Functional Ecology. 2018;32:2509–2519. wileyonlinelibrary.com/journal/fec  | 2509© 2018 The Authors. Functional Ecology © 2018 British Ecological Society

Received:26April2018  |  Accepted:30July2018DOI: 10.1111/1365-2435.13195

R E S E A R C H A R T I C L E

Resveratrol supplementation reduces oxidative stress and modulates the immune response in free‐living animals during a viral infection

Manrico Sebastiano1  | Marcel Eens1 | Simone Messina1  | Hamada AbdElgawad2 |  Kévin Pineau3 | Gerrit T. S. Beemster2  | Olivier Chastel4 | David Costantini1,5,6

1DepartmentofBiology,BehaviouralEcologyandEcophysiologyGroup,UniversityofAntwerp,Antwerp,Belgium;2DepartmentofBiology,IntegratedMolecularPlantPhysiologyResearch(IMPRES),UniversityofAntwerp,Antwerp,Belgium;3Grouped'EtudeetdeProtectiondesOiseauxenGuyane(GEPOG),Cayenne,FrenchGuiana;4Centred’EtudesBiologiquesdeChizé(CEBC),UMR7372‐CNRS/UniversityofLaRochelle,LaRochelle,France;5UMR7221CNRS/MNHN,MuséumNationald'HistoireNaturelle,SorbonneUniversités,Paris,Franceand6InstituteofBiodiversity,AnimalHealthandComparativeMedicine,UniversityofGlasgow,Glasgow,UK

CorrespondenceManricoSebastiano,DepartmentofBiology,BehaviouralEcologyandEcophysiologyGroup,UniversityofAntwerp,Antwerp,Belgium.Email:manrico.sebastiano@uantwerpen.be

Funding informationFWO;CNRS;UniversityofAntwerp;CEBC

HandlingEditor:DanaHawley

Abstract1. Dietqualitymayhaveanimportanteffectontheregulationofoxidativestatusandtheimmunesystemduringaninfectiousdisease.However,therelationshipamongintakeofspecificdietarymolecules,anindividual’soxidativestatusandtheoccurrenceandprogressofaviraldiseaseremainsalmostunexploredinfree‐liv-ingorganisms.

2. Here,westudyawild,long‐livedanimal,theMagnificentfrigatebirdFregata mag‐nificens toinvestigate:(a)thedifferencesinanumberofphysiologicaltraits(bio-markers of blood oxidative status, corticosterone (CORT), immunity andinflammation)betweensickandhealthynestlings;and(b)whetherexperimentallyincreasedintakeofresveratrol(apolyphenolwithantioxidantandantiviralprop-erties)affects thesephysiologicalmarkersduringtheprogressofasevereviraldisease.

3. Birdswithvisibleclinicalsignsshowedhigheroxidativedamage,haemolysisandhaemagglutination scores and lower antioxidant defences in comparison withbirdswithout clinical signs. At the end of the experiment, supplemented birdsshowedthefollowing:(a)increasedplasmahaptoglobinlevelsandcirculatinganti-oxidantdefences;(b)reducedgenerationoflipidoxidativedamage;and(c)negligi-ble to no influence on immunemarkers, baseline CORT levels and activity ofantioxidantenzymes.

4. Ourworkillustrateshowtheavailabilityofspecificorganicmoleculesinthedietmayconstraintheindividuals’capacitytocopewithviralinfectionsinfree‐livinganimals.

K E Y W O R D S

antioxidantdefencesofbirds,avianglucocorticoid,avianinfectiousdiseases,Frigatebird,immuneresponse,oxidativestress,stresshormones,wildanimals

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1  | INTRODUC TION

Infectiousagentsareanimportantselectiveforce,potentiallyreduc-ingsurvivalandreproductionofthehostand,eventually,resultinginpopulationdeclines (Preeceet al., 2017;Smith,Sax,&Lafferty,2006).Awell‐documentedinfectiousdiseaseisrepresentedbytheWestNilevirus(WNV),avirusthatismostlyfoundinbirdsbecausetheyaresuspectedtobethemostimportantamplifyinghosts.WNVhasbeenfoundin63birdspecies1yearafteritsfirstappearancein1999(Kramer&Bernard,2001)andhascausedmassivemortalityinmanycorvidspecies(McLean,2006).Similarly,avianinfluenzavirus(AIV)hascausedworld‐widesevereoutbreaksinpoultry,wildbirdsandhumans(Chatziprodromidouetal.,2018).

Theassociationbetweendiseasesandimmunedefences(Savageet al., 2016) or the selection of immune traits during outbreaks(Legagneuxetal.,2014)hasbeenofgreat interest inevolutionaryecologyandphysiology,yetthepathophysiologicalmechanismsun-derlyingtheimpactofinfectiousdiseasesonwildliferemainpoorlydocumented. An approach that simultaneously quantifies physio-logicalstressandimmunestatuswouldbeidealtodeterminehowan organism is affected by a given pathogen (Hawley & Altizer,2011). For instance, estimating stress hormone corticosterone(CORT) levels during infectionmay be relevant because exposuretostressorsstimulatesCORTrelease(Sapolsky,Romero,&Munck,2000).AlthoughCORTcoordinates the stress–response (Sapolskyetal.,2000),highlevelsand/orchronicreleaseofCORTareknowntosuppresstheimmuneresponse(Bourgeon&Raclot,2006;Gao,Sanchez,&Deviche,2017)andtoincreasetheimpactofvirusinfec-tioninbirds(Owen,Nakamura,Coon,&Martin,2012).

Similarly,oxidativestress isknownto limit immunefunction inbirds(Catoni,Schaefer,&Peters,2008).Thus,oxidativestressmaycontributetothespreadof infectiousdiseases(deCrommenacker,Richardson,Koltz,Hutchings,&Komdeur,2012;Kelesetal.,2010).Forinstance,Marek’sdiseaseincreasesdamagetoDNA,lipidsandproteins in chickens (Keles et al., 2010), suggesting that oxidativestressmightpartiallyexplainanorganism’svulnerabilitytoviraldis-eases(Li,Feng,&Sun,2011)andviralreplication(Costantini,Seeber,etal.,2018;Lietal.,2011).Measuringbothantioxidantdefencesandoxidativedamage,whichrespectivelyreflecttheabilitytomountaprotective response to an adverse condition and the deleteriouseffects the animals undergo (Beaulieu & Costantini, 2014), mightthereforeprovevaluable to infer the individual’s capacity to copewithapathogen.

