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1 qutic Toxicology 162 (2015) ontents lists ville t ScienceDirect qutic Toxicology journl homepge: Trnscriptionl nd iochemicl mrkers in trnsplnted Perc flvescens to chrcterize cdmium- nd copper-induced oxidtive stress in the field Michel. Defo, Louis erntchez, Peter G.. mpell, Ptrice outure, Institut Ntionl De L Recherche Scientifique (INRS), entre Eu Terre Environnement, 490 De L ouronne, Quéec, Q G1K 99, nd Institut De iologie Intégrtive Et Des Systèmes (IIS), Université Lvl, Quéec, Q G1V 06, nd rticle info strct rticle history: Received 17 Jnury 2015 Received in revised form 19 Ferury 2015 ccepted 20 Ferury 2015 ville online 21 Ferury 2015 Keywords: Wild fish Metl pollution Reciprocl trnsplnttion iomrkers Plsticity trits Genomic rection norm Despite recent progress chieved in elucidting the mechnisms underlying locl dpttion to pollution, little is known out the evolutionry chnge tht my e occurring t the moleculr level. The gol of this study ws to exmine ptterns of gene trnscription nd iochemicl responses induced y metl ccumultion in clen yellow perch (Perc flvescens) nd metl depurtion in contminted fish in mining nd smelting region of nd. Fish were collected from reference lke (lke Opstic) nd d, u nd Zn contminted lke (lke Dufult) locted in the Rouyn-Nornd region (Qc, nd) nd cged for one or four weeks in their own lke or trnsplnted in the other lke. Free-rnging fish from the sme lkes were lso collected. Kidney d nd u concentrtions in clen fish cged in the contminted lke incresed with the time of exposure, ut metl depurtion did not occur in contminted fish cged in the clen lke. fter 4 weeks, the mjor retinoid metolites nlysed, the percentge of free dehydroretinol (droh) nd the retinol dehydrogense-2 (rdh-2) trnscription level in liver decresed in clen fish trnsplnted into the metl-contminted lke, suggesting tht metl exposure negtively impcted retinoid metolism. However, we oserved n increse in lmost ll of the retinoid prmeters nlysed in fish from the metl-impcted lke cged in the sme lke, which we interpret s n dpttion response to higher mient metl concentrtion. In support of this hypothesis, liver trnscription levels of microsoml glutthione-s-trnsferse-3 (mgst-3) nd glucose-6-phosphte dehydrogense (g6pdh) were enhnced in clen fish trnsplnted into the metl-contminted lke nd this up-regultion ws ccompnied y n increse in the ctivities of corresponding enzymes, involved in ntioxidnt response. However, lthough in the sme fish the trnscription level of u/zn superoxide dismutse (u/zn sod) ws lso incresed, this did not led to chnge in the ctivity of the SOD enzyme, suggesting tht this upregultion ws imed t mintining SOD-relted ntioxidnt cpcities. In contrst, the trnscription level of the ct gene, which did not chnge in contminted fish, did not compenste for the decrese of T ctivity. fter 4 weeks of exposure, some plstic responses of the clen fish were oserved when they were trnsplnted in the metl-contminted lke. However, proly s consequence of the prior 80 yers of exposure to metls, the contminted popultion showed limited plstic response in the expression of the mjority of the cndidte genes tested, when they were trnsplnted in the reference lke. The overll findings of this field investigtion highlight how yellow perch moleculrly nd iochemiclly responded to sudden or reltively long-term exposure (4 weeks) to cocktil of metls Elsevier.V. ll rights reserved. 1. Introduction orresponding uthor. Tel.: ; fx: It is rodly ccepted tht nthropogenic ctivities re ffecting ll ecosystems. mong these ctivities, pollution sources including metl mining nd smelting hve contriuted to drstic chnges in environmentl conditions tht could potentilly led to chnges in X/ 2015 Elsevier.V. ll rights reserved.

2 40 M.. Defo et l. / qutic Toxicology 162 (2015) iodiversity (Eisler, 2004). nimls my use three routes (dispersion, phenotypic plsticity or evolutionry dpttion) to etter respond to these stressors or shifts in environmentl conditions (Hnsen et l., 2012). In fvourle ecologicl nd physicl conditions such s n sence of physicl rriers, they will disperse to new loctions. However, in most cses the dispersl of fish to suitle nery hitts is hrd to chieve, nd therefore they rect vi phenotypic plsticity to express prticulr trits (Merilä nd Hendry, 2014) or vi evolutionry dpttion if chnges in environmentl conditions occur over the long term (Hnsen et l., 2012). In some cses, popultions fil to dpt to nthropogenic stress. For instnce, sulphur dioxide emissions from se metl smelters hve led to the rpid cidifiction of lkes nd consequent species extinctions (Gonzlez et l., 2013). Generlly, studies deling with the development of plstic trits hve een crried out for only one point in time, therey ignoring the time dependency of chnges in gene expression, the intensity of expression nd the nture of genes (sme or not) tht were involved in the plstic development etween two time points (uin-horth nd Renn, 2009). When ssessing the mechnisms of ction of pollutnts, it is criticl to evlute the time point when the differences in gene expression occurred, ecuse it cn offer vlule tool to identify genes tht trigger plstic development (uin-horth nd Renn, 2009). Wheres, dptive stress response genes my e involved in short exposure periods, dditionl sets of genes my ecome involved s the exposure time increses, in order to mintin niml homeostsis (Denslow et l., 2007). In support of this hypothesis, using cdn microrry on flounders (Pltichthys flesus) exposed to different slinities in oth short-term (1 dy) nd long-term (50 dys) reciprocl-trnsplnttion experiments etween the North se nd the ltic se, Lrsen et l. (2007) demonstrted tht the numer of genes significntly nd differentilly expressed incresed with exposure time, indicting plstic response to locl chnge in environmentl conditions (Lrsen et l., 2007). ompred to most other fish species, the yellow perch (Perc flvescens) is considered s iomonitor ecuse it fulfils severl criteri (Giguère et l., 2004). It is undnt nd widely distriuted cross North mericn freshwter ecosystems nd is prticulrly relevnt in metl toxicology studies ecuse it tolertes metls such s cdmium (d) nd copper (u) t high concentrtions (Hontel et l., 1995; Hontel et l., 1992; Rjotte nd outure, 2003). This species possesses efficient iochemicl defence mechnisms (e.g. incresing metllothionein concentrtions with incresing metl levels in its tissues; (Giguère et l., 2005), which llow it to live in metl impcted res. Nevertheless, metl concentrtions in yellow perch tissues hve een correlted with iologicl impcts such s the metolic imlnce of vitmin 2 (Defo et l., 2012), impirment of metolic cpcities (outure nd Kumr, 2003), poor condition nd overll helth (outure et l., 2008; Rjotte nd outure, 2002) nd elevted metllothionein concentrtion (Giguère et l., 2005). In recent investigtion on the mechnisms of d nd Ni toxicity in yellow perch, we identified trnscriptionl nd functionl signtures specific to d nd Ni