Comparative Biochemistry and Physiology, Part A 173 (2014) 52–60

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Comparative Biochemistry and Physiology, Part A

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Influence of domestication process on immune response to repeatedemersion stressors in Eurasian perch (Perca fluviatilis, L.)

J. Douxfils a, S. Lambert a, C. Mathieu a, S. Milla a, S.N.M. Mandiki a, E. Henrotte a, N. Wang a, M. Dieu b, M. Raes b,C. Rougeot c, P. Kestemont a,⁎a University of Namur (UNamur), Research Unit in Environmental and Evolutionary Biology (URBE), Rue de Bruxelles, 61, B-5000 Namur, Belgiumb University of Namur (UNamur), Research Unit in Cellular Biology (URBC)-NARILIS, Rue de Bruxelles, 61, B-5000 Namur, Belgiumc University of Liège, Aquaculture Research and Education Centre (CEFRA), Chemin de la Justice, B-5000 Tihange, Belgium

⁎ Corresponding author at: Unité de Recherches enEvolutive (URBE), Université de Namur, Rue de BruxelleTel.: +32 81 72 43 63; fax: +32 81 72 43 62.

E-mail address: patrick.kestemont@unamur.be (P. Kes

http://dx.doi.org/10.1016/j.cbpa.2014.03.0121095-6433/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 January 2014Received in revised form 12 March 2014Accepted 19 March 2014Available online 25 March 2014

Keywords:DomesticationEmersionFishImmunityProteomicsStress

Domesticationmight be a possible way to reduce the physiological response to long-term stressors and deleteri-ous effects on immunity. The present study aimed to evaluate the chronic immune response induced by repeatedemersions and the possible impact of domestication by comparing farmed Eurasian perch with short (F1) andlong (F4) captive-life history. In the first experiment, fish were exposed to a single emersion and physiologicalstress response was measured in the short term to characterize fish sensitivity to the tested stressor. Serum cor-tisol and glucose elevatedwithin 6 h post-stress and splenosomatic index (SSI) decreasedwithin 48 h, indicatingthat the specieswas affected by emersion stressor. In the second experiment, F1 and F4 generationswere submit-ted to repeatedwater emersions (3 times/week during 44 days). On day 9, 18 and 44, samplingswere performed48 h post-stressor to highlight any sustained disruption of immune system. Serum cortisol, glucose, SSI and lyso-zyme activitywere evaluated and serumproteomewas analyzed using 2D-DIGE. Any of the tested variableswereaffected by repeated emersions and proteomic analysis only revealed that alpha-2 macroglobulins (a2Ms) wereup-regulated in the serum of stressed individuals. Domestication also resulted in the up-regulation of five a2Misoforms and down-regulation of complement C3 and Ig light chain proteins, independently of any stressor expo-sure. In conclusion, the results suggested that repeated emersions are not severe stressors for Eurasian perch,probably explaining why domestication had no influence on fish responses. Changes associatedwith domestica-tion are highly complex and certainly need further investigations.

© 2014 Elsevier Inc. All rights reserved.

1. Introduction

To be effective, an aquaculture production system aims to minimizeinfection with pathogens. However, husbandry practices present manysituations where stress can directly or indirectly compromise the im-mune status of fish and increase their susceptibility to naturally-occurring pathogens (Ashley, 2007; Segner et al., 2012). Stress isconsidered as a primary contributing factor leading to impaired healthin cultured fish and there is the need to reduce stress responsivenessin captive populations.

Farmed animals are continuously restricted to holding facilities andthey are not able to evade inauspicious situations (flight response). Con-sequently, only animals with favorable phenotype will be able to accli-mate to captivity conditions, survive and reproduce. Such natural

Biologie Environnementale ets, 61, B-5000 Namur, Belgium.

temont).

selection processmay lead to adaptation to captivity, namely to domes-tication (Price, 1999; Morgan and Thromborg, 2007). It is assumed thatreduction in stress responsiveness may be an important part of domes-tication process although evidence for such change in fish and othervertebrates is limited (Mignon-Grasteau et al., 2005; Awata et al.,2011). Reduction of stress responsiveness to commonly-imposed hus-bandry stressors may result in a lower vulnerability to starvation,injuries or diseases and this may lead to an improved fitness(Mignon-Grasteau et al., 2005; Awata et al., 2011). Consequently,stress-resistant animals may be naturally selected along domestication(Awata et al., 2011). Influence of domestication on stress responsive-ness has not been deeply studied so far but reduction of stress sensitiv-ity in fish undergoing domestication has already been suggested on thebasis of behavioral (lower flight distance and panic movements) andphysiological (lower heart rate response, attenuated and/or delayedcortisol response) observations (Johnsson et al., 2001; Begout Anrasand Lagardère, 2004; Verbeek et al., 2008; Zuberi et al., 2011). Anotherconsideration is that the level of cortisol, an important actor of the pri-mary stress response, has a genetic component and some fish may be

53J. Douxfils et al. / Comparative Biochemistry and Physiology, Part A 173 (2014) 52–60

predisposed to constantly display high or low blood cortisol level in re-sponse to stressors (Barton, 2002; Vandeputte and Prunet, 2002).More-over, cortisol response has been shown to be moderately to highlyheritable in carp Cyprinus carpio (h2 = 0.60) and rainbow troutOncorhynchus mykiss (h2 = 0.41 to 0.56) (Pottinger and Carrick, 1999;Barton, 2002; Vandeputte and Prunet, 2002). Additionally, inter-individual divergence in stress coping strategy/style has been observedin mammals and in several fish families such as cichlids, salmonids,sticklebacks and tropical species (Overli et al., 2007). Coping strategyhas been defined as a “coherent set of behavioural and physiological stressresponses, which is consistent over time and which is characteristic to acertain group of individuals” (Koolhas et al., 1999). In a population, cop-ing strategy can be interpreted as a continuum between two extremes(proactive and reactive) (Ruiz-Gomez et al., 2011). Interestingly, it hasbeen demonstrated that coping strategies have a strong genetic basis(perhaps in combination to epigenetic factors in early life) allowing sta-ble responses over time and differential adaptation to environmentalconditions (Koolhas et al., 1999; Overli et al., 2007). In fish, heritablecoping strategies (cortisol production, anxiety-like behavior, cognitivefunction, training) have been demonstrated in rainbow trout selectedfor high (HR) or low (LR) stress responsiveness (first to fourth genera-tions) (Overli et al., 2007; Ruiz-Gomez et al., 2011). In view of theabove, natural and/or artificial selection on thebasis of differential stressresponses may be part of the domestication process in fish species andthis may contribute to improve health and performances of individualsalong generations.