Importantly,dietaryantioxidantsmight impact thehost’s ca-pacity to cope with an infectious disease because antioxidantsreduce immunopathology associated with the immune/inflam-matoryresponse(Dhinaut,Balourdet,Teixeira,Chogne,&Moret,2017); theyoccur in limitedsupply for free‐ranginganimals,po-tentiallyconstrainingtheircapacitytocopewithoxidativestress(Catoni et al., 2008;Costantini, Angeletti, et al., 2018) andmaylimit steroid synthesis (Ozdemir, Ozudogru, Imik, Can, & Sunar,2011). Accordingly, antioxidants can inhibit the replication ofseveralviruses(i.e.,felineimmunodeficiencyvirusMortolaetal.,

1998; influenzavirusHanetal.,2000;duckenteritisvirusXuetal., 2013; herpes simplex virus Civitelli et al., 2014). Moreover,dietary antioxidants exhibit antiviral effects that are apparentlynotdirectlyconnectedtotheirantioxidantproperties(e.g.,Abba,Hassim,Hamzah,&Noordin,2015).However, to thebestofourknowledge,experimentalsupplementationofmoleculeswithan-tioxidant and antiviral properties to study the impact on oxida-tivestatus,CORTlevels,andimmunityduringaviralinfectionhasneverbeencarriedoutinfree‐livinganimals.

Here, we studied nestlings of Magnificent frigatebird (Fregata magnificens) coping with a severe viral disease. We investigatedwhether birds with visible clinical signs (i.e., skin crusts) showedan alteration of their physiological traits (bloodoxidative balance,immunestatus,CORTlevels,andinflammation)incomparisonwithbirdswithoutclinicalsigns.Wethentestedwhethersupplementa-tionofresveratrol,apolyphenolwithbothantioxidantandantiviralactivity(Abbaetal.,2015),improvesthephysiologicaltraits,short‐termprogress of the disease and survival perspectives of supple-mentedbirds.OurexperimentwascarriedoutonaprotectedislandinFrenchGuiana,whereoutbreaksofviral infectionsoccuryearly,causing85%to95%nestlingmortality(fieldobservations).Bacterialcultures,viralscreeningandmicroscopicevaluationofskinsamplesexcluded thepresenceofectoparasites, avianpoxvirus (deThoisyetal.,2009)andavianinfluenza(unpublishedresults),butdetectedherpesvirusDNAinbodycrusts(deThoisyetal.,2009).Recentworkfoundupto10millioncopiesofherpesviralDNAinnestlingswithclinicalsignsofthedisease,suggestingthatherpesvirusreplicationisinvolvedintheappearanceofclinicalsigns(Sebastiano,Eens,AbdElgawad, et al., 2017). This population offers an unprecedentedopportunity to investigate thepotential connectionbetween viraldisease progression and diet quality employing an experimentalapproach,becauseclinicalsignsareassociatedwithseveralphysio-logicalbiomarkersofoxidativestressandinflammation(Sebastiano,Eens,AbdElgawad, et al., 2017; Sebastiano, Eens,Angelier, et al.,2017),andwecanexperimentallyfeedfrigatebirdsinthewild.

2  | MATERIAL AND METHODS

2.1 | Sample collection

The fieldwork was carried out in 2016 on Grand ConnétableIsland,aprotectedareaofftheNorthernAtlanticcoastofSouthAmerica (French Guiana, 4°49’30N; 51°56’00W), which hostsapproximately 1,300 reproductive pairs of frigatebirds (GEPOGfield observations). Most frigatebird pairs in this colony startbreeding between the end of November and the beginning ofDecember. Consequently, all nestlings were approximately ofthesameage(~4monthsold)whencaptured(seealsoStatisticalAnalysis section).A total of26nestlingswithout visible clinicalsignsand34sicknestlingsshowingvisibleclinicalsignswereran-domly chosen at different sites of the island (Figure 1). Visibleclinical signs of the disease include crusts on the head and the

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body, hyperkeratosis on eyes and the consequent thickeningofthecornea(deThoisyetal.,2009).All60nestlingswerecapturedat thenestbyhandon7June (D1).Within3minaftercapture,2mlofbloodwascollected from thebrachialveinusingahep-arinizedsyringeanda25Gneedle.Immediatelyafterwards,eachbirdwasringedwithanaluminiumringforindividualrecognitionandthebeak‐headdistancewasmeasuredtocontrolfortheageofnestlings.Thisfirstsampleofbloodwasusedtotestanypre-treatmentdifferenceamonggroups.

TheexperimentstartedonD2.Weadministeredpillsoftrans-Resveratrol (seeSupporting InformationAppendixS1) to theex-perimental groups (12 of 26 healthy nestlings and 18 of 34 sicknestlings,respectively),whiletheremainingindividuals(14healthyand16 sicknestlings, respectively; i.e., control groups)were ad-ministeredanemptypillasaplacebotoaccountfortheeffectofhandling.Wechoseresveratrolbecauseofitsstrongantiviralac-tivityagainstherpesvirus(Abbaetal.,2015;Sebastiano,Chastel,deThoisy,Eens,&Costantini,2016),whichactively replicates insicknestlingfrigatebirds(Sebastiano,Eens,Angelier,etal.,2017).Pillsweredippedinfishoil (Craftycatcher, Ipswich,UK)tofacil-itateswallowing.Theadministrationofpillswascarriedoutninetimes(D2–4,8th–10thofJune;D12–14,18th–20thofJune;D21–23,27th–29thofJune).OnD24,theexperimentendedandasec-ondsampleof2mlofbloodwastakenfromthesameindividuals.Blood sampleswere kept coldwhile in the field and centrifugedwithin less than 2hr to separate plasma (used for CORT, oxida-tivestressbiomarkers,inflammatoryandimmunemarkers)andredbloodcells.Bothsamplesofplasmaandredbloodcellswerethenkept indry iceuntil theendofthefieldworkand,uponarrival inthelaboratory,werekeptina−80°Cfreezer.Twopicturesofeachbirdweretakenfromthesamedistanceandsamepositionatthestartandtheendoftheexperiment(pre‐andpost‐treatment),toscoreclinicalsignsofthediseaseandassessifvisibleclinicalsignsincreased or decreased during the experiment (see the specificsectionbelow).

2.2 | Molecular analyses

Allanalyseswereperformedusingestablishedprotocols forver-tebrates. The determination of nonenzymatic antioxidants was

performedusing reduced (GSH) andoxidized (GSSG)glutathionein red blood cells. High‐performance liquid chromatography(HPLC)withelectrochemicaldetection (Reversed‐PhaseHPLCofShimadzu,HaiZhongLu,Shanghai)wasappliedfollowingSinhaetal. (2014),andconcentrationswereexpressedasμmol/gof freshweight.WealsocalculatedtheGSH/GSSGratioasametricofoxi-dativebalance.Furthermore,thenonenzymaticantioxidantpoweroferythrocytes (TAC, an indexof circulatingnonenzymatic anti-oxidants) was estimated following Benzie and Strain (1996) andexpressedasμmolTrolox/gof freshweight.Theenzymaticanti-oxidant capacitywasmeasuredusing three different biomarkersin redbloodcells.Superoxidedismutase (SOD)activitywasesti-matedbymeasuringtheinhibitionofnitrobluetetrazoliumreduc-tion at 560nm andwas expressed as U/mg protein perminute.Catalase activity (CAT)wasmeasured bymonitoring the rate ofdecomposition of hydrogen peroxide (H2O2) at 240nm and wasexpressedasμmolH2O2/mgproteinperminute.Glutathioneper-oxidase(GPX)activitywasdeterminedbymeasuringthedecreasein NADPH absorbance at 340nm and was expressed as μmol NADPH/mgproteinperminute.Damagetobiomoleculeswasas-sessedbyquantifyingtheplasmalevelofthiobarbituricacidreac-tivesubstances(TBARS),whichreflectlipidperoxidation,andthelevelofproteincarbonyls in redbloodcellsasameasureofoxi-dativedamagetoproteins.Resultsareexpressed,respectively,asnmolofmalondialdeyde (MDA)equivalents/ml (Hodges,DeLong,Forney, & Prange, 1999) and nmol/mg protein (Levine,Williams,Stadtman,&Shacter,1994).