exposure. The results indicte tht metls, prticulrly d, ffect the trnscription level of genes nd the ctivity of enzymes involved in oxidtive stress response nd vitmin metolism (Defo et l., 2014). In n erlier study in evolutionry ecotoxicology, negtive correltions etween heptic concentrtions of cdmium, ut not copper, nd genetic diversity hve een reported in wild yellow perch living in metlimpcted res (ourret et l., 2008). Results from nother study crried out in the copper nd gold mining region of Rouyn-Nornd hve shown tht metls, especilly d, induce chnges in the yellow perch genome y selecting lleles tht increse the fitness of perch inhiting metl-impcted lkes (élnger-deschênes et l., 2013). oth studies support the hypothesis tht pollution cn trigger nturl selection. Gene expression cn lso e directly informtive out popultion dptive genetic divergence (Lrsen et l., 2007). When ttempting to test hypotheses imed t identifying possile ecologicl drivers, trits nd genes involved in the dpttion to chnging environmentl conditions, one pproch involves the comprison of the trnscription levels of genes mong different exposure conditions (ôté et l., 2014; Hoffmnn nd Dorn, 2007; Meier et l., 2014). mong nturl popultions, vrition in gene expression hs een shown to ply prominent role in ecologicl dpttion (Derome nd erntchez, 2006; Pvey et l., 2011; St-yr et l., 2008). The expression of mrn ws reported to explin c. 40% of the vrince in protein expression (reu et l., 2009) mening tht chnges in gene trnscription levels do not necessrily involve modifictions in the concentrtions of the corresponding enzymes (Giulini et l., 2013; Regoli et l., 2011). It is therefore importnt to tke into ccount functionl nd genomic informtion when designing genomic studies (Furlong, 2011) insted of simply discussing functionl outcomes (e.g. chnge in enzymes ctivities) on the sis of trnscriptomic dt (Nikinm et l., 2013). hnge in response to the interctions etween n orgnism nd it environment cn e oserved t different levels including morphologicl, ehviorl or physiologicl. The generic term of genomic rection norm is generlly used to descrie the reltionship etween trit nd environment for given genotype, or to define the extent of plsticity of tht trit (uin-horth nd Renn, 2009). In eroic orgnisms, oxygen, in its norml configurtion (O 2 ) is somewht unrective; however, it cn e trnsformed into rective oxygen species (ROS) in response to chnging environmentl or metolic conditions (Gliszczyńsk-Świglo, 2006; Scndlios, 2005). ROS production cn led to cellulr oxidtive stress, itself inducing lipid peroxidtion, DN nd protein dmge nd other dverse effects (hen et l., 2013; Leonrd et l., 2004). Metls hve different modes of generting or incresing cellulr ROS (Defo et l., 2014; Lushchk, 2011). In order to counterlnce oxidtive dmge, gene trnscription levels of cellulr ntioxidnt response nd the ctivities of enzymes involved in these metolic pthwys increse in response to higher cellulr ROS (Uren Wester et l., 2013). Studies hve reported tht yellow perch living in metl impcted res possess efficient iochemicl defence mechnisms (Giguère et l., 2005). oth non-enzymtic ntioxidnt iomrkers such s vitmin metolites nd enzymtic ntioxidnt iomrkers including superoxide dismutse (SOD), ctlse (T), glutthione-s-trnsferse (GST) nd glucose-6-phosphte dehydrogense (G6PDH) interct together to minimize the dmging effects of ROS in fish (Scndlios, 2005). In previous lortory study where yellow perch were seprtely exposed to queous d (7.0 nmol/l nd 71 nmol/l) nd Ni (1020 nmol/l nd 10,200 nmol/l) for 6 weeks, we oserved tht the enzyme ctivities of liver retinyl ester hydrolse nd lecithin dehydroretinyl cyl trnsferse incresed in fish exposed to higher d. However, no mesurle effect ws oserved in fish exposed to Ni either t lower or higher Ni concentrtion (Defo et l., 2014). mong ntioxidnt defences, increses in liver T nd G6PDH ctivities were oserved in fish exposed to higher d, however no effects on liver microsoml GST nd SOD ctivity were reported (Defo et l., 2014). Exposure to Ni did not ffect the ctivities of these enzymes except for T where n increse in liver T ctivity ws oserved t the higher Ni concentrtion. In contrst to the responses in liver, muscle T nd GST ctivities decresed or remined unchnged with metl exposure, except t low d concentrtions, where the ctivities incresed (Defo et l., 2014). Levesque et l. (2002) reported tht the liver G6PDH ctivity ws low in perch inhiting the most metl-contminted lkes, in summer, ut this ctivity remined

3 M.. Defo et l. / qutic Toxicology 162 (2015) Fig. 1. Study re nd lkes smpled. The reference lke is indicted in green nd the metl-contminted lke in red. The cross refers to the point source of metl contmintion in the Rouyn-Nornd region of Quéec. high compred to tht of their homologs cught in fll of the sme yer (Levesque et l., 2002). In the present reciprocl trnsplnttion field experiment using cged fish, trnscriptionl nlysis coupled with iochemicl nlyses were employed to investigte the effects of metl exposure on retinoid metolism nd oxidtive stress response iomrkers in yellow perch. Our specific ojectives were: (i) to exmine temporl chnges in liver trnscription levels of set of toxicologicllyrelevnt genes involved in the oxidtive stress response in yellow perch exposed up to four weeks to metls; (ii) to compre gene expression levels with corresponding iochemicl endpoints (enzyme ctivities nd retinoid storge) in order to determine if metl-induced chnges in gene trnscription levels led to significnt physiologicl effects; (iii) to compre responses of popultion from clen site to those t site where the popultion hs een exposed to contminnts for 80 yers nd could hve therefore developed dpttion to cope with the contminted environment; nd (iv) to determine t wht time scle chnges in the metolic response pper s result of metl ccumultion or depurtion. 2. Mterils nd methods 2.1. Study re, fish smpling nd reciprocl trnsplnttions Two lkes were selected long the polymetllic concentrtion grdient in the Rouyn-Nornd region (Quéec-nd) (Fig. 1). Since 1927, copper smelter hs een in opertion in this region nd metl concentrtions (d, u nd Zn) in lkes situted downwind from the smelter re mrkedly higher thn in lkes locted upwind (onhm-rter et l., 2006). Lke Dufult is closely situted, c. 2 km, downwind from the smelter nd ws chosen s the metl-contminted lke (on) due to historicl tmospheric Tle 1 Totl queous nd sediment concentrtions of trce metls d, u nd Zn in the study. omprtment Wter Sediments Metl [d] (nm) [u] (nm) [Zn] (nm) [d] ( mol/g dw) [u] ( mol/g dw) [Zn] ( mol/g dw) References Opstic (Ponton, 2010) (Giguère et l., 2004) (Giguère et l., 2006) < (orgmnn et l., 2004) < (roisetière et l., 2006) (Fortin et l., 2010) (Kremer et l., 2008) (Ponton, 2010) (Ponton, 2010) Dufult (Giguère et l., 2004) (Giguère et l., 2006) (orgmnn et l., 2004) (roisetière et l., 2006) (Fortin et l., 2010) (Kremer et l., 2008) Lke smpled in 2010 y Dominic Ponton, INRS-ETE, unpulished dt.