Our model fish, the Eurasian perch, was chosen because of its recenthistory under intensive farming conditions, suggesting that domestica-tion is certainly still in its infancy. Pertaining to stress sensitivity, it hasalready been shown that Eurasian perch is responsive to severaltype of stressors (acute or chronic/repeated) in captivity (Acereteet al., 2004; Jentoft et al., 2005; Strand et al., 2007; Milla et al., 2010;Douxfils et al., 2011a,b, 2012; Rimoldi et al., 2012). Moreover, immunemodulations have already been observed in stressed individuals inseveral studies (Milla et al., 2010; Douxfils et al., 2011b, 2012). InEurasian perch, stress response to an acute emersion stressor hasbeen characterized by a significant cortisol and glucose elevation(Acerete et al., 2004;Milla et al., 2010) and by short-term immunemod-ulations in spleen (total immunoglobulin level, lysozyme activity andmRNA level, hepcidin mRNA level) (Milla et al., 2010). However, thephysiological and immune effects of repeated exposure to emersionare still unknown. Moreover, the influence of domestication on thestress response to aquaculture stressors has not been deeply studiedso far.

The present study aimed to complement the first data already ob-tained (Douxfils et al., 2011a,b, 2012) on the interaction between do-mestication, stress and immune system in Eurasian perch confrontedto captivity stressors. More specifically, the objective is to determinethe chronic immune responses to repeated emersions and to investi-gate whether such effects are affected by domestication process bycomparing fish from filial 1 (F1) and filial 4 (F4) generations. Usualstress indicators (plasma cortisol and glucose levels), spleno-somaticindex and serum lysozyme activity were measured concomitantlywith a 2D-DIGE proteomic analysis of serum in order to observe thebiological effects at both physiological and molecular levels. A prelimi-nary experiment was first conducted to characterize the sensitivity ofperch to the model stressor tested since the method used to get fishout of the water was different from the one described by Milla et al.(2010).

Fig. 1. Scheme of the way we obtained our captive F1 and F4 generations. Dotted arrowsindicate capture of wild fish at the onset of the spawning period. Bold arrows indicate re-production giving birth to the following progeny.

2. Materials and methods

The present protocol (FUN06/067) has been carried out in agree-ment with the local Ethics Committee for Animal Experiments in accor-dance with the European guidelines concerning animal welfare.

2.1. Fish populations, domestication level and culture conditions

Two filial generations (Filial 1: F1 versus Filial 4: F4) of one-year oldjuveniles were compared in this study. Fish from the fourth generation(F4) originated from the reproduction of the third captive generation ofbreeders (F3) maintained under natural conditions in outdoor tanks(1.6 m3/4 m2) in a recirculating system. Those F3 breeders originatedfrom successive reproductions of wild individuals (F0) from theMeuse River and their related offspring (F1, F2) in captivity. To obtainthe first generation of captive fish (F1) used in the present study, anoth-er group of wild breeders (F0′) was captured from the Meuse River atthe onset of the spawning period and allowed reproducing synchro-nously to the F3 breeders in the same photo-thermal conditions(Fig. 1). Indeed, important mortality systematically occurred in cap-tured F0 breeders following first reproduction in captivity so that wewere forced to capture new F0 parents (F0′ fish in this case) each timewe needed a novel F1 group. Moreover, using F0 breeders which havebeen maintained for several years in captivity and that may have accli-mated to captive-life conditionsmay lead in differences in reproductiveperformances with time and thus divergence in the resulting offspring(when compared to generations obtained from the first spawning ofcaptured F0 breeders). Therefore, the F1 and F4 generations used inthis study were respectively derived from different groups of wild fish(F0′ and F0) captured at an identical location (i.e. the “Tailfer” backwa-ter) on the Meuse River in Belgium, suggesting adherence to the samepopulation. From the fingerling stage, the two generations (F1, F4)of juveniles were reared under similar standardized conditions(recirculating system, 4 m2 tanks, 23 °C, photoperiod LD 16:8, O2 N

6 ppm) in order to avoid the influence of different rearing surroundingson the fish biology. At the start of the experiment, the initial mean bodyweight was 63.5 ± 19.3 g and 78.6 ± 28.6 g for F1 and F4 fishrespectively.

2.2. Stress experiments and fish sampling

2.2.1. Preliminary experimentIn order to evaluate the sensitivity of the species to the tested emer-

sion stressor, Eurasian perch juveniles (only F4 generation)were loadedonto 100 L tanks (n=25per tank; 24 tanks). Fishwere exposed to a sin-gle water emersion challenge i.e. a complete tank draining resulting inair exposure for 30 s before the tank was filled again with recirculatingwater (12 tanks). Control groupswhichwere not submitted to emersionwere also included in the experiment (12 tanks). Samplings took placeat 1, 6, 24 and 48h post-stress in such away that each tankwas sampledonly once (triplicate tanks for each condition and time). At each sam-pling time, 5 fish were rapidly captured in each triplicate tank fromboth the stressed and the control condition (3 × 5 stressed fish; 3 × 5control fish), anesthetized in an ethyl 3-aminobenzoate methane sul-fonic acid salt (98% purity) (MS-222, Sigma) solution (180 mg/L) andweighed. Blood was collected by caudal vein puncture within 5 min toavoid the release of handling-induced cortisol and was allowed to clotat 4 °C prior to centrifugation (7500 g, 5 min). Aliquoted serum wasthen stored at −20 °C until assayed for cortisol and glucose. Fish werethen rapidly euthanized by spinal cord rupturing. Spleen was dissected

54 J. Douxfils et al. / Comparative Biochemistry and Physiology, Part A 173 (2014) 52–60

and weighed for determination of spleno-somatic (SSI) as follows:100 × spleen weight / total fish weight.