Plasma haptoglobin concentration was quantified using acommercially available assay (PHASE Haptoglobin assay; TrideltaDevelopment Ltd), and concentrations were expressed as mg/ml.Theplasmaconcentrationofnitricoxide (NO)wasestimatedfromtheconcentrationofthestableendproductsofnitricoxideoxida-tion(i.e.,nitrateandnitrite)andexpressedinμmol/L.Innatehumoralimmunity was determined by the haemolysis‐haemagglutinationassayasdescribed inMatson,Ricklefs,andKlasing (2005).Finally,the plasma concentration of corticosterone was measured by ra-dioimmunoassayfollowingLormée,Jouventin,Trouve,andChastel(2003)andexpressedasng/ml.

Detailedprotocols areprovided in theSupporting InformationAppendixS1.

F I G U R E 1  Nestlings’classificationbasedonvisibleclinicalsignsofthedisease:(a)“nosigns,”(b)“mild”and(c)“severe”

(a) (b) (c)

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2.3 | Bird classification based on clinical signs

Toclassifythesampledbirdsbasedontheseverityofvisibleclini-calsigns,pictureswereanalysedandblindlyscoredtwicebythesameperson (1weekapart). Scores ranged from0 (absoluteab-senceofclinicalsigns)to10(birdfullycoveredbycrusts),includinghalf scores. Reproducibility between the two scores (calculatedfrom variance components derived from a one‐way analysis ofvariance,accordinglytoLessells&Boag,1987)ofeachindividualwassignificantlyhighbothbefore(r=0.96,F=49.67,p<0.0001)and after (r=0.93,F=31.64,p<0.0001) the treatment. An av-eragescorewas,therefore,calculatedandusedtofurtherdividenestlings into three studygroups tobeused for statistical com-parisons: healthy group (hereafter “no signs,” average score <1),nestlingswithfewcrustsontheneckandaroundtheeyes(hereaf-ter“mild,”averagescore≥1or<4)andverysicknestlingsshowingmorewidespreadand thicker crusts (hereafter “severe,” averagescore≥4;forreference,seeFigure1).Thisclassificationenabledus to detect birds that changed group over the progress of thedisease(whichmatchesachangeinthevisibleclinicalsigns),thusto identify: (a) birds that never showed the appearance of clini-calsigns,hereafter“alwayshealthy”;(b)birdsthatshowedtheap-pearanceofclinicalsigns,hereafter“newsick”;(c)birdsthathadanimprovementofvisibleclinicalsigns,hereafter“bettercondition”;and(d)birdsthatdidnotchangetheirstatus,hereafter“samese-verity.”Finally,noneofthesickbirdsshowedanincreasedsever-ityofclinicalsigns,probablybecausetheworseningofanalreadycriticalconditioncoincidedwith thedeathof thebird (hereaftercalled“didnotsurvive”).

2.4 | Statistical analyses

Twogenerallinearmodelswereusedtoanalysepretreatmentgroupdifferences:(a)amongbirdsclassifiedontheseverityofclinicalsigns(nosigns,mildandsevere);and(b)amonggroupsbasedonthepro-gressofthedisease(alwayshealthy,newsick,bettercondition,sameseverityanddidnotsurvive).Athirdlinearmodel(whichincludedall34sicknestlingasindependentobservations,thusnotdividedintogroups)wasusedtoinvestigatewhetherthescoresassignedtotheseverityofclinicalsignsofeachnestling(includedasacontinuousvariable)wereassociatedwiththeanalysedbiomarkers.

Twolinearmixedmodelswitharepeatedmeasuresdesignwereused to assess the effects of the treatment: (a) between the twogroupsbasedonthepresenceorabsenceofvisibleclinicalsignsofthediseaseatthefirstsamplingperiod(MODEL1);and(b)amonggroupsbasedontheprogressof thedisease (fourgroups: “alwayshealthy,”“newsick,”“bettercondition”and“sameseverity”;MODEL2).Mixedmodelsonlyincludednestlingsforwhichwehadtwomea-surements(pre‐andpost‐treatment),implyingthatnestlingsthatdidnotsurviveor thatwerenot foundat thesecondsamplingperiodwerenot included.Aspreliminary analysis showed that thehead‐beakdistance(usedasaproxyoftheage)wassimilarbetweenhealthyandsick(ttest;t=−1.85;p=0.07)andbetweensupplementedand

unsupplemented nestlings (t test; t = 1.85; p=0.07), this variablewasnotfurtherincludedinstatisticalanalyses.Thereducedmodelwasobtainedby sequentially removingnonsignificant interactionsfrom the fullmodel starting from the three‐way interaction. PosthocTukeytestswereusedtoexplorefurthersignificantinteractions.

Finally,ageneralizedlinearmodelusingabinomialerrorvarianceandalogitlinkfunctionwasusedtoassesswhetherthetreatmentinfluenced theprobabilityof theappearanceof clinical signs,of adecrease in visible clinical signsof thedisease, or death.Detailedinformationondata transformation and setupof the linearmixedmodelscanbefoundintheSupportingInformationAppendixS1.

AllanalyseswereperformedusingR(v.3.3.1,RCoreTeam,2013).

3  | RESULTS

3.1 | Bird classification based on clinical signs

Before theexperiment,wehada totalof26birdswithoutclinicalsignsand34sickbirds(13mildand21severelyaffected).Overthecourseoftheexperiment,ofthe26birdsclassifiedashealthybeforetheexperiment,wehadatotalof11birds(foursupplemented)thatnever showed the appearance of clinical signs, hereafter “alwayshealthy”;11birds(foursupplemented)thatshowedtheappearanceofclinical signs,hereafter “newsick.”Of the34birdsclassifiedassickbeforetheexperiment,eightbirds(foursupplemented)hadanimprovement of visible clinical signs, hereafter “better condition”;18birds(ninesupplemented)didnotchangetheirstatus,hereafter“same severity”; seven birds (four supplemented) died, hereaftercalled“didnotsurvive.”Fourhealthyandonesickbird (allsupple-mented)werenotfoundattheendoftheexperiment.