4 42 M.. Defo et l. / qutic Toxicology 162 (2015) Fig. 2. Experimentl design of ll exposure conditions: 1Ref-Op, 1Ref-Du, 1on-Op, 1on-Du, 4Ref-Op, 4Ref-Du, 4on-Op, 4on-Du, 0Ind-Op nd 0Ind-Du (see Tle 2 for detils of the experimentl design). deposition of reltively high levels of metls such s d, u nd Zn (Tle 1). This hs resulted in incresed levels of metls in the wter column, sediments nd in prey. Lke Opstic, locted c. 30 km upwind of the metl emission site, ws selected s the reference lke (Ref.) ecuse of the reltively low metl concentrtions in the wter column, sediments nd prey. Furthermore erlier work hs shown tht Zn concentrtions re tightly controlled in yellow perch (e.g. Giguère et l., 2004, 2005). t the time of first smpling (My 22, 2012), lke Opstic nd lke Dufult wter physico-chemicl properties were, respectively: ph (7.81 ± 0.03 nd 7.53 ± 0.07); oxygen (10.11 ± 0.72 mg/l nd 9.01 ± 0.15 mg/l) nd temperture (17.6 ± 1.7 nd 17.2 ± 2.5 ). Vlues re expressed s mens ± stndrd error. Other wter qulity vriles were not mesured in this study; however, some dt were otined from previous studies crried out in 2010 in oth lkes (Dominic Ponton INRS-ETE, unpulished dt). The mjor ction nd nion concentrtions found in lke Opstic nd lke Dufult were: c. (1 nd 0.39 mmol/l); Mg (0.11 nd 0.11 mmol/l); N (0.13 nd 0.17 mmol/l); K (0.025 nd mmol/l); l (0.09 nd 0 mmol/l); SO 4 (0.06 nd 9 mmol/l). The cge experiments were crried out in spring 2012 ccording to the pproch developed in Kremer et l. (2005,); Kremer et l. (2005,), with slight modifiction. In rief, plnktivorous juvenile yellow perch (2.47 ± 0.07 g; 6.57 ± 0.07 cm; men ± SE) were collected etween My 22 24, 2012 either in lke Opstic, herefter Op or in lke Dufult, herefter Du, nd reciproclly trnsplnted into cges locted in oth lkes. The cge wlls were mde of 5 mm mesh netting, llowing free movement of wter nd food into the cges. totl of 8 cges were used (Fig. 2), creting 4 condition regimes for ech exposure period (1 nd 4 weeks). The reciprocl-trnsplnttion experiment conditions re explined in Tle 2. lso, t the eginning of the trnsplnttion phse nd t 1 nd 4 weeks, free-rnging fish were collected from ech lke. However, only metl nlyses were performed on free-rnging fish smpled t 1 nd 4 weeks. The effect of cging itself, which compres fish cged in their own lke with free-rnging fish, ws exmined in nother study (Julie Grsset, INRS-ETE, unpulished Tle 2 Exposure condition, percentge of mles nd yellow perch mortlity in the reciprocl trnsplnttion experiment. Opstic is the reference lke nd Dufult is the contminted lke. ondition Percentge of mles Percentge of mortlity Explntion 0Ind-Op 10 Free-rnging indigenous lke Opstic fish smpled t the eginning of experiment 0Ind-Du 10 Free-rnging indigenous lke Dufult fish smpled t the eginning of experiment 1Ind-Op 10 Free-rnging indigenous lke Opstic fish smpled t 1week 1Ind-Du 5 Free-rnging indigenous lke Dufult fish smpled t 1 week 1Ref-Op Fish from the reference lke cged in the reference lke for one week 1Ref-Du Fish from the metl-contminted lke cged in the reference lke for one week 1on-Op Fish from the reference lke cged in the metl-contminted lke for one week 1on-Du Fish from metl-contminted lke cged in metl-contminted lke for one week 4Ind-Op 31 Free-rnging indigenous lke Opstic fish smpled t 4 week 4Ind-Du Free-rnging indigenous lke Dufult fish smpled t 4 week 4Ref-Op Fish from the reference lke cged in the reference lke for four weeks 4Ref-Du Fish from the metl-contminted lke cged in the reference lke for four weeks 4on-Op Fish from the reference lke cged in metl-contminted lke for four weeks 4on-Du Fish from metl-contminted lke cged in the metl-contminted lke for four weeks The numer prefix in the condition column indictes the time of exposure (weeks).

5 M.. Defo et l. / qutic Toxicology 162 (2015) Tle 3 Nucleotide sequences of specific prtil coding sequence (primer) pirs nd PR products used in RT-qPR nlysis. Gene revitions Nme nd functions Specific primers PR products ˇ-ctin Reference gene F: 5 - GTTTGTTT-3 From Pierron et l. (2014) R: 5 - GGGGGTTGTT-3 rdh-2 Photoreceptor ssocited retinol dehydrogense 2 F: 5 -GTGGTGTT (retinoid nd oxidtive stress metolism) R: 5 - TGGGG-3 ct tlse (oxidtive stress metolism) F: 5 - GTTTTTTGTTGGTG R: 5 - GTGGTTTG-3 mgst-3 Microsoml glutthione S trnsferse-3 (oxidtive stress F: 5 - TTTTGTGGTT metolism) R: 5 - TGTTGTTTGTT-3 g6pdh Glucose 6 phosphte dehydrogense (pentose phosphte F: 5 - GGGGTGTTTGGTGT nd oxidtive stress metolism) R: 5 - TTTGTGTGGGTGGGG-3 sod u/zn superoxide dismutse (oxidtive stress metolism) F: 5 -TGGGGGGGGGTTT R: 5 -TGTGTGGTTTGT-3 F: forwrd primer. R: reverse primer. dt). Ech cge initilly contined fish, nd t the end of ech exposure period the remining fish (up to mximum of 20) were smpled from ech cge. The percentge of fish mortlity in ech cge is reported in Tle 2. Lengths nd weights were recorded nd sex determined. Livers were dissected for moleculr nd iochemicl nlyses, kidneys for metl nlyses nd pyloric cæc for iometric nlyses. Tissues were immeditely frozen nd stored in liquid nitrogen in the field. Upon rrivl in the lortory, smples were plced in freezer t 80 where they were kept until used for nlyses. The fish mnipultion protocol ws pproved y the Ministère des Ressources Nturelles et de l Fune du Quéec nd y the omité Institutionnel de Protection des nimux (IP) of INRS lcultion of fish condition index (Kn) nd corrected pyloric cæc weights The reltive condition fctor (Kn) ws clculted using the Eq. Kn = (Wf/L 3.16 ) 100 (where Wf is fish weight, L is the totl fish length) (Guthier et l., 2009). The corrected pyloric cæc weights were clculted ccording to the method descried elsewhere (Guthier et l., 2011) with 2.6 g s the stndrd fish weight nd 0.92 s the llometric exponent nlyses of kidney metl concentrtions, liver quntittive RT-PR, determintion of retinoid nd protein concentrtions nd enzyme ctivities dmium, u nd Zn were nlyzed in kidney tissues fter digestion in trce metl grde nitric cid ccording to Pierron et l. (2009), using n inductively coupled-plsm tomic emission spectrophotometer (IP-ES). Internl stndrds were within 10% of nominl vlues in ll cses nd men metl recoveries from the reference mteril nlyzed (TORT-2, loster heptopncres, Ntionl Reserch ouncil of nd, Ottw, ON) were 94.8 ± 0.4% for d, 85.3 ± 1.3% for u nd 89.0 ± 0.9% for Zn (men ± S.E.). RN isoltion, cdn synthesis nd nlysis of liver gene trnscription levels were performed in ccordnce with methods nd technicl stndrdiztion procedures cited in Defo et l. (2014). The prtil coding sequences (primers) used in this study re listed in Tle 3. ll the iochemicl prmeters, including liver retinoid concentrtions nd enzyme ctivities (T, SOD, G6PDH nd GST) nlyzed in this study were conducted using the protocol descried y Defo et l. (2014), without modifiction Dt nlysis To test for differences in iometric prmeters, metl concentrtions, enzyme ctivities nd retinoid concentrtions mong experimentl conditions, we used one-wy nlysis of vrince (NOV), fter checking the ssumption of normlity (Levene test, p > 0.05). We lso used NOV to evlute the differences in gene trnscription levels mong conditions nd to determine the degree of plsticity (genomic rection norm) mong experimentl conditions. When the proility ws significnt (p < 0.05), multiple comprison tests (Tukey HSD test) were used to identify differences etween two conditions. When the ssumption of normlity ws not met, nd when log 10 or ox-cox (Peltier et l., 1998) trnsformtion of the dt ws unle to normlize the distriution, non-prmetric pired Wilcoxon tests were pplied. For vitmin determintion, sttisticl nlyses were performed without considering sex s n explntory vrile. proility of p < 0.05 indictes sttisticlly significnt difference from the control experiment condition. Results were expressed s mens ± stndrd error (SE). The reltionships etween individul kidney metl concentrtions nd liver gene trnscription level, iochemicl nlysis or iometric prmeters were investigted using the non-prmetric Spermn rnk correltion test. nlyses were performed using JMP 9.0 softwre. 3. Results 3.1. Kidney metl ccumultion dynmics Kidney metl concentrtions reflected the metl contmintion sttus of the two studied lkes. t the eginning of the experimenttion (t = 0), metls ccumulted in kidneys of free rnging-fish from the reference lke Opstic (0Ind-Op) (Fig. 3) were 4.6 nd 1.3 times lower for d nd u, respectively, thn those for fish in the metl-impcted lke Dufult (0Ind-Du) (Fig. 3). This trend ws confirmed in indigenous free-rnging fish smpled t t = 1 week. Kidney metl concentrtions in free-rnging fish collected t t = 4 weeks in the metl-impcted lke (Fig. 3) were lso significntly higher (p < 0.05) for d, u nd Zn (4.5, 1.9 nd 1.5 times, respectively) thn those in fish from the reference lke (Fig. 3). Metl ccumultion ptterns in yellow perch kidneys vried over the trnsplnttion period. Kidney d concentrtion of fish from reference lke Opstic cged in metl-impcted lke Dufult (4on-OP) incresed over time in comprison to reference fish cged in lke Opstic (4Ref-Op) (Fig. 3). The increse ws not significnt fter 1 week of exposure (1on-Op), ut significnt (p < 0.05) increse ws oserved t the end of experiment (4on- Op) (Fig. 3), lthough the d concentrtion ws lower thn tht of fish living nturlly in lke Dufult (4 Ind-Du) (Fig. 3). similr ut more pronounced trend ws lso seen for u (Fig. 3). No trend in kidney Zn ccumultion ws oserved in cged fish (Fig. 3). Unlike the results otined in the contmintion experiment, kidney d, u nd Zn concentrtions of fish from the metl-impcted lke trnsplnted into the reference lke were not different from those in fish tht remined cged in lke Dufult (Fig. 3).

6 44 M.. Defo et l. / qutic Toxicology 162 (2015) Kidney d nd u concentrtion (μmol.g dry weight-1) c d u Zn αβ e β d αβ d β α αβ α Kidney Zn concentrtion (μmol.g dry weight-1) 0Ind-Op 1Ind-Op 1Ref-Op 1on-Op 4Ind-Op 4Ref-Op 4on-Op Exposure condition Kidney d nd u concentrtion (μmol.g dry weight-1) d u Zn β β β β β α α β Kidney Zn concentrtion (μmol.g dry weight-1) 0Ind-Du 1Ind-Du 1Ref-Du 1on-Du 4Ind-Du 4Ref-Du 4on-Du Exposure condition Fig. 3. Totl cdmium, copper nd zinc concentrtions in the kidney of indigenous yellow perch smpled fter 1 nd 4 weeks for the contmintion experiment (Fig. 3; lke Opstic specimens) or for the depurtion experiment (Fig. 3; lke Dufult specimens). Free-rnging perch from lke Opstic (Ind-Op) nd lke Dufult (Ind-Du), respectively, represent control fish for contmintion nd depurtion experiments. on-op nd on-du denote fish cged in lke Dufult, while Ref-Op nd Ref-Du represent fish cged in lke Opstic. The numer prefix in the exposure conditions indictes the time of exposure (weeks). rs re mens ± stndrd error (SE) (11 N 20/condition). Mens designted with different letters for given metl re significntly different (p-vlue < 0.05). Different letters (cpitl letters for d, lower cse letters for u nd greek letters for Zn) represent comprisons of kidney metl concentrtions mong exposure conditions Fish condition index (Kn) nd corrected pyloric cæc weights In the metl contmintion experiment (lke Opstic to lke Dufult), nlysis of iometric prmeters reveled little difference mong the experimentl conditions. Indeed trnsplnted fish (1on-Op nd 4on-Op) displyed higher condition fctors thn their corresponding control fish (1Ref-Op) nd (4Ref-Op), respectively (Tle 4). However, no chnge in pyloric cæc weights ws oserved either t 1 week or 4 weeks in trnsplnted fish compred to control fish (Tle 4). Trnsferring fish from the contminted lke to the reference lke(lke Dufult lke Opstic) did not significntly ffect the iometric prmeters nlysed, except for the fish trnsplnted for 4 weeks (4Ref-Du), which displyed higher condition fctors thn control fish (4on-Du) (Tle 4). significnt