2.2.2. Exposure to repeated emersion stressorFour weeks prior to the start of the experiment, F1 and F4 fish were

transported from the Aquaculture Research and Education Center(CEFRA) of the University of Liège (Belgium) to our experimental facil-ities, loaded into twelve 100 L tanks (6 tanks per generation; 45 fish pertank) and allowed to acclimate to these new housing conditions for4 weeks. Then, F1 and F4 fishwere exposed in triplicate tanks to repeat-ed (3 times/week during 44 days) water emersion stressors. For eachgeneration, triplicate tanks in which fish were not stressed throughoutthe experimental periodwere also used as the control condition. Duringthe experiment, fish were fed to apparent satiation once a day in theearly morning but were not fed on days during which they werestressed. Fish were sampled on days 9 (i.e. after 4 emersion challenges),18 (i.e. after 7 emersion challenges) and 44 (i.e. after 15 emersion chal-lenges). Fish were not directly sampled following emersion stressor but48 h-post exposure (Fig. 2) because we wished to evaluate the chronic/sustained immune response tomultiple stressors in the long term inde-pendently of the short-term effect induced by the last emersion. At eachsampling date, 5 fish per tankwere collected, anesthetized as previouslystated and weighted. Blood was collected, treated and stored as ex-plained above until assayed for cortisol, glucose, lysozyme activity and2D-DIGE proteomic analysis. Fish were then rapidly euthanized by spi-nal cord rupturing and spleen was dissected and weighed for determi-nation of SSI. No fish died throughout the experimental period,neither in the control nor in the stressed groups.

2.3. Glucose and cortisol assays

Serum glycemiawas determined colorimetrically based on a glucoseoxidase/peroxidase method described by Trinder (1969). For cortisolassay, sera from 5 fish per tank were uniformly pooled together ateach sampling time. Cortisol concentration was then assayed induplicate using a cortisol ELISA kit (DRG, EIA-1887) and following themanufacturer's instructions. Cortisol was determined in one assay; theintra-assay coefficient of variation was 3.6% and the assay dynamicrange was between 0 and 800 ng·mL−1.

2.4. Serum lysozyme activity

Serum samples were used for lysozyme assay according to themethod of Siwicki and Studnicka (1987) and Fatima et al. (2007)modified by Douxfils et al. (2011b).

2.5. Statistical analysis

For both experiments, physiological and immune data are expressedas the mean ± standard deviation (SD). Replicate tank was consideredas the statistical unit (n = 3). Normality and variance homogeneitywere tested according to Kolmogorov–Smirnov and Bartlett tests re-spectively. “Ln” transformation was performed for cortisol and glucosevalues from the single emersion experiment and “log” transformationwas performed for cortisol values for the repeated emersion experimentin order to fit with variance homogeneity requirements. For the singlestress experiment, data were analyzed using a fixed two-way ANOVA.

Fig. 2. Experimental scheme of the repeated emersion protocol and sampling schedule.White sand then, tank re-filling). Samplings were performed on days 9, 18 and 44, 48 h after the last s

For the multiple stress experiment, data were analyzed separately foreach sampling time (D11, D20 and D46) according to a fixed two-wayANOVA, taking domestication and stressor occurrence as two-level fac-tors. When significant (p b 0.05), means were compared according tothe LSD post hoc test. The results were analyzed with the Statistica™software for windows (StatSoft, USA).

2.6. 2D-DIGE proteomic analysis

2.6.1. Protein extraction and CyDye minimal labelingProteomic analysis was carried out on serum sampled on day 44. For

each tank, sera from 4 fish (same sampled fish as for the physiologicalanalyses explained above) were uniformly pooled by pairs. Therefore,each tank was represented by two pooled samples and proteomic anal-ysis was conducted on 24 pooled samples (12 tank corresponding to 2generations of fish, stressed or not, in triplicates). Sera samples werethen 6-fold diluted in DLA buffer (7 M urea, 2 M thiourea, 4% CHAPS,30mMTris, pH 8.5) and centrifuged for 15min at 12,000 g. Protein con-centration of each diluted sera pool was determined using the Bradfordprotein assay with BSA as a standard. Using appropriate amounts of 50mMNaOH, pH of sampleswas adjusted to 8.5 and protein concentrationwas measured again with the Bradford protein assay. For CyDyelabeling, 200 pmol of fluorescent CyDye (GE Healthcare) was added to25 μg proteins. Cy3 and Cy5 were both used to label replicates of eachexperimental condition. A mixed sample composed of equal amountsof all sera pools (n = 24) was labeled with Cy2 and was used as an in-ternal standard. Labeling was performed on ice in the dark andquenched with 1 mM lysine for 10 min on ice. For each gel (n = 12),the three labeled mixtures were combined and total proteins (75 μg)were mixed with an equal volume of reduction buffer (7 M urea, 2 Mthiourea, 2% CHAPS, 2% DTT, 2% IPG4–7 buffer) for 15 min at roomtemperature.

2.6.2. Protein separation by 2D-DIGEPrior to electrofocusing, IPG strips (pH 4–7; 24 cm; GE Healthcare)

were passively rehydrated overnight into 450 μL of a standard rehydra-tion solution (7M urea, 2 M thiourea, 2% CHAPS, 0.28% DTT, 0.5% IPG4–7

buffer and a trace amount of bromophemol blue). The twelve samplesets containing labeled mixtures were cup-loaded onto the stripsand isoelectric focusing was run on an Ettan™ IGPphor II unit (GEHealthcare) with the following electrophoresis conditions: 20 °C, 300V for 3 h, 1000 V for 6 h, 8000 V for 3 h, and finally 8000 V for 6 hfor a total of 68,000 V-h. Afterward, focused IPG strips were reduced(1% DDT) and alkalized (2.5% iodoacetamide) in equilibration buffer(6 M urea, 30% glycerol, 2% SDS, 0.002% bromophemol blue, 50 mMTris pH 8.8) just before loading on 10% 24 cm, 1-mm-thick, SDS-PAGEgels. The strips were overlaid with 0.5% agarose in SDS running buffer(25 mM Tris, 192 mM glycine, 0.1% SDS) and run at a constant voltage(0.5watt per gel) during 17 h at 15 °C in an Ettan™DALTsix electropho-resis unit (GEHealthcare) until the blue dye front reached the bottom ofthe gels.