3.2 | Pretreatment basal differences among groups

Birdswithno clinical signs showed (a) significantly lower reducedglutathione, oxidized glutathione and oxidative damage to lipidsthanbirdswithsevere(t>2.76,p<0.02;Figure2a–c)clinicalsigns;and(b)significantlylowerreducedglutathioneandoxidativedam-agetolipidsthanbirdswithmild(t>2.59,p<0.03,Figure2a,c)clin-icalsigns.Oxidativedamagetolipidswasalsosignificantlyhigherinbirdswithsevereclinicalsignsthaninbirdswithmildclinicalsigns(t=2.58,p=0.03,Figure2c).New sickbirdshadatendencytohavepretreatment loweroxidizedglutathionethanbirdsthatwereinabetter condition at theendof theexperiment (t=2.80,p = 0.052; Figure3a).Finally,pretreatmentoxidativedamagetolipidswassig-nificantly lowerboth inbirdsthatwerealways healthy and in new sickbirdsthaninbirdsthat(a)thatwereinabetter conditionattheendoftheexperiment;(b)showedthesame severity ofclinicalsignsattheendoftheexperiment;or(c) did not survive(t>2.90,p < 0.04; Figure3b).

Amongmarkersofimmunity,theinflammationproteinhaptoglobinwassignificantlyhigherinbirdswithsevereclinicalsignsthanbirdswithmildclinicalsigns(t=3.75,p<0.01,Figure2d)orwithno clinical signs (t=−3.93,p<0.01,Figure2d).Furthermore,higherhaptoglobinlevels

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werefoundinbirdsthatdid not survive (t>2.95,p<0.04,Figure3c).Birdswithno clinical signsshowed(a)significantlylowerhaemaggluti-nationthanbirdswithmild (z=−3.88,p<0.01,Figure2e)andsevere (z=−3.19, p<0.01; Figure 2e) clinical signs; and (b) lower haemoly-sisthanbirdswithsevereclinicalsigns(z=−2.72,p=0.02;Figure2f).Haemagglutinationwasalsohigherinbirdsthatdid not survive and in birdswiththesame severityofclinicalsignsthannew sickbirds(z>3.49,p<0.01;Figure3d),orbirdsthatwerealways healthy(z>2.98,p < 0.02; Figure3d)overthecourseoftheexperiment.

Noneof theotherbiomarkersofoxidative status, immunityandbasalplasmaCORTshowedasignificantassociationwiththediseasestatus(Table1).Finally,noneofthebiomarkersshowedasignificantrelationshipwhen clinical signswere used as a continuous variable,with the exception of haptoglobin (i.e., haptoglobin increases with

increasing clinical signs; F=30.47, p<0.01; Supporting InformationFigureS1).

3.3 | Effect of resveratrol administration

We then examinedwhether administration of resveratrol wouldaffecttheoxidativestatusofthebirds.Circulatingnonenzymaticantioxidants increased in supplemented birds with no clinical signs (t =−3.94, p<0.01; Figure 4, Supporting Information TableS1). Specifically, this increase in nonenzymatic antioxidants onlyoccurred in supplemented birds that were always healthy duringthe experiment (t =−3.70, p=0.03; Figure 5). Oxidative damageto lipids did not change in supplemented birds (Figure 4), whileit strongly increased in unsupplemented sick birds (t =−6.19,

F I G U R E 2  Pretreatmentdifferencesamongbirdsclassifiedontheseverityofvisibleclinicalsigns(nosigns,n=26;mild,n=13;severe,n=21)of:(a)reducedglutathione(μmol/goffreshweight);(b)oxidizedglutathione(μmol/goffreshweight);(c)oxidativedamage(nmolMDAequivalents/ml);(d)haptoglobin(mg/ml);(e)haemagglutinationscore;and(f)haemolysisscore.Dataareshownasmean±standarderror.Valuesthatdonotsharethesameletteraresignificantlydifferentfromeachother

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p<0.01; Figure 4, Supporting Information Table S1), indicatingthatresveratrolpreventedincreasedproductionofoxidativedam-age. This increase in oxidative damage to lipids occurredmostly

inbirdsthatshowedthesame severityofclinicalsignsduringtheexperiment (t =−4.91,p<0.01;Figure5,Supporting InformationTableS2).

F I G U R E 3  Pretreatmentdifferencesamongbirdsclassifiedbasedontheprogressofthedisease(alwayshealthy,n=11;newsick,n = 11; bettercondition,n=8;sameseverity,n=18;didnotsurvive,n=7)of:(a)oxidizedglutathione(μmol/goffreshweight);(b)oxidativedamage(nmolMDAequivalents/ml);(c)haptoglobin(mg/ml);and(d)haemagglutinationscore.Dataareshownasmean±standarderror.Valuesthatdonotsharethesameletteraresignificantlydifferentfromeachother

TA B L E 1  Linearmodelsexplainingwhetherthepre‐treatmentlevelofaspecificbiomarkerdifferedamonggroupsclassifiedontheseverityofclinicalsigns(nosigns,mildandsevere)ortheprogressofthedisease(alwayshealthy,newsick,sameseverity,bettercondition,anddidnotsurvive)

Biomarker

Grouping factor

Severity of clinical signs Progress of the disease

F‐value p‐value F‐value p‐value

Carbonyls 2.07 0.14 0.74 0.57

Catalase 0.48 0.62 0.80 0.53

Corticosterone 0.32 0.73 0.28 0.89

Glutathioneperoxidase(GPX) 1.84 0.17 0.88 0.48

GSH/GSSGratio 0.30 0.74 0.78 0.54

Haptoglobin 10.1 <0.01 5.16 <0.01

Hemagglutination 9.78 <0.01 6.18 <0.01

Hemolysis 3.05 <0.06 1.84 0.14

Nitricoxide(NOX) 0.20 0.82 1.55 0.20

Non‐enzymaticantioxidants(TAC) 1.07 0.35 0.93 0.45

Oxidativedamage(TBARS) 22.2 <0.01 8.83 <0.01

Oxidizedglutathione(GSSG) 4.33 0.02 2.71 0.04

Reducedglutathione(GSH) 5.18 <0.01 2.45 0.07

Superoxidedismutase(SOD) 0.89 0.41 0.72 0.58

Note.Thelinearmodelontheprogressofthediseaseincludedallnestlingsexcludingthefiveindividualsthatwerenotfoundatthesecondsamplingperiod. p‐valuesareinboldwhenpost‐hoccomparisonsweresignificant.