7 M.. Defo et l. / qutic Toxicology 162 (2015) Tle 4 Numer of fish in ech smpling group (N), reltive condition index (Kn), corrected pyloric cæc weight nd mjor vitmin. concentrtions in liver of indigenous freernging yellow perch from lke Opstic (0Ind-Op) nd lke Dufult (0Ind-Du) nd in yellow perch cged in lke Dufult (on-op nd on-du) or in lke Opstic (Ref-Op nd Ref-Du) smpled fter 1 nd 4 weeks (see Section 2.1). Vlues re men ± SE nd mens within columns designted with different letters re significntly different (p-vlue < 0.05). iometric prmeters Retinoid metolism ondition N Reltive index condition (Kn) orrected pyloric cæc wet weight (mg) [droh] [droh-plmitte] [Totl vitmin esters] 0Ind-Op 20 2 ± ± ± ± c ± c 0Ind-Du ± ± ± ± c ± c 1Ref-Op 20 1 ± ± 0.9 c ± ± c ± c 1Ref-Du 20 3 ± ± ± ± c ± c 1on-Op ± ± ± ± 0.03 c ± c 1on-Du 8 2 ± ± ± ± c 37 ± c 4Ref-Op 17 5 ± ± 0.4 cd ± c 50 ± ± Ref-Du ± ± 0.8 cd ± ± ± on-Op ± ± 0.3 d ± c ± c 24 ± c 4on-Du 20 5 ± ± 0.4 d ± ± ± ϒ N =6. The numer prefix in the condition column indictes the time of exposure (weeks). Expressed in mol/g liver wet weight. reltionship ws oserved etween Kn nd kidney u concentrtions ( = 3; p = ) nd etween pyloric cæc weights nd kidney u concentrtions ( = 0.19; p = 0.014), for ll fish pooled hnges in liver retinoid levels during metl ccumultion nd depurtion s expected, ll-trns-3,4-didehydroretinoids were the predominnt form of vitmin found in yellow perch liver. fter 4 weeks of metl exposure, the concentrtion of dehydroretinyl plmitte (droh-plmitte) nd the min form of vitmin storge significntly decresed in fish from lke Opstic cged in lke Dufult (4on-Op) in comprison to control fish (4Ref-Op) (Tle 4). The percentge of free dehydroretinol (droh) lso significntly decresed in fish from lke Opstic trnsplnted in lke Dufult (4on-Op) in comprison to control fish (4Ref-Op) (Fig. 4; left pnel). There were no significnt differences mong ny of the forms of vitmin nlysed in fish from the metl-impcted lke tht were cged in their own lke or in the reference lke (Tle 4 nd Fig. 4; right pnel), except for the concentrtion of free liver droh, which decresed in fish cged in lke Opstic fter 4 weeks (4Ref-Du) (Tle 4) Response of moleculr iomrkers of oxidtive stress during metl ccumultion nd depurtion In the metl ccumultion experiment, liver rdh-2 trnscription levels were lower in fish exposed to metls compred to controls (1Ref-Op nd 4Ref-Op), nd the decrese ws significntly more pronounced fter 4 weeks of exposure compred to 4Ref-Op) (Fig. 4; left pnel). Liver ct trnscription levels were not significntly different in fish exposed to metls compred to controls (Fig. 4; left pnel). However, liver mgst-3 (Fig. 4; left pnel), g6pdh (Fig. 4D; left pnel) nd u/zn sod (Fig. 4E; left pnel) trnscription levels were significntly higher in fish exposed to metls compred to controls, nd the increses were more pronounced fter 4 weeks of exposure. In the metl depurtion experiment, liver ct (Fig. 4; right pnel), mgst-3 (Fig. 4; right pnel) nd u/zn sod (Fig. 4E; right pnel) trnscription levels hd decresed fter 1 week. However no chnge in liver rdh-2 (Fig. 4; right pnel) nd g6pdh (Fig. 4D; right pnel) trnscription levels ws oserved compred to controls. fter 4 weeks, the trnscription levels of the mesured genes were no longer different, except for liver rdh-2, which hd trnscription level 1.9 times higher in fish exposed to low metls (4Ref-Du) thn in control fish (4on-Du) (Fig. 4; right pnel). Positive correltions were oserved etween kidney u concentrtions nd u/zn sod, mgst-3 nd g6pdh trnscription levels; correltions were not significnt with kidney d concentrtions (Fig. 5). We chose not to present correltions with Zn ecuse no significnt results were otined etween the studied prmeters nd kidney Zn concentrtions hnges in oxidtive stress relted enzyme ctivities during metl ccumultion nd depurtion fter 4 weeks, the ctivity of liver T hd decresed in fish trnsplnted from lke Opstic to lke Dufult (4on-Op) (Fig. 4; left pnel). In contrst, the ctivities of liver GST (Fig. 4; left pnel) nd G6PDH (Fig. 4D; left pnel) were significntly higher in fish tht hd een exposed to high metl concentrtions (4on- Op), reltive to the control (4Ref-Op). Liver SOD ctivity hd not incresed significntly s consequence of metl exposure fter 4 weeks (Fig. 4E; left pnel). No effect of metl depurtion ws detected on the ctivity of the nlysed enzymes when fish were trnsplnted from lke Dufult to lke Opstic (4Ref-Du), reltive to the corresponding control (4on-Du) (t = 4 weeks; Fig. 4 E; right pnel). lthough gene trnscription levels were not correlted with kidney d concentrtions, the corresponding iochemicl prmeters nlysed were positively correlted except for liver SOD ctivity, for which negtive ut significnt correltion ws oserved (Fig. 5) Metl genomic rection norm: response to chnging environment Differentils in gene trnscription levels showing the representtion of trit vlues in reltion to chnges in environmentl conditions (clen fish cged in the contminted lke, contminted fish cged in the clen lke, or free rnging fish cged in their own lke) were estimted fter 1 week (Fig. 6 left pnel) nd 4 weeks (Fig. 6 right pnel). mong the five genes nlysed, the results clerly indicte tht ptterns of genomic rection norms vry depending on fish origins nd smpling time. fter 1 week none of the genes nlysed were trnscriptionlly induced or inhiited in either the metl contmintion or the metl depurtion experiments (Fig. 6 left pnel). With regrd to three of the

8 46 M.. Defo et l. / qutic Toxicology 162 (2015) Fig. 4. Liver gene trnscription level (men ± SE) of rdh-2 (), ct (), mgst-3 (), g6pdh (D) nd u/zn sod (E) nd corresponding iochemicl nlysis of indigenous yellow perch smpled fter 1 nd 4 weeks for the contmintion experiment (lke Opstic specimens; left pnel) or for the depurtion experiment (lke Dufult specimens; right pnel). Free-rnging perch from lke Opstic (Ind-Op) nd lke Dufult (Ind-Du), respectively, represent control fish for contmintion nd depurtion experiments. on-op nd on-du denote fish cged in lke Dufult, while Ref-Op nd Ref-Du represent fish cged in lke Opstic (see Section 2.1). The numer prefix in the exposure condition indictes the time of exposure (weeks). Mens designted with different letters for given prmeter re significntly different (p-vlue < 0.05; 6 N 8/condition). Different letters (cpitl letters for gene trnscription level nd lower cse letters for iochemicl nlysis) represent comprisons of ech prmeter mong exposure conditions. ND: not determined.