2.6.3. Software image analysis and statistical testsCyDye-labeled gels were scanned with a Typhoon 9400 scanner

(GE Healthcare) at CyDye-specific wavelengths (488 nm, 532 nm and633 nm for Cy2, Cy3 and Cy5 respectively). Resolution was 100 μm.Image analysis was carried out using the Decyder v5.0 software

tars indicate exposure to emersion stressor (rapid tank draining followed by 30 s emersiontressor was applied.

Fig. 3. Serum cortisol level (ng·mL−1) in control and stressed juvenile perch fol-lowing a single emersion stressor. Values are the mean ± SD and significant differences(p b 0.05) are highlighted by different lower case letters. Hours indicated each of the sam-pling time following stressor exposure. CTRL = control fish, STRESS = stressed fish.

Fig. 4. Serum glucose level (mg·mL−1) in control and stressed juvenile perch following asingle emersion stressor. Values are the mean ± SD and significant differences (p b 0.05)are highlighted by different lower case letters. Hours indicated each of the sampling timefollowing stressor exposure. CTRL = control fish, STRESS = stressed fish.

55J. Douxfils et al. / Comparative Biochemistry and Physiology, Part A 173 (2014) 52–60

(GEHealthcare). At first, theDIAmodule co-detected and quantified theprotein spots in each image using the internal standard sample as a ref-erence to normalize the data. Second, the BVAmodule allowedmultiplegel comparison and ratio calculations by performing a gel-to-gelmatching of the internal standard spot map on each gel. Data were an-alyzed by a two-way ANOVA (p b 0.05).

2.6.4. Mass spectrometry and protein identificationPreparative gels loaded with 250 μg of proteins frommixed samples

were run following the protocol described above except that they werepost-stainedwith Krypton™ protein stain (No. 46630, Pierce) followingthe manufacturer's protocol. Preparative gels were scanned with theTyphoon 9400 scanner (GE Healthcare). Spots were excised using theEttan™ Spot Picker (GE Healthcare). The gel pieces were twice washedwith distilledwater and then shrunkwith 100% acetonitrile. The proteo-lytic digestionwas performed by the addition of 3 μL ofmodified trypsin(Promega) suspended in 50 mM NH4HCO3 cold buffer. Proteolysis wasperformed overnight at 37 °C. The supernatantwas collected and the el-uates were kept at— 20 °C prior to analysis. Peptides were analyzed byusing a nano-LC–ESI-MS/MSmaXis UHR-TOF spectrometer coupled to a2D-LC Dionex UltiMate 3000 (Bruker, Bremen, Germany). The digestswere separated by reverse-phase liquid chromatography using a75 μm × 150 mm reverse phase Dionex column (Acclaim PepMap 100C18) in an Ultimate 3000 liquid chromatography system. Mobilephase Awas 95% of 0.1% formic acid inwater and 5% acetonitrile. Mobilephase B was 0.1% formic acid in acetonitrile. The digest (15 μL) wasinjected, and the organic content of themobile phase was increased lin-early from 5% B to 40% in 35min and from 40% B to 100% B in 5min. Thecolumn effluent was connected to an ESI nano Sprayer (Bruker). In sur-vey scan,MS spectrawere acquired for 0.5 s in themass to charge (m/z)range between 50 and 2200. The three most intense peptide ions 2+ or3+ were sequenced. The collision-induced dissociation (CID) energywas automatically set according tom/z ratio and charge state of the pre-cursor ion. MaXis and Dionex systems were piloted by Compass HyStar3.2 (Bruker). Peak lists were created using DataAnalysis 4.0 (Bruker)and saved as XML file for use with ProteinScape 2.0 (Bruker) with Mas-cot 2.2 as search engine (Matrix Science). Enzyme specificity was set totrypsin, and themaximumnumber of missed cleavages per peptide wasset at one. Carbamidomethylation was allowed as fixed modification,and oxidation of methionine and Gln-pyro-Glu was allowed as variablemodification. Mass tolerance for monoisotopic peptide window was7 ppm and MS/MS tolerance window was set to 0.05 Da. The peaklists were searched against the full NCBInr database (12852489 se-quences downloaded on February 2011). Scaffold (version Scaffold-2_06_01, Proteome Software Inc., Portland, OR) was used to validateMS/MS based peptide and protein identifications. All MS/MS sampleswere analyzed using Mascot (Matrix Science, London, UK; version 2.2)and X! Tandem (The GPM, thegpm.org; version 2007.01.01.). Peptideidentifications were accepted if they could be established at greaterthan 95% probability as specified by the Peptide Prophet algorithm(Keller et al., 2002). Protein identifications were accepted if they couldbe established at greater than 99% probability and contained preferen-tially more than one identified peptide. Protein probabilities wereassigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003).When a protein spot could not be assigned to one single protein identi-fication or if similar peptides were shared between several proposedproteins, all the protein hits were grouped to fit with the parsimonyprinciple.

3. Results

3.1. Stress response to single emersion

Cortisol values ranged from 9.24± 0.18 to 57.28± 24.5 ng·mL−1. Asignificant cortisol rise (p b 0.01) was observed at 1 h post-stressfollowed by a return to concentrations similar to that of the control

fish from 6 h post-stress onwards (Fig. 3). Within control groups,cortisol levels were similar over the 48-h period, with the exception ofa lower cortisol concentration observed at the 6-h sampling (significantdifferences with cortisol level at 1-h sampling and 48-h sampling —

p level b 0.05).Glucose values ranged from575.9±67.4 to 1243.7± 331.9 μg·mL−1

and significant differences were observed between control and stressedfish at 1 and 6 h(s) post stress (p b 0.01 and 0.001 respectively) (Fig. 4).