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Among immune markers, the plasma concentration of hap-toglobin increased in supplemented new sick birds (birds thatshowedtheappearanceofclinicalsignsattheendoftheexperi-ment,t =−3.86,p=0.03;Figure5,SupportingInformationTableS2). The concentrationof nitric oxidedidnot change in supple-mentedbirds,whileitsignificantlydecreasedinunsupplementedbirds(t=2.96,p<0.01,SupportingInformationTablesS1andS2).Other biomarkers of immunity and stresswerenot significantlyinfluencedbythetreatmentconsideringbothbirdsdividedbasedon the presence/absence of clinical signs (MODEL 1) and birdsdivided on the progress of the disease (MODEL 2; SupportingInformationTablesS1andS2).

3.4 | Effects of resveratrol administration on the progress of the disease

Whenwecompared supplementedandunsupplementedbirds, nosignificantdifference in theprogressof thediseasewasdetected.Resveratrolsupplementationdidnotinfluencetheprobabilityofde-velopingclinicalsigns(z=0,p=1;exactsamenumberofindividualsineachgroup),nordiditinfluencetheprobabilityofmortalityand/orreductioninvisibleclinicalsigns(z=0.19,p=0.85).

4  | DISCUSSION

Ourstudyisthefirsttomeasuretheeffectofresveratroladministra-tiononoxidativestatus,inflammation,immunityandCORTlevelsinawildvertebratefacingaseverevirusoutbreak.Beforethestartof

theexperiment,thereweresignificantdifferences intheoxidativeand immunestatusesbetweensickandhealthybirds inour studypopulation.Therewasalsoastrongincreaseinlipidoxidativedam-ageduringtheprogressofthedisease.Theexperimentalpartofourstudydemonstratedthatresveratrolsupplementationincreasedan-tioxidantdefencesandlimitedthegenerationoflipidoxidativedam-ageduringtheprogressofthedisease.

Theanalysisofseveralbiomarkerspriortotheantioxidanttreat-mentenabledustodiscoverthataviraldiseasecanaffectdiversefunctionalpathways.Viraldiseasesareknowntoaffecttheoxida-tivestatus(Durgut,Ataseven,Sagkan‐Ozturk,&Ozturk,2013;Keleset al., 2010) and immunity (Staley&Bonneaud, 2015) of exposedanimals. Accordingly, we found that nestlings with visible clinicalsigns showedapronouncedalterationof their immunestatusandcellularoxidativebalance incomparisonwithbirdswithoutclinicalsigns.Contrarytoourexpectation,baselineCORTlevelsdidnotdif-feramongthedifferentgroups.CORTalsodidnotincreaseduringtheprogressofthedisease,whichisinagreementwithourpreviousresults (Sebastiano, Eens, Angelier, et al., 2017). By dividing birdsaccording to the severity of clinical signs and the progress of thediseaseoverthecourseoftheexperiment,instead,wefoundnovelfindings.Birdswithoutclinicalsignsshowed lowerhaemolysisandhaemagglutinationscores,resultsthathadnotpreviouslyemerged(Sebastiano,Eens,Angelier,etal.,2017).

Supplementationwithresveratroldidnotaffecthaemolysisandhaemagglutinationscores,whichmightsuggest that theseparticu-laraspectsof the immunesystemplayaminor role incopingwitha viral infection in frigatebirds (Sebastiano, Eens, Angelier, et al.,2017).Incontrast,nestlingssupplementedwithresveratrolshowed

F I G U R E 4  Effectofresveratroladministrationonthelevelsof(left) circulatingnonenzymaticantioxidants(μmolTrolox/goffreshweight),and(right)oxidativedamagetolipids(nmolofMDAequivalents/ml)levels.Asterisksindicatesignificantdifferences:*p < 0.05; **p<0.01;***p<0.001.Dataareshownasmean±standarderror

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anelevatedproductionofnitricoxideduringtheprogressofthedis-ease,whilenitricoxideproductiondrasticallydecreasedinunsupple-mentedbirds.Previousstudieshavefoundthatcellsfromknockoutmice lacking nitric oxide production showed a lower antimicrobialactivityduringSalmonella infection (Vazquez‐Torres, Jones‐Carson,Mastroeni,Ischiropoulos,&Fang,2000),indicatingthatnitricoxideisimportanttodefeatpathogens.Previousstudieshaveshownthatdietary antioxidants can act as immunostimulants in birds (Catonietal.,2008;Hooda,Tyagi,Tyagi,&Sharma,2005).Thus,thestim-ulatingeffectofresveratrolontheproductionofnitricoxidemightunderlieitsantiviralactivitybecausenitricoxidecontrolsthefunc-tionofnatural killer cells andcytokines that areessential to com-batpathogens(Bogdan,Rollinghoff,&Diefenbach,2000).Althoughourresultssupportapotentialroleofresveratrolinpromotingnitricoxideproductioninawildbird(Gülçin,2010),itisunclearwhytherewasadecrease innitricoxide inunsupplementedbirds thatnevershowed the appearance of clinical signs. Similarly to nitric oxide,synthesisofhaptoglobinincreasesduringinfectionstoprotectfromoxidativedamage(MacKellar&Vigerust,2016).Innestlingssupple-mented with resveratrol, haptoglobin was upregulated during theearlystageoftheinfection.Incontrast,inthosenestlingsthatwerenotgivenresveratrol,haptoglobinwashigherinbirdsatanadvancedstageofthedisease.Thisisinagreementwithpreviousresults(Asasi,Mohammadi, Boroomand, Hosseinian, & Nazifi, 2013; Sebastiano,Eens,Angelier,etal.,2017)andsuggeststhathaptoglobinproductionisupregulatedatanadvancedstageofthedisease,butresveratrolstimulatedtheproductionofhaptoglobinatanearlierphaseofthedisease.Theconsequencesofthischangeinhaptoglobinproductionforindividualsurvivalareunclearandneedfurtherinvestigation.

Supplementationoforganicmoleculeswithantioxidantproperties(e.g., vitamins, polyphenols) may reduce oxidative stress (Costantini,2014).However,anincreasedintakeofthesecompoundsmayalsointer-ferewiththeendogenousantioxidantsystems,leadingtoadecreaseinenzymaticantioxidantactivity(Wang,Chien,&Pan,2006)andexpres-sionofantioxidantgenes(Selmanetal.,2006).Wefoundanincreaseinnonenzymaticantioxidantcapacityinerythrocytes,whichoccurredinbirdsthatnevershowedtheappearanceofclinicalsigns,suggestingthatresveratrolhadantioxidanteffects.Todate,thehighantioxidantactivityof resveratrolhasbeen linkedwith itscapacity to induceglutathionesynthesis(Bellaver,Souza,Souza,&Quincozes‐Santos,2014;Kodeetal.,2008),butourresultsdonotshowsuchrelationship,implyingthatthismechanismneedsfurtherassessment.