9 M.. Defo et l. / qutic Toxicology 162 (2015) Liver g6pdh (RQ) Trnscription level Enzyme ctivity 0Ind-Op 1Ref-Op 1on-Op 4Ref-Op 4on-Op Exposure condition Liver G6PDH (U gprotein-1) D Liver g6pdh (RQ) Trnscription level Enzyme ctivity ND 0Ind-Du 1Ref-Du 1on-Du 4Ref-Du 4on-Du Exposure condition Liver G6PDH (U g protein-1) Liver u/zn sod (RQ) Trnscription level Enzyme ctivity 0Ind-Op 1Ref-Op 1on-Op 4Ref-Op 4on-Op Exposure condition Liver SOD (U mg protein-1) E Liver u/zn sod (RQ) Trnscription level Enzyme ctivity ND 0Ind-Du 1Ref-Du 1on-Du 4Ref-Du 4on-Du Exposure condition Liver SOD (U mg protein-1) Fig. 4. (ontinued.) nlysed genes (u/zn sod, mgst-3 nd g6pdh), it ppers tht fish from lke Dufult showed reduced response reltive to fish from lke Opstic where no difference in expression ws oserved etween the rering environments; wheres, this ws the cse for lke Dufult perch. fter 4 weeks the nlysis of the depurtion effect reveled cler response only with rdh-2 gene, where the gene incresed in expression (Fig. 6; right pnel). However, in the metl contmintion experiment this gene ws under-expressed in comprison with their homologs tht remined cged in clen lke (Fig. 6; right pnel). tlse gene expression ws not trnscriptionlly induced or inhiited in fish from lke Opstic cged in lke Dufult (Fig. 6; right pnel). In contrst, more pronounced nd progressive response ws noticed t the end of the experiment for u/zn sod, mgst-3 nd g6pdh in fish from lke Opstic cged in lke Dufult (Fig. 6 E, respectively, right pnel). 4. Discussion We hve inevitly collected nd nlysed only specimens found live t the end of ech experiment. ging induced high rte of mortlity, especilly fter four weeks (Tle 2). Nevertheless, survivors ppered helthy nd their condition index nd feeding ctivity illustrted y the corrected pyloric cæc weights were comprle to vlues in free-rnging fish. Mortlity in cges ws likely due to fctors other thn contmintion, since there ws no evidence tht it ws higher in fish cged in the contminted lke. In the sence of predtors, dominnce nd competition for food resources in restricted environments like cges or quri led to differentil growth rtes mong individuls incresing over time, which cn induce selection for helthier, fster-growing individuls. Regrdless of the cuses of mortlity, our dt suggest tht lthough survivors my differ from

10 48 M.. Defo et l. / qutic Toxicology 162 (2015) S permn correltion coefficient (ρ) S permn correltion coefficient (ρ) Gene trnscription level iochemicl nlysis d T SOD GST G6PDH DROH Gene trnscription level iochemicl nlysis Metolic pthwys u T SOD GST G6PDH DROH Metolic pthwys Fig. 5. Spermn correltion coefficients () etween kidney d nd u concentrtions nd (i) gene trnscription level of liver ct, u/zn sod, mgst-3, g6pdh nd rdh-2, (ii) corresponding iochemicl nlysis of indigenous free-rnging yellow perch from lke Opstic (0Ind-Op) nd lke Dufult (0Ind-Du) (oth control lkes for contmintion nd depurtion experiments t the strt of the experiment or 0 week, respectively) nd in yellow perch cged in lke Dufult (on-op nd on-du) or in lke Opstic (Ref-Op nd Ref-Du) smpled fter 1 nd 4 weeks. (60 N 80), p < their popultion of origin in terms of vriility of the iomrkers exmined, fish smpled in cges remin comprle mong themselves Effects of metl contmintion nd depurtion on kidney metl concentrtion lthough we did not mesure the concentrtions of dissolved metls in wter or sediment in this study, mpell et l. (2008) reported 22-fold difference in queous d concentrtion (the min metl responsile for deleterious effects on qutic orgnisms) etween the two study lkes. Others uthors (Tle 1) hve reported mjor differences in queous metl concentrtions etween the reference lke Opstic nd the contminted lke Dufult, rnging roughly from 22 to 70-fold for d, 2 to 6-fold for u nd 58 to 78-fold for Zn. Importnt differences were lso oserved in the sediment metl concentrtions of the two lkes, with fctors of 28, 52, 29.4 etween lke Dufult nd lke Opstic for d, u nd Zn, respectively. In this study, liver tissue ws prioritized for iochemicl nd moleculr nlysis nd kidney for metl nlysis. Previous studies hve reported good correltion etween liver nd kidney metl concentrtions in indigenous yellow perch nd demonstrte tht kidney metl contmintion is suitle surrogte for estimting metl ccumultion (outure et l., 2008,; Kremer et l., 2005,). In response to the differences in mient metl concentrtions, kidney metl (especilly d) ccumultion ws much lower in fish living in the reference lke (lke Opstic) thn in fish from the metl-impcted lke (lke Dufult) (Fig. 3 nd ), thus confirming the contmintion sttus of oth study lkes. Unsurprisingly, metl-nïve fish cged in the contminted lke hd incresed their renl d nd u concentrtions fter 4 weeks of exposure. Unlike the cse for u, kidney d concentrtions in trnsplnted fish hd not reched levels similr to those of indigenous cged fish from lke Dufult, indicting tht stedy-stte ws not reched during the experimentl period. dmium, non-essentil metl, my hve ccumulted more slowly in comprison to u or Zn, which re oth essentil metls. Our study grees with tht of Kremer et l. (2005), who oserved n increse in kidney d concentrtions fter yellow perch where trnsplnted from lke Opstic to lke Dufult for 30 dys. However, in their study, no cler temporl trend in kidney u concentrtions ws oserved. No significnt loss of metls from the kidney for either smpling period ws oserved in fish cught in the metl-impcted lke nd held within the reference lke (Fig. 3). lthough not surprising, this result indictes tht the iologicl depurtion times of kidney d, u nd Zn re longer thn 30 dys. To our knowledge, the only other study reporting metl depurtion in indigenous fish is tht of Kremer et l. (2005). Juvenile yellow perch living in metlimpcted lke (high d, u nd Zn) were held for up to 75 dys in cges within reference lke (low d, u nd Zn). Gill nd gut d concentrtions decresed rpidly, ut the iologicl hlf lives of d in the kidney nd liver were 52 nd 75 dys, respectively. The depurtion of excess u lso occurred more rpidly in the liver nd gut compred to the kidney nd gills (Kremer et l., 2005). Our study comined with theirs suggests tht metl depurtion rtes re metl-nd tissue-specific. lthough our experiment took plce within single seson (spring), it is importnt to note the increse in kidney metl concentrtions in free-rnging fish tht occurred over the experimentl period, prticulrly in fish collected t t = 4 weeks. However, decrese in kidney d nd u concentrtions ws oserved in freernging fish smpled t t = 1 week in comprison to those collected t the eginning of the experiment. Food vilility likely plyed role in metl uptke during our smpling period. Indeed, fish from the reference lke cged in the metl-impcted lke displyed higher vlues of Kn, metric ssocited with recent feeding ctivity, nd higher pyloric cæc weights, reflecting higher feeding rte nd food conversion efficiency (ergot et l., 1981; Guthier et l., 2008), compred to their homologs cged in the reference lke. The positive reltionship oserved etween Kn nd kidney u concentrtions in our study further supports the ide tht tissue metl ccumultion during the cging experiment ws likely enhnced due to incresed feeding ctivity ( = 3; p = ). However, we cnnot rule out tht the increse in metl ccumultion is due to higher queous metl exposure or to n increse in the proportion of trophiclly ville metl in prey (Luom nd Rinow, 2005).

11 M.. Defo et l. / qutic Toxicology 162 (2015) Opstic 1.4 Opstic Liver rdh-2 (RQ) Dufult Liver rdh-2 (RQ) Dufult Ref.-lke ont.-lke Ref.-lke ont.-lke Opstic Opstic Liver ct (RQ) Ref.-lke ont.-lke Dufult Liver ct (RQ) Ref.-lke ont.-lke Dufult Liver u/zn sod (RQ) Ref.-lke ont.-lke Opstic Dufult Liver u/zn sod (RQ) Ref.-lke Opstic Dufult ont.-lke Liver mgst-3 (RQ) Ref.-lke ont.-lke Opstic Dufult D Liver mgst-3 (RQ) Ref.-lke Opstic Dufult ont.-lke Liver g6pdh (RQ) Ref.-lke ont.-lke Opstic Dufult E Liver g6pdh (RQ) Ref.-lke Opstic Dufult ont.-lke Fig. 6. Genomic rection norm of rdh-2 (), ct (), u/zn sod (), mgst-3 (D) nd g6pdh (E) of indigenous yellow perch from lke Opstic nd lke Dufult cged in lke Dufult (ont.-lke) or in lke Opstic (Ref.-lke) nd smpled fter 1 week (left pnel) nd 4 weeks (right pnel) of exposure. Mens designted with different letters re significntly different (p-vlue < 0.05). pitl letters represent comprison etween fish from lke Dufult cged in contminted nd references lkes, nd lowercse letters indicte comprison etween fish from lke Opstic cged in reference nd contminted lkes. Vlues (expressed in reltive quntifiction) re men nd stndrd errors (SE).