SSI values ranged from 0.062± 0.012 to 0.090± 0.016% and a glob-al stress effect was observed (p b 0.01), all sampling times considered,values being lower in stressed individuals in comparison with their re-spective controls (Fig. 5).

3.2. Physiological and immune responses of fish from distinct generations torepeated emersions

In the current study, serum cortisol level ranged from 4.7 to24.9 ng·mL−1 and stressed F1 fish displayed a lower (p b 0.01) basalcortisol level compared to the corresponding control fish on day 9while no differencewas recorded among the F4 groups (Table 1). Glyce-mia varied between 385 and 727 μg·mL−1 but levels were not signifi-cantly different between treatments. In this study, SSI ranged from0.046 to 0.064% and these values were not affected by repeated emer-sion stressors (Table 1).

Fig. 5. SSI values (%) in control and stressed juvenile perch following a single emersionstressor. Values are the mean ± SD. Asterisks indicated a global effect of thestressor throughout the experimental period with respect to un-stressed fish. Hoursindicated each of the sampling time following stressor exposure. CTRL = control fish,STRESS = stressed fish.

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Serum lysozyme activity ranged from 315 to 603 U·mL−1·min−1.No significant differences were detected in relation to repeated emer-sions and between the two generations (Table 1). The 2D-DIGE analysis(see Fig. 6 for a representative 2D-gel) conducted on serumat the end ofthe experimental period only revealed that alpha-2 macroglobulinswere more abundant following exposure to repeated emersions (spotnos. 240 and 248) whatever the generation considered (Table 2).

Domestication did not seem to modulate the physiological and im-mune response to repeated emersions, both F1 and F4 generationsdisplaying similar values either regarding the above-mentioned vari-ables or the serumprotein profile. Regardless of repeated stressor expo-sure, the 2D-DIGE analysis revealed that several alpha-2macroglobulins(spot nos. 234, 240, 248, 254 and 256) were significantly up-regulatedwhile the abundance of a complement C3 protein (spot no. 1192) andof an Ig light chain (IgL) (spot no. 1324) was down-regulated in theF4 generation, independently of any stressor exposure (Table 2).

4. Discussion

4.1. Eurasian perch is responsive to an acute emersion stressor

In order to characterize perch responsiveness to the model stressortested, the physiological response in terms of cortisol and glucose levels(two commonly measured stress indicators) as well as spleen

Table 1Stress indicators, morphological indices and immune variables evaluated followingrepeated emersion in two generations (F1 and F4) of Eurasian perch juvenile.

Cortisol(ng/mL)

Glucose(μg/mL)

SSI (%) Lysozyme activity(U/mL/min)

Day 9F1 CTRL 14.7 ± 8.0 570 ± 108 0.057 ± 0.004 520 ± 43F1 EMER 4.7 ± 3.5* 580 ± 84 0.057 ± 0.006 603 ± 84F4 CTRL 5.1 ± 1.6 465 ± 29 0.057 ± 0.006 539 ± 54F4 EMER 15.3 ± 7.1 550 ± 17 0.053 ± 0.004 535 ± 90

Day 18F1 CTRL 16.3 ± 11.4 486 ± 128 0.050 ± 0.012 366 ± 47F1 EMER 15.3 ± 14.0 727 ± 251 0.061 ± 0.003 350 ± 18F4 CTRL 11.8 ± 8.6 584 ± 50 0.055 ± 0.007 371 ± 71F4 EMER 17.7 ± 2.3 721 ± 122 0.046 ± 0.013 345 ± 24

Day 44F1 CTRL 6.7 ± 1.5 385 ± 68 0.056 ± 0.004 325 ± 41F1 EMER 24.9 ± 24.0 470 ± 49 0.064 ± 0.012 355 ± 56F4 CTRL 6.1 ± 4.0 458 ± 50 0.055 ± 0.006 335 ± 20F4 EMER 6.0 ± 6.0 450 ± 57 0.059 ± 0.012 315 ± 32

Asterisks indicate significant differences related to repeated emersion exposure.CTRL = control treatment; EMER = repeated emersion.N = 3.

contraction was evaluated following exposure to a single emersionstressor. The results indicated that Eurasian perch was responsive tothe tested stressor. Indeed, cortisol level increased 1 h after stressor ex-posure and hyperglycemia was observed at 1 and 6 h post-stress. Theseresults are similar to those obtained by Milla et al. (2010) on Eurasianperch submitted to a single netting stressor for 1min.We also observedthat cortisol values of un-stressed groups at the 6-h sampling werelower than thosemeasured for the other un-stressed groups throughoutthe experimental period. One possible explanation would be circadianfluctuations in cortisol secretion as it has beenwell established inmam-mals and some fish species. Despite such diurnal variation, cortisol levelin all un-stressed groups however remained in the range of un-stressedEurasian perch (i.e. below 30 ng·mL−1) (Acerete et al., 2004; Millaet al., 2010; Douxfils et al., 2011a,b, 2012). Following stressors such assevere exercise or environmental hypoxia, it has been demonstratedthat fish spleen can contract to supply blood with additional erythro-cytes and that this happens very rapidly (within minutes) (Yamamotoet al., 1985; Kita and Itazawa, 1989; Douxfils et al., 2012). Such processis part of themechanisms aiming to improve blood oxygen carrying ca-pacity in order to face the increased energy demand associated withstressor exposure (Pichavant et al., 2002; Shimps et al., 2005; Chabotand Claireaux, 2008; Terova et al., 2008) as well as providing bloodcells for preparing immune response. In the current experiment, we ob-served that fish were particularly agitated when water level was low(during tank draining and re-filling) and also during total air exposure.Therefore, SSI has been measured as a valuable indirect indicator ofspleen contraction and red blood cell release. Exposure to emersionsignificantly reduced spleen volume over the 48-h period, probablyreflecting an effect of the stressor on oxygen requirement. Milla et al.(2010) reported comparable results in the same species submitted toa single 1-min air exposure through capture and netting suggestingthat this phenomenon is commonly observed in Eurasian perch follow-ing exposure to an acute stressor.