Resveratrolisalsoknowntohaveaverystronginhibitionpoweragainst lipid peroxidation (Gülçin, 2010), and its administration

F I G U R E 5  Effectofresveratroladministrationonthelevelsof(a)haptoglobin(mg/ml),(b)circulatingnonenzymaticantioxidants(μmolTrolox/goffreshweight)and(c)oxidativedamagetolipids(nmolofMDAequivalents/ml).Asterisksindicatesignificantdifferences:*p<0.05;**p<0.01;***p<0.001.Dataareshownasmean±standarderror

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preventedanincreaseinoxidativedamagetolipids.Thisresultmighthave fundamental implications for theprogressof thediseasebe-cause(a)highlevelsofoxidativedamageareassociatedwithreducedshort‐term survival probability (Sebastiano,Eens,AbdElgawad, etal.,2017);(b)birdsthatwerenaturallyrecoveringfromclinicalsignsdidnotshowanincreaseinoxidativedamage;and(c)acellconditionofoxidativestressappearstofacilitatevirusreplication,whilecellsthatareable toupregulateantioxidantdefencesand limitdamagecansurviveviralinfections(Qiangetal.,2006).Thisresult,however,doesnotenableustoassesswhethertheprotectionfromoxidativedamagewasduetotheantioxidantortheantiviralactivityofresver-atrol,andfurtherstudiesarewarrantedtoclarifythismechanism.

Increased production of CORT is another mechanism activatedinanimals facingan infectionbecause it inducesanumberofphysi-ological changes (e.g., mobilization of stored energy, stimulation ofimmunefunction)thatsustainthebodyfunctionandmaintaincellularhomoeostasis(Sapolskyetal.,2000).However,ourresultsprovidenosupportforthisrelationshipbetweenvirus‐inducedstressandCORTrelease,butthehighvariationinCORTlevelsamongindividualscouldarise from exposure to strong environmental stressors (Martinet &Blanchard, 2009; Sebastiano, Bustamante, et al., 2016, 2017 ), andclearlydeservesfurtherinvestigations.

The antiviral and antioxidant effects of resveratrol might alsoresult in lifeextensionaspreviously suggestedbyameta‐analyticstudy (Hector,Lagisz,&Nakagawa,2012).However,wecouldnotdetect the effect of resveratrol supplementation on the survivalprobabilityofbirds.Thismighthavebeenduetotherelativelyshorttreatmentperiodcomparedwiththerapidprogressofthedisease.Alonger‐termtreatmentwouldproveusefultounderstandwhetherresveratrolincreasessurvivalprobabilitiesofsickbirdsandwhetheritcanpreventtheappearanceofclinicalsigns.Thismighthavefun-damentalimplicationsforthelong‐termviabilityofthispopulation,whichisnowatriskduetothemassivemortalityeventsofnestlings.Inlong‐livedspecieswithlowfecundity,astheMagnificentfrigate-bird,evenasmallrateofnestlingmortalitycanindeedhaveimport-ant negative demographic effects (Finkelstein, Doak, Nakagawa,Sievert,&Klavitter,2010).

5  | CONCLUSIONS

Wehaveprovidedexperimental support to thehypothesis that di-etary compoundsmayconstrain thecapacityoforganisms to copewithaviraldisease.Ourworkshowsthattheeffectsofthesedietaryorganicmoleculesmaycomethroughbothantioxidantprotectionandantiviralproperties.Itwillbeimportanttoexpandourstudytootherspeciesandenvironmentalconditionstofurtherassesstheconditionsunderwhichthequalityofdietmayaffectthecapabilityofanimalstocopewithaviraldisease.Althoughourwork focusedon theef-fectsofapolyphenol(i.e.,resveratrol),naturallyoccurringdietsmayberichinmanyothercompoundsthatshowsimilarproperties(e.g.,carotenoids,vitamins)tothoseofpolyphenols,butwhoseeffectsonviraldiseasesinfree‐ranginganimalshavenotbeenexploredsofar.

ACKNOWLEDG EMENTS

We thank the associate editor and two reviewers for providingvaluablecommentsonourwork.WealsothanktheCEBC(Centred’EtudesBiologiquesdeChizé),SENTINELprojectfundedbyCNRS,theUniversityofAntwerpandtheFWO(FondsWetenschappelijkOnderzoek)forfundingfieldoperationsandlaboratoryanalyses,theGEPOG(Grouped’EtudeetdeProtectiondesOiseauxenGuyane)and DEAL Guyane, for their logistic support and access to theGrandConnétableNatureReserve.Thisworkwas also supportedbyapostdoctoralfellowshipfromtheFWO(12U8918N)toHamadaAbdElgawad.Weareespeciallygrateful toAlainAlcide,AmandineBordin and Jérémie Tribot for their help in the field, to JasmijnDaans,DannyHuybrecht,CharlineParenteauandColetteTrouvé,fortheirhelpwithlaboratoryanalysesandHanAsardforprovidingtheinfrastructurefortheoxidativestressanalyses.

CONFLIC T OF INTERE S T

Allauthorsdeclarenoconflictofinterest.

AUTHORS’ CONTRIBUTIONS

D.C.,O.C.andM.S.designedthestudy;M.E.,D.C.,O.C.,G.B.andK.P.coordinateddifferentphasesofthestudy;M.S.,S.M.andK.P.collected the samples;M.S., S.M. and H.A. performed laboratoryanalyses;M.S.analysedthedataandwrotethemanuscriptwiththecontributionofallauthors.

DATA ACCE SSIBILIT Y

Data are deposited in the Dryad Digital Repository: https://doi.org/10.5061/dryad.r75251m(Sebastianoetal.,2018).

ORCID

Manrico Sebastiano http://orcid.org/0000‐0002‐9186‐0772

Simone Messina http://orcid.org/0000‐0001‐6034‐7450

Gerrit T. S. Beemster http://orcid.org/0000‐0001‐6014‐053X

R E FE R E N C E S

Abba,Y.,Hassim,H.,Hamzah,H.,&Noordin,M.M.(2015).Antiviralac-tivityof resveratrolagainsthumanandanimalviruses.Advances in Virology,2015,1–7.https://doi.org/10.1155/2015/184241

Asasi,K.,Mohammadi,A.,Boroomand,Z.,Hosseinian,S.A.,&Nazifi,S.(2013).Changesofseveralacutephasefactorsinbroilerchickensinresponsetoinfectiousbronchitisvirusinfection.Poultry Science,92,1989–1996.https://doi.org/10.3382/ps.2012‐02902

Beaulieu, M., & Costantini, D. (2014). Biomarkers of oxidative status:Missingtoolsinconservationphysiology.Conservation Physiology,2,cou014.https://doi.org/10.1093/conphys/cou014

Bellaver,B.,Souza,D.G.,Souza,D.O.,&Quincozes‐Santos,A. (2014).Resveratrolincreasesantioxidantdefensesanddecreasesproinflam-matorycytokinesinhippocampalastrocyteculturesfromnewborn,

2518  |    Functional Ecology SEBASTIANO ET Al.

adultandagedWistarrats.Toxicology in Vitro,28,479–484.https://doi.org/10.1016/j.tiv.2014.01.006