12 50 M.. Defo et l. / qutic Toxicology 162 (2015) Effect of metl contmintion on liver retinoid metolism In the present field study, the heptic retinoid concentrtions of fish originting from lke Opstic decresed fter 4 weeks exposure to metls, suggesting tht exposure to high d nd u levels negtively impcted yellow perch liver retinoid metolism. lthough this result contrsts with previous study where we reported positive correltions etween kidney d concentrtions nd liver dehydroretinoids (free nd esterified form) in wild fish collected in metl impcted lkes (Defo et l., 2012), it is supported y our recent lortory study where heptic retinoid concentrtions hd decresed fter 6-week exposure to wterorne d nd Ni (Defo et l., 2014). fter 4 weeks, exposure to elevted queous d nd u concentrtions cused down-regultion of the trnscription level of liver rhd-2, gene encoding for the synthesis of enzymes involved in the oxidtion of retinol to retinldehyde for retinoic cid iosynthesis (Lee et l., 2009). This result contrsts with our recent study where we reported tht liver rhd-2 ws mrkedly up-regulted in fish chroniclly exposed in cocktil of metls in the field (M.. Defo, INRS-ETE, unpulished dt). The discrepncy etween these studies could e due to multiple fctors such s the exposure time (4 weeks here versus chronic exposure); the nture of fish smpled (cptivity ginst free-rnging fish) nd the interctions of multiple stressors found in the nturl environment (for exmple diet qulity nd vilility) or individul iometric prmeters such s fish sex, size nd ge, tht proly ffected retinoid metolism (Defo et l., 2014). We lso oserved tht down-regultion of this gene ws coupled with decrese in the percentge of liver free droh. This synchronous rection t the moleculr nd iochemicl levels indictes tht metls negtively impct retinoid metolism t oth levels of iologicl orgniztion. This result is in greement with our previous studies where we reported negtive correltions etween kidney d concentrtions nd the percentge of liver free droh in fish inhiting metl-impcted lkes (Defo et l., 2012). However, the current investigtion lso demonstrted tht when perch living nturlly in metl-impcted lke were cged in the sme lke, their liver retinoid concentrtions (free droh, drohplmitte nd totl vitmin esters) nd the percentge of free liver droh were ll significntly higher thn those of metl-nïve perch cged in the sme metl-impcted lke for 4 weeks. This oservtion of higher retinoid prmeters in the metl-contminted fish cged in their own lke suggests compenstory mechnism in order to counterct the direct effects of metl induced oxidtive stress (Defo et l., 2012). It is likely tht more thn 80 yers of metl pollution hve led to the selection of trits tht enhnced the retinoid metolic cpcity of wild perch. Our present finding is in greement with our previous report where we demonstrted tht dult perch from d-impcted lkes displyed significntly higher concentrtions of liver droh nd droh-esters thn fish from reference lkes (Defo et l., 2012). Lke Dufult perch my hve cquired some tolernce to metls through physiologicl cclimtiztion or dpttion. For exmple, recent study showed tht metl contmintion hs driven rpid dptive evolution y fvoring llelic selection of genes tht could increse yellow perch fitness in metl-impcted res (élnger-deschênes et l., 2013). lso, higher nucleoside diphosphte kinse (NDPK) ctivity, n indictor of iosynthetic cpcities, ws reported in yellow perch inhiting metl-impcted lke in the region of Sudury (Rjotte nd outure, 2002). In support of these higher physiologicl cpcities, fish from lke Dufult cged in the sme lke exhiited higher liver eroic cpcities (higher cytochromec-oxidse ctivity) thn did fish from lke Opstic held cptive in lke Dufult for 4 weeks (M.. Defo, INRS-ETE, unpulished dt) Effect of metl depurtion on liver trnscriptionl nd iochemicl mrkers nlysed In contrst to the yellow perch from lke Opstic cged in the contminted lke Dufult, fish from lke Dufult trnsplnted in Opstic did not show chnges in trnscriptionl nd iochemicl response fter 1 nd 4 weeks, except for liver rdh-2 where the exposure to low queous d nd u concentrtions cused n up-regultion of the trnscription level of this gene. Uptke rtes, tissue ioccumultion levels nd depurtion rtes of metls re importnt prmeters tht define metl toxicity. The results indicte tht depurtion time in this study (4 weeks) ws not enough to significntly limit the production of ROS, resulting in no chnges in the trnscription levels nd in the ctivities of the ntioxidnt enzymes, compred to the control group (4on-Du). In support of this, no significnt loss of metls from the kidney for either smpling period ws oserved in fish cught in the metl-impcted lke nd held within the reference lke. Our results suggest tht rhd-2 trnscription level ws up-regulted proly in order to ctivte the synthesis of retinoid metolites involved in fish growth. In support of this hypothesis, we found tht fish trnsplnted from the metl-contminted lke to the clen lke displyed higher condition fctors nd hevier pyloric cæc weights thn their corresponding control fish (4on-Du), lthough not significntly for the ltter prmeter Reltionships etween gene trnscription levels nd functionl responses This is the first study exmining the time course of the effects of metl exposure on trnscriptionl nd functionl regultion in metl-nïve wild perch cged in contminted lke. We hve identified sets of genes tht were differently expressed nd consistently ssocited with iochemicl responses to metl (especilly d) stress, suggesting tht the trnscriptome nd iologicl function of fish cn e significntly ltered in response to d exposure in the wild. lthough ll genes nlysed re involved in ntioxidnt defense mechnisms, the responses of individul genes ws vrile depending on the durtion of metl exposure. onsistent with the hypothesis tht d induced oxidtive stress nd therefore might increse the level of cellulr ROS, n increse in the trnscription level of most of the genes studied ws oserved. omining moleculr nlysis nd iochemicl responses, our study hs demonstrted tht genes involved in the response to metl-induced oxidtive stress were differentilly expressed in fish from lke Opstic following 4 weeks of cclimtiztion to cocktil of metls in lke Dufult, compred to their homologs cged in lke Opstic. Moreover we oserved differences in the ctivities of the corresponding enzymes. Specificlly, fter 4 weeks, yellow perch trnsplnted into the metl-impcted lke displyed high trnscription levels of liver g6pdh, mgst-3, u/zn sod, presumly in order to reduce the level of ROS generted in the metl-exposed fish down to levels noted in the reference fish. Furthermore, the up-regultion of some of the studied genes resulted in n increse in enzyme ctivities (e.g. GST nd G6PDH), indicting synchronous rection t the moleculr nd iochemicl levels to counterct the oxidtive stress engendered y metl exposure. Typiclly, trnsltionl responses re expected to follow the trnscriptionl responses (Nikinm nd Rytkönen, 2011). Our results suggest tht in metl-contminted environments regultion of the relevnt metolic pthwys might e chieved t oth levels of iologicl orgniztion. Our results re in greement with our previous lortory study where we reported consistent reltionships etween liver mgst-3 nd g6pdh trnscription levels (up-regultion) nd corresponding enzyme ctivities (incresing) in yellow perch exposed to d (Defo et l., 2014).