4.2. Exposure to repeated emersions does not severely affect the immunesystem of Eurasian perch

Chronic effects of repeated emersion stressors on the immune sys-temwere evaluated at physiological (serum lysozyme activity) andmo-lecular (serum proteome) levels in order to evaluate whether this typeof frequent disturbances may affect the immune status of Eurasianperch in the long-term. Moreover, a generation with a longer captivelife history (F4) was compared to a wild-close generation (F1) (bothof them sharing an identical populational origin in the natural environ-ment) in order to assess the potential influence of domestication pro-cess on this response. Physiological indicators (cortisol, glucose andSSI) were measured as well over the 44-day experimental period. Asshown in the previous section, the preliminary experiment demonstrat-ed that serum cortisol level rises shortly after exposure to a single emer-sion stressor. In this long-term experiment, samplings were howeversystematically performed 48 h after the last stressor was applied be-cause we wished to evaluate the chronic/sustained immune effects ofmultiple stressors in the long term independently of the short-term re-sponse induced by the last emersion. Therefore, the reported cortisoland glucose values were not related to the immediate acute responseto emersion (cortisol and glucose peaks decreased to resting levelswithin 6 h post-stress — cfr preliminary experiment). Regarding thesecortisol values at 48 h, no clear stress-related differences were observedthroughout the experimental period, suggesting that the hypothalamo–pituitary–interrenal (HPI) axis has not been chronically sensitized/disturbed by the repeated stressor. Glycemia was not significantly af-fected by the tested stressor aswell indicating that the energeticmetab-olism was not deeply disrupted by this stressor. The present study alsorevealed that SSI values did not differ significantly throughout theexperiment. It might be suggested that spleen did not contract or atleast to a lesser extent following repeated emersion stressor (other

Fig. 6. Representative 2D-gel of serum proteome in Eurasian perch. Numbers assigned by the Decyder software indicate protein spots with significant changes in intensity.

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compensatory mechanisms may have been developed with time) and/or that spleen recovered its normal size within 48 h post-stressor, pos-sibly through re-sequestration of circulating cells (Houston et al., 1996).In conclusion, our results showed that the effects of multiple emersionswere not more important than those induced by single emersion asshown by the preliminary experiment. The laps of time between emer-sion stressorsmay have been sufficient for thefish to rapidly regain nor-mal physiological status or fish may have habituated to the repeateddisturbance.

Pertaining to the immune status of Eurasian perch, serum lysozymeactivity in repeatedly-stressed fish did not significantly differ from thatof the respective unstressed groups. However, few differences wererevealed in serum by 2D-DIGE analysis at the end of the experimentalperiod. Indeed, two alpha-2 macroglobulin molecules (a2Ms) wereup-regulated in fish subjected to the repeated disturbance. These pro-teins, present in both vertebrates and invertebrates, are non-specificplasma protease inhibitors mediating the clearance of endogenousand exogenous proteases (Mutsuro et al., 2000; Onara et al., 2008). Infish, a2Ms regulate protein degradation and participate to innate de-fense against invading pathogens in blood (Funkestein et al., 2005; Ma

Table 2List of identified proteins differentially regulated in the serum of Eurasian perch juvenile (F1 a

Spot no. Accession no.a Protein name Species M

234 gi 225907442 Alpha-2-macroglobulin Perca flavescens 1240 gi 225907442 Alpha-2-macroglobulin Perca flavescens 5

248 gi 225907442 Alpha-2-macroglobulin Perca flavescens 5

254 gi 225907442 Alpha-2-macroglobulin Perca flavescens 5256 gi 225907442 Alpha-2-macroglobulin Perca flavescens 41192 gi 31559515 Complement component C3 Platichthys flesus 21324 gi 158602736 Immunoglobulin light chain Epinephelus coioides 2

a NCBI database accession number.b Refers to the number of sequenced peptides exclusively assigned to the protein.c CTRL, control treatment; EMER, repeated emersion.d Positive and negative values indicates up- and down-regulation respectively.e p b 0.05.f p b 0.01.g p b 0.001.

et al., 2010). In trout (Oncorhynchus spp.) and brook charr Salvelinusfontinalis, it has been demonstrated that high a2M concentration inplasma was associated with an increased resistance to parasites andbacteria (Woo, 2001, 2003; Chuang et al., 2008).

In our study, immune variables have only beenmeasured in blood. Itwould however be interesting to extend the analysis to other internalorgans (spleen, liver or head kidney) and physical barriers (mucus,gills and gut) to get an overall view of the fish immune status. Indeed,stress-induced immune modulations were shown to be tissue-specific,notably in Eurasian perch (Milla et al., 2010). Moreover, Pruett (2003)reviewed that significant changes in immune variables did not neces-sarily impact the overall resistance to disease. Therefore, including abacterial challenge experiment would also be necessary to better reflectthe stress-related changes in immune status.

4.3. Influence of domestication process on stress and immune responses inEurasian perch

Domestication level did not affect the stress and immune responsesto repeated emersions, in terms of cortisolemia, glycaemia, spleen

nd F4 generations) following exposure to repeated emersions.