Benzie, I. F. F., & Strain, J. J. (1996). The Ferric Reducing Ability ofPlasma (FRAP) as a measure of “Antioxidant Power”: The FRAPAssay.Analytical Biochemistry,239,70–76.https://doi.org/10.1006/abio.1996.0292

Bogdan,C.,Rollinghoff,M.,&Diefenbach,A. (2000).Theroleofnitricoxideininnateimmunity.Immunological Reviews,173,17–26.https://doi.org/10.1034/j.1600‐065X.2000.917307.x

Bourgeon,S.,&Raclot,T. (2006).Corticosteroneselectivelydecreaseshumoral immunity in female eiders during incubation. Journal of Experimental Biology, 209, 4957–4965. https://doi.org/10.1242/jeb.02610

Catoni,C.,Schaefer,H.M.,&Peters,A.(2008).Fruitforhealth:Theef-fectofflavonoidsonhumoralimmuneresponseandfoodselectionin a frugivorous bird. Functional Ecology,22, 649–654. https://doi.org/10.1111/j.1365‐2435.2008.01400.x

Chatziprodromidou, I. P., Arvanitidou, M., Guitian, J., Apostolou, T.,Vantarakis,G.,&Vantarakis,A. (2018).Globalavian influenzaout-breaks2010–2016:Asystematicreviewoftheirdistribution,avianspecies and virus subtype. Systematic Reviews, 7, 17. https://doi.org/10.1186/s13643‐018‐0691‐z

Civitelli,L.,Panella,S.,Marcocci,M.E.,DePetris,A.,Garzoli,S.,Pepi,F.,…Angiolella,L.(2014).Invitroinhibitionofherpessimplexvirustype1 replication byMentha suaveolens essential oil and itsmain com-ponentpiperitenoneoxide.Phytomedicine,21,857–865.https://doi.org/10.1016/j.phymed.2014.01.013

Costantini,D.(2014).Oxidative stress and hormesis in evolutionary ecology and physiology: A marriage between mechanistic and evolutionary ap‐proaches.Berlin:Springer.

Costantini,D.,Angeletti,D.,Strinati,C.,Trisolino,P.,Carlini,A.,Nascetti,G., & Carere, C. (2018). Dietary antioxidants, food deprivationand growth affect differently oxidative status of blood and brainin juvenile European seabass (Dicentrarchus labrax). Comparative Biochemistry and Physiology Part A Molecular Integrative Physiology,216,1–7.

Costantini,D.,Seeber,P.A.,Soilemetzidou,S.‐E.,Azab,W.,Bohner, J.,Buuveibaatar,B.,…Greenwood,A.D.(2018).Physiologicalcostsofinfection:Herpesvirusreplicationislinkedtobloodoxidativestressinequids.Scientific Reports,8,10347.

de Thoisy, B., Lavergne, A., Semelin, J., Pouliquen, J.‐F., Blanchard,F., Hansen, E., & Lacoste, V. (2009). Outbreaks of disease pos-sibly due to a natural avian herpesvirus infection in a colony ofyoung magnificent Frigatebirds (Fregata magnificens) in FrenchGuiana. Journal of Wildlife Diseases, 45, 802–807. https://doi.org/10.7589/0090‐3558‐45.3.802

Dhinaut,J.,Balourdet,A.,Teixeira,M.,Chogne,M.,&Moret,Y.(2017).Adietarycarotenoidreducesimmunopathologyandenhanceslongev-itythroughanimmunedepressiveeffectinaninsectmodel.Scientific Reports,7,12429.https://doi.org/10.1038/s41598‐017‐12769‐7

Durgut,R.,Ataseven,V.S.,Sagkan‐Ozturk,A.,&Ozturk,O.H. (2013).Evaluation of total oxidative stress and total antioxidant status incowswithnaturalbovineherpesvirus‐1infection.Journal of Animal Science,91,3408–3412.https://doi.org/10.2527/jas.2012‐5516

Finkelstein,M.E.,Doak,D.F.,Nakagawa,M.,Sievert,P.R.,&Klavitter,J.(2010).Assessmentofdemographicriskfactorsandmanagementpriorities:Impactsonjuvenilessubstantiallyaffectpopulationviabil-ityofalong‐livedseabird.Animal Conservation,13,148–156.https://doi.org/10.1111/j.1469‐1795.2009.00311.x

Gao,S.,Sanchez,C.,&Deviche,P.J.(2017).Corticosteronerapidlysup-pressesinnateimmuneactivityinthehousesparrow(Passer domesti‐cus).Journal of Experimental Biology,220,322–327.

Gülçin, İ. (2010).Antioxidantpropertiesofresveratrol:Astructure–ac-tivity insight. Innovative Food Science & Emerging Technologies, 11,210–218.https://doi.org/10.1016/j.ifset.2009.07.002

Han,S.N.,Meydani,M.,Wu,D.,Bender,B.S.,Smith,D.E.,Vina,J.,…Meydani,S.N. (2000).Effectof long‐termdietaryantioxidantsup-plementation on influenza virus infection. Journals of Gerontology. Series A, Biological Sciences and Medical Sciences, 55, B496–B503.https://doi.org/10.1093/gerona/55.10.B496

Hawley,D.M.,&Altizer,S.M.(2011).Diseaseecologymeetsecologicalimmunology:Understandingthelinksbetweenorganismalimmunityandinfectiondynamicsinnaturalpopulations.Functional Ecology,25,48–60.https://doi.org/10.1111/j.1365‐2435.2010.01753.x

Hector,K.L.,Lagisz,M.,&Nakagawa,S.(2012).Theeffectofresvera-trolonlongevityacrossspecies:Ameta‐analysis.Biology Letters,8,790–793.https://doi.org/10.1098/rsbl.2012.0316

Hodges, M. D., DeLong, M. J., Forney, F. C., & Prange, K. R. (1999).Improvingthethiobarbituricacid‐reactive‐substancesassayfores-timating lipid peroxidation in plant tissues containing anthocyaninandotherinterferingcompounds.Planta,207,604–611.https://doi.org/10.1007/s004250050524

Hooda,S.,Tyagi,P.K.,Tyagi,P.K.,&Sharma,D.(2005).Effectofhigherdietary concentrationof alpha tocopherolongrowthperformanceand immune response in growing quails. Indian Journal of Poultry Science,40,32–35.