13 M.. Defo et l. / qutic Toxicology 162 (2015) lthough we reported high trnscription level of liver u/zn sod in fish originting from lke Opstic nd trnsplnted into lke Dufult, there ws no mesurle effect of metls on SOD ctivity, suggesting tht regultion of liver trnscription is only sufficient to mintin SOD ctivity. It is not surprising tht gene trnscription levels of some ntioxidnts increse wheres no chnge is reported t the functionl level (Giulini et l., 2013). s mentioned ove, there is dely etween trnscription nd trnsltion in eukryotic cells nd multiple regultion steps re involved in trnscription nd trnsltion (Regoli et l., 2011). Unlike the responses of liver GST nd G6PDH ctivities, liver T ctivity hd decresed with metl exposure fter 4 weeks; however, no chnge in ct trnscription level ws oserved. These results suggest tht, for this prmeter, the response t the ctlytic level is more sensitive to metl-induced oxidtive stress thn is the trnscriptionl response when wild perch re exposed to d nd u in the field for reltively long time. decrese in T ctivity is often compensted for y n increse in other ntioxidnt enzyme ctivities, s n lterntive mechnism for ROS removl (Regoli et l., 2011); ntioxidnt defence enzymes ct co-opertively or synergisticlly in order to fford n optiml protection ginst oxidtive stress (gnyukov et l., 2006). The present results contrst with our previous lortory oservtions of good greement etween liver ct trnscription level (upregultion) nd T ctivity (increse) when perch were exposed to d for 6 weeks (Defo et l., 2014). The difference etween the two studies could e due to the multiple stress fctors tht operte in the nturl environment. Indeed some oxidtive stress iomrkers my e more sensitive to metl pollution due to synergistic or comined effects of fctors, such s higher temperture, tht my exert their influence in nturl environment (Lpointe et l., 2011) Time scle of chnges in metolic responses nd metl genomic rection norm This field experiment hs dded temporl dimension to our previous study in which we reported the impct of incresing metl concentrtions on retinoid metolism in wild perch (Defo et l., 2012). lthough multiple stressors co-exist in nturl environments, we hypothesized tht the results of our present field mnipultion experiment would proly e comprle to those of our recent lortory study, in which we exposed perch to n environmentlly relevnt concentrtion of d for six weeks. Indeed, s in our previous lortory experiment, we found tht liver trnscription levels of some ntioxidnt defence genes (mgst-3, g6pdh) were enhnced nd tht this up-regultion ws ccompnied y n increse in the ctivities of the corresponding enzymes (Defo et l., 2014). In ddition to the intensity of contmintion, it is importnt to determine the time point t which dverse effects of pollutnts my occur in order to etter understnd the impcts of metl contmintion in nturl popultions. In this reciprocl-trnsplnttion experiment, plstic responses were generlly oserved fter 4 weeks of exposure for ll of the genes nlysed, except for ct for which the response might tke more thn 4 weeks. However, the responses of the trnscription levels of liver mgst-3 nd u/zn sod egn fter 1 week. This result suggests tht the numer of genes involved in oxidtive stress responses my vry over time (Denslow et l., 2007). Indeed, 1 week of exposure to d nd u my e insufficient to induce chnges in the ctivities of ntioxidnt enzymes nlysed. Studies hve reported specific genomic rection norms in response to chnges of environmentl conditions such s temperture in rown trout (Meier et l., 2014) or slinity in eels (ôté et l., 2014). To our knowledge, our s is the first study reporting genomic rection norm to metls using reciprocl trnsplnt experiments directly in the nturl environment. lthough loss of plsticity ws oserved in the contminted popultion, our results support n importnt role for phenotypic plsticity in explining yellow perch dpttion to metl-induced oxidtive stress when clen fish from lke Opstic were trnsferred into lke Dufult. nlysis of metl contmintion effect showed cler plstic effect for lmost ll genes nlysed t the end of the experiment. Similr ptterns of plsticity (progressive) were oserved in the expression of u/zn sod, mgst-3 nd g6pdh. This degree of plsticity could e linked to the extent to which fish re comtting oxidtive stress. However, rdh-2 expression showed different ptterns of plsticity, suggesting tht yellow perch used different strtegies to cope with metl-induced oxidtive stress. Finlly, ct expression did not show ny vrition etween the two groups of cged fish, indicting tht ct is the gene with the lest plstic expression mong those studied. The different genomic rections oserved etween lke Opstic yellow perch trnsplnted into lke Dufult nd those cged in their own lke of origin, fter 4 weeks, possily reflect n dptive plstic response to the chnge in the environment. This is likely the cse since they were cuslly linked with plstic responses oserved t the iochemicl level, except for the ctlse pthwy. Since ll of the genes nlysed re involved in the cellulr response to oxidtive stress, our study supports the ide tht the genomic response of nïve perch to metl exposure is strongly oriented towrds comtting oxidtive stress. In contrst to the yellow perch from lke Opstic cged in the contminted lke Dufult, fish from lke Dufult trnsplnted in Opstic did not show pronounced plstic response fter 4 weeks. This difference in genomic rection norms for t lest three genes supports the hypothesis of genetic differences etween the two popultions. lso, the two previous studies indicted tht incresed metl exposure led to n importnt reduction in diversity (élnger-deschênes et l., 2013; ourret et l., 2008). Such loss of diversity could perhps impct on the potentil for mintining plstic response. Overll then, our results support the hypothesis tht more thn 80 yers of metl exposure my hve driven rpid evolutionry chnge in the lke Dufult popultion genome (élnger-deschênes et l., 2013; ourret et l., 2008). To generlize this hypothesis would require the nlysis of more popultions. Other fctors, such s epigenetic effects (induction of phenotypic vrition without DN sequence ltertion) (Pierron et l., 2014; Richrds, 2006), my lso explin the oserved differences in gene expression etween the two fish groups (Pierron et l., 2014). Indeed, lthough the yellow perch popultions used in this study were similr in ody weight nd length nd originted from the sme region with similr nturl mcro-environments, epigenetic mechnisms could exert crucil influence on phenotypic plsticity nd on the interctions etween the fish nd their environment, therey modifying gene expression nd contriuting to environmentlly-induced phenotypic chnges (ngers et l., 2010). 5. onclusion This study indictes tht 4-week exposure period in contminted environment is sufficient for metl-nïve juvenile perch to significntly increse their kidney d nd u concentrtions. However, more time is necessry to depurte metls tht hve ccumulted over the course of their lives. Our results clerly show tht four-week exposure of fish from the clen lke to metls negtively ffected indictors of retinoid metolism nd incresed oxidtive stress relted prmeters, t oth the moleculr nd iochemicl levels. lso, this study revels tht perch from clen popultions my prtly djust to contmintion y plstic responses nd tht such responses occur within the first 4 weeks of exposure; the contminted popultion tended to express