atching peptidesb Theoretical pI/Mw (kDa) Treatmentc Fold changed

5.2/57 F4 vs F1 1.42e

5.2/57 EMER vs CTRLF4 vs F1

1.42e

1.82g

5.2/57 EMER vs CTRLF4 vs F1

1.46f

1.93g

5.2/57 F4 vs F1 1.63f

5.2/57 F4 vs F1 1.34e

6.6/30 F4 vs F1 −1.40e

5.1/26 F4 vs F1 −1.83g

58 J. Douxfils et al. / Comparative Biochemistry and Physiology, Part A 173 (2014) 52–60

contraction and serum protein profile. Our previous studies (Douxfilset al., 2011a,b, 2012) however suggested that domestication may mod-ulate the stress responsiveness of Eurasian perch depending on thestressor characteristics. Indeed, it seemed that Eurasian perch of thefirst captive-reared generation (F1) displayed an increased sensitivityof the hypothalmo–pituitary–interrenal axis and a significant cellularstress response (HSP70) in comparison to F4 individuals whenconfronted to chronic confinement (Douxfils et al., 2011a). Moreover,a decrease in the abundance of some immune actors has been observedin F1 confined fishwhile only immune stimulationswere highlighted inF4 confined ones, suggesting a better immune status of domesticatedfish exposed to chronic confinement (Douxfils et al., 2011b). Converse-ly, when exposing Eurasian perch to single and repeated sublethalhypoxia, it was observed that both F1 and F5 generations displayedsimilar physiological and immune modulations, thereby suggestingthat domestication may also not be associated with a strong divergencein stress responsiveness (Douxfils et al., 2012). To elucidatewhydomes-tication was associated with a better tolerance to chronic confinementbut not to repeated hypoxia or emersions, we suggest that it may de-pend on the stressor capacity to severely threaten internal homeostasis.Actually, stress response is first an adaptive mechanism to promote thebest chance of survival towards hostile situations. Low-level stress re-sponses may not constitute a systematic advantage and the preserva-tion of optimal stress responses may be necessary for immediatesurvival under severe conditions. This might be particularly true duringexposure to harsh physico-chemical, mechanical or thermal stressorswhich constitute real threats to homeostasis. Sublethal hypoxiamay be-long to this stressor category. On the other hand, certain stressors arenot endangering physical health but are rather associated with cogni-tive stress (i.e. fear, anxiety and apprehension of possible danger)which can be minimized through repetition and experience by animals(Tort, 2011). Under such stressing situations, reduction in stress sensi-tivity may not be deleterious since individuals are not really in danger.On the contrary, reduction in stress responsiveness may be profitablesince energy expenditure associated with allostatic load would beavoided. Chronic confinementmay be included in the list of such cogni-tive stressors. Based on the results from the current study, it can be sug-gested that domestication did not influence fish responsiveness to thetested stressor since the responses were very low and thus, repeatedemersions do not seem to be a significant stressor for Eurasian perch.Another hypothesis to explain such divergent results between our stud-iesmight be related to natural selection process in captivity. It should bementioned that artificial selection based on specific traits was not per-formed in any of our studies. But, natural selection, as can be observedfrom the wild, may have occurred in captivity, particularly during thefirst generations. Natural selection in captivity is an environmentally-driven process that eliminates animals unable to reproduce and favorsanimals with a better fitness which can wean a high proportion ofyoungs in the environment provided by humans (Mignon-Grasteauet al., 2005). Natural selection can be involved to different extents andin various directions along domestication, depending on the particularenvironmental conditions and human interventions. This means thatdomesticated/farmed animals may display behavioral, morphologicaland physiological differences in comparison to the original wild popula-tion and that divergencemay occur between diverse domesticated pop-ulations (e.g. F4) depending on differential experience of their breeders(e.g. F0, F1, F2, F3) during lifetime and related selection (Vandeputteand Launey, 2004; Mairesse et al., 2007).

In the present study, domestication modulated the abundanceof several serum proteins (i.e. up-regulation of five a2Ms, down-regulation of C3 and IgL proteins in F4 individuals), independently ofany stressor exposure. In agreementwith past studies, these results fur-ther support that immune proteins are highly represented in the pro-tein profile modulated along domestication process (Douxfils et al.,2011b; Douxfils et al., 2012). Observation of five protein spots on the2D-gels and identified as a2Ms is not surprising since previous studies

reported a2Ms to be resolved as two protein bands (α and β chains)by reducing SDS-PAGE electrophoresis (Mutsuro et al., 2000; Chuanget al., 2008). So it is highly probable that the spots identified as a2Msin the current study representα and/or β chains, not the entire protein.Additionally, several authors reported up to four a2M isoforms in theserum of the common carp C. carpio (Mutsuro et al., 2000; Onaraet al., 2008). According to Onara et al. (2008), the presence of multiplea2M isoforms is not surprising since this is common to all members ofthe thioester-containing protein family (i.e. C3, C4, C5 and a2M).Hence, this may explain why several a2M spots were present of the2D gels and indicate a positive modulation of several isoforms. Domes-tication also down-regulated the abundance of a C3 protein and an Iglight chain. However, we cannot conclude on the physiological effectsof such changes without further characterization of these proteins. In-deed, various C3 isoforms have been reported in teleosts and it hasbeen demonstrated that these molecules can highly differ in their bind-ing and/or haemolytic capacity (e.g. evidence for an isoformwith no rolein complement-induced hemolysis) (Lambris et al., 1993; Zarkadis et al.,2001; Holland and Lambris, 2002; Boshra et al., 2006). Moreover, C3proteolysis produces several cleavage products (e.g. C3a, C3b, C3f,iC3b, etc.) diverging in their specific immuneactivities. Similarly, severalIg light chain isotypes have been reported in teleosts (Espelid andGrontvedt, 2003; Okamoto et al., 2003; Zhang et al., 2003; Edholmet al., 2011). Differences in the relative abundance and antigen bindingcapacity of these Ig light chain isotypes have also been documented(Partula et al., 1996; Timmusk et al., 2000; Edholm et al., 2011).

At this stage of the study, it is not possible to determine the causes ofsimultaneous up-regulation of a2Ms and down-regulation of IgL and C3along domestication. Nevertheless, since immunoglobulins are pro-duced by B-lymphocytes while C3 and a2M proteins are mainly pro-duced by hepatocytes (Funkestein et al., 2005; Boshra et al., 2006;Onara et al., 2008; Saurabh et al., 2010), we may suggest a differentialimpact of domestication on these cell types. It is also difficult to con-clude on the immune consequences of these changes in a2M, Ig lightchains and C3 abundance as contradictory modulations were observedand much work is needed to better characterize these proteic frag-ments. Bacterial challengewould also be interesting as survival capacityto infection is generally the ultimate targeted outcome.

4.4. Comparative response to emersion, hypoxia or confinement stressor inEurasian perch

In this section,wewould like to present a brief overview of the over-all stress responsiveness of Eurasian perch to diverse aquaculturestressors and summarize the main results of our prior studies. Indeed,our experiments have been carried out in similar rearing facilities andglobally used the same analytical techniques. Comparing the results ofsuch standardized experiments is particularly interesting since discrep-ancies between studies of similar topic are often due to divergence inexperimental design and analyses.

