Shifting gers: enzymtic evidence for the energetic dvntge of switching diet in wild-living fish. Cn. J. Fish. Aqut. Sci. 59, Smith, P.K., Krohn, R.I., Hermnson, G.T., Mlli, A.K., Grtner, F.H., Provenzno, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., Mesurement of protein using Bicinchoninic cid. Anl. Biochem. 15, Soengs, J.L., Aldegunde, M., 22. Energy metolism of fish rin. Comp. Biochem. Physiol. B 131, Somero, G.N., 21. The physiology of climte chnge: how potentils for cclimtiztion nd genetic dpttion will determine winners nd losers'. J. Exp. Biol. 213, Wester, K.A., 23. Evolution of the coordinte regultion of glycolytic enzyme genes y hypoxi. J. Exp. Biol. 26, Zhou, B.S., Wu, R.S.S., Rndll, D.J., Lm, P.K.S., Ip, Y.K., Chew, S.F., 2. Metolic djustments in common crp during prolonged hypoxi. J. Fish Biol. 57,