We conducted several experiments on stress response in theEurasian perch including stressors with relatively different characteris-tics, i.e. chronic confinement (Douxfils et al., 2011a,b), single/repeatedhypoxia (Douxfils et al., 2012) and single/repeated emersion (thispaper). According to the classification proposed by Tort (2011), chronicconfinement can be viewed as a mild long-term social and cognitivestressor while sub-lethal hypoxia can be regarded as a severe environ-mental and physico-chemical disturbance. Finally, water emersion canbe described as a multiple-component stressor with physico-chemical,mechanical and cognitive characteristics. Here above, we would like todiscuss the main results of these previous studies independently of do-mestication process (data of “wild-close” F1 fish only). All studies con-sidered, it seems that Eurasian perch did not show any cortisolelevation during exposure to long-term or repeated stress (i.e. duringchronic confinement, single/repeated hypoxia for 4 h, and chronic re-sponse to repeated emersion). Cortisol only increased in the very

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short-term (between 20min to less than 6 h) during exposure to acutestressors (i.e. netting + anesthesia, single emersion). Our data sug-gested that this hormone may not constitute a very sensitive stressmarker in Eurasian perch. Fish may either show no cortisol elevationor only a short-lasting response, precluding easy detection of thesestressing conditions. Despite the lack of cortisol and glucose responseto most of the tested stressors, we systematically observed up or down-regulation of immune proteins and/or activities in stressed Eurasianperch. For instance, serum and spleen immune modulations (increasedlysozyme and respiratory burst activities, increased immunoglobulinconcentration, lower abundance of complement C3 and transferrin)were reported in both the short- and long-term during chronic confine-ment (Douxfils et al., 2011b). To our knowledge, these are the first re-sults demonstrating a significant influence of chronic confinement,consisting in reduction of the tank size, on the immune system of fish.Indeed, studies evaluating the effects of chronic confinement are scarcein the literature (e.g. Barnett and Pankhurst, 1998; Fanouraki et al.,2007). Indeed, many studies arguing that they evaluated confinementstress have assessed the physiological effects of crowding rather thanconfinement because tank size reduction was associated with an in-crease in stocking density. But water volume and the number of fishper tank play important roles in stress response because these parame-ters influence the fish swimming behavior and social interactions. Inthis respect, crowding and confinement are rather different stressors.Similarly, during hypoxia, significant effects (hyperglycemia, spleencontraction, increased abundance of several complement C3 proteins)were observed despite the absence of a serum cortisol response. Follow-ing repeated exposure to the hypoxia stressor, hyperglycemia loweredand transferrin level elevated in the serum, suggesting the developmentof acclimation mechanisms to this recurrent disturbance. However,subsequently to repeated hypoxia, Eurasian perch displayed down-regulation of some C3molecules together with a reduction in serum ly-sozyme activity. This may reflect reallocation of energy and resourcesaway from the immune system in favor of the acclimation process.Very few physiological and immune effects were observed following re-peated emersion as only α-2-macroglobulins were up-regulated in theserumof stress individuals after 44 days. These effectswere less numer-ous than during exposure to repeated hypoxia but this can be partly dueto different laps of time between stressor application and fish sampling(48 h and 4 h post-emersion and post-hypoxia respectively).

All in all the result of our studies suggest that Eurasian perch can beaffected by common husbandry practices and that immune modula-tions (both stimulatory and inhibitory) might be frequently associatedwith exposure to stressors, either in the short- or in the long-term. Inour studies, the effects of stressors were mainly investigated in blood(occasionally liver and spleen) and bacterial challenges were not sys-tematically conducted. However, stress-induced alterations in some im-mune functions can occurwithout any deleterious consequences for theoverall host resistance to disease (Pruett, 2003; Small and Bilodeau,2005). Indeed, even if stressed individuals exhibit immune depressionin some internal organs or in circulating blood, this may not necessarilyresult in an increased susceptibility to disease as long as fish are able tomaintain physical integrity and optimal immune functions at the prima-ry barriers that prevent pathogens from invasion. Therefore, we assumenot only immune actors but defensemechanisms in other organs and inthe fish primary barriers should also be studied and experimental infec-tion conducted to compare disease susceptibility of stressed and un-stressed fish.

5. Conclusions

The current results revealed that Eurasian perchwas responsive to asingle emersion but that repeated exposure to this stressor did not seemto further impact fish physiology and immune system. Indeed, chroniceffects of repeated emersions were really low (increased abundance ofa2Ms in serum at the end of the experimental period), suggesting that

repeated emersion was not a severe stressor to Eurasian perch and/orthat acclimation may occur with time. Domestication did not affectstress responsiveness and immune response to the tested stressor,probably due to its inability to strongly disturb fish homeostasis overweeks. The beneficial effects of domestication on stress and immune re-sponses seem to be linked to cognitive (which can be learned by theanimal) or mild stressors that do not constitute a real danger for imme-diate fish survival.

Effects of domestication seem to be relatively complex and thesemay be further influenced,within a considered species, by the particularrearing system and conditions in captivity so that generalizing the re-sults to all domestication cases does not seempossible. Further researchon the field is now needed to discriminate between global and study-specific changes associated with domestication.

Acknowledgments

The study was supported by the Belgian National Fund for ScientificResearch (FNRS) (Fellowship to J. Douxfils: F.C. 75186, projects no.1.1.767.07.F and 1.1.676.09.F), by the Funds for Collective FundamentalResearch (FRFC) in Belgium (Project no. 2.4570.06) and finalized duringthe Interuniversity Attraction Poles (IAP) “AquaStress” project (P7/31)(funding by the Belgian Science Policy Office — BELSPO). C. Rougeot isa member of the AFFISH-RC of the University of Liège. Thanks are dueto A. Evrard from URBE, Edouard Delaive and Catherine Demazy fromthe URBC-Narilis for their valuable help in fish care and maintenance,proteomic analysis and in-gel trypsin digestion. The proteomic and MSfacility of the URBC-Narilis is supported by the FNRS.

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