Keles,H.,Fidan,A.F.,Cigerci,I.H.,Kucukkurt,I.,Karadas,E.,&Dundar,Y.(2010).IncreasedDNAdamageandoxidativestressinchickenswithnaturalMarek'sdisease.Veterinary Immunology and Immunopathology,133,51–58.https://doi.org/10.1016/j.vetimm.2009.07.003

Kode, A., Rajendrasozhan, S., Caito, S., Yang, S. R., Megson, I. L., &Rahman, I. (2008). Resveratrol induces glutathione synthesis byactivation of Nrf2 and protects against cigarette smoke‐mediatedoxidative stress in human lung epithelial cells.American Journal of Physiology‐Lung Cellular and Molecular Physiology, 294, L478–L488.https://doi.org/10.1152/ajplung.00361.2007

Kramer,L.D.,&Bernard,K.A.(2001).WestNilevirusinfectioninbirdsandmammals.Annals of the New York Academy of Sciences,951,84–93.https://doi.org/10.1111/j.1749‐6632.2001.tb02687.x

Legagneux, P., Berzins, L. L., Forbes, M., Harms, N. J., Hennin, H. L.,Bourgeon, S., … Burness, G. (2014). No selection on immunologi-calmarkers in response to a highly virulent pathogen in anArcticbreeding bird. Evolutionary Applications, 7, 765–773. https://doi.org/10.1111/eva.12180

Lessells, C. M., & Boag, P. T. (1987). Unrepeatable repeatabili-ties: A common mistake. The Auk, 104, 116–121. https://doi.org/10.2307/4087240

Levine,R.L.,Williams,J.A.,Stadtman,E.R.,&Shacter,E.(1994).Carbonylassaysfordeterminationofoxidativelymodifiedproteins.Methods in Enzymology,233,346–357.

Li,X.,Feng,J.,&Sun,R.(2011).OxidativestressinducesreactivationofKaposi's Sarcoma‐associated herpesvirus and death of primary ef-fusionlymphomacells.Journal of Virology,85,715–724.https://doi.org/10.1128/JVI.01742‐10

Lormée,H.,Jouventin,P.,Trouve,C.,&Chastel,O.(2003).Sex‐specificpatterns inbaseline corticosteroneandbody condition changes inbreedingRed‐footedBoobiesSula sula. Ibis,145, 212–219.https://doi.org/10.1046/j.1474‐919X.2003.00106.x

MacKellar,M.,&Vigerust,D.J.(2016).Roleofhaptoglobininhealthanddisease: A focus on diabetes.Clinical Diabetes: A Publication of the American Diabetes Association,34,148–157.https://doi.org/10.2337/diaclin.34.3.148

Martinet,V.,&Blanchard,F. (2009).Fisheryexternalitiesandbiodiver-sity:Trade‐offsbetweentheviabilityofshrimptrawlingandthecon-servationofFrigatebirdsinFrenchGuiana.Ecological Economics,68,2960–2968.https://doi.org/10.1016/j.ecolecon.2009.06.012

Matson,K.D.,Ricklefs,R.E.,&Klasing,K.C.(2005).Ahemolysis‐hem-agglutination assay for characterizing constitutive innate humoralimmunityinwildanddomesticbirds.Developmental and Comparative Immunology,29,275–286.https://doi.org/10.1016/j.dci.2004.07.006

     |  2519Functional EcologySEBASTIANO ET Al.

McLean,R.G.(2006)West Nile virus in North American birds.OrnithologicalMonographsNo.60,pp.44–64.AmericanOrnithologists'Union.

Mortola,E.,Okuda,M.,Ohno,K.,Watari,T.,Tsujimoto,H.,&Hasegawa,A.(1998).Inhibitionofapoptosisandvirusreplicationinfelineimmu-nodeficiency virus‐infected cells byN‐acetylcysteine and ascorbicacid. Journal of Veterinary Medical Science, 60, 1187–1193. https://doi.org/10.1292/jvms.60.1187

Owen,J.C.,Nakamura,A.,Coon,C.A.C.,&Martin,L.B. (2012).TheeffectofexogenouscorticosteroneonWestNilevirus infection inNorthernCardinals (Cardinalis cardinalis).Veterinary Research,43,9.https://doi.org/10.1186/1297‐9716‐43‐34

Ozdemir,D.,Ozudogru,Z.,Imik,H.,Can,M.,&Sunar,M.(2011).Effectsof dietary antioxidant supplementation on the adrenal glands inquails(Coturnix coturnix japonica)rearedunderheatstress.Revue De Medecine Veterinaire,162,8–12.

Preece, N. D., Abell, S. E., Grogan, L.,Wayne, A., Skerratt, L. F., vanOosterzee, P., … Epstein, J. H. (2017). A guide for ecologists:Detectingtheroleofdiseaseinfaunaldeclinesandmanagingpop-ulationrecovery.Biological Conservation,214,136–146.https://doi.org/10.1016/j.biocon.2017.08.014

Qiang, W., Kuang, X., Liu, J., Liu, N., Scofield, V. L., Reid, A. J., …Wong,P.K. (2006).Astrocytes survivechronic infectionandcy-topathic effects of the ts1 mutant of the retrovirus Moloneymurine leukemia virus by upregulation of antioxidant defenses.Journal of Virology, 80, 3273–3284. https://doi.org/10.1128/JVI.80.7.3273‐3284.2006

RCoreTeam(2013).R: A language and environment for statistical comput‐ing.Vienna,Austria:RFoundationforStatisticalComputing.

Sapolsky,R.M.,Romero,L.M.,&Munck,A.U. (2000).Howdogluco-corticoids influence stress responses? Integrating permissive, sup-pressive,stimulatory,andpreparativeactions.Endocrine Reviews,21,55–89.https://doi.org/10.1210/er.21.1.55

Savage,A.E.,Terrell,K.A.,Gratwicke,B.,Mattheus,N.M.,Augustine,L., & Fleischer, R. C. (2016). Reduced immune function predictsdiseasesusceptibility in frogs infectedwithadeadly fungalpatho-gen. Conservation Physiology, 4, cow011. https://doi.org/10.1093/conphys/cow011

Sebastiano,M.,Bustamante,P.,Costantini,D.,Eulaers,I.,Malarvannan,G.,Mendez‐Fernandez,P.,…Chastel,O.(2016).Highlevelsofmer-curyandlowlevelsofpersistentorganicpollutantsinatropicalsea-bird inFrenchGuiana, theMagnificent frigatebird,Fregata magnifi‐cens. Environmental Pollution,214,384–393.

Sebastiano,M., Bustamante, P., Eulaers, I.,Malarvannan, G.,Mendez‐Fernandez, P., Churlaud, C.,…Chastel,O. (2017). Trophic ecologydrivescontaminantconcentrationswithinatropicalseabirdcommu-nity.Environmental Pollution,227,183–193.

Sebastiano, M., Chastel, O., de Thoisy, B., Eens, M., & Costantini, D.(2016). Oxidative stress favours herpes virus infection in verte-brates: Ameta‐analysis.Current Zoology, 62, 325–332. https://doi.org/10.1093/cz/zow019

Sebastiano,M., Eens,M., Abd Elgawad,H., de Thoisy, B., Lacoste, V.,Pineau,K.,…Costantini,D.(2017).Oxidativestressbiomarkersareassociatedwithvisibleclinicalsignsofadiseaseinfrigatebirdnest-lings.Scientific Reports,7,1599.

Sebastiano,M.,Eens,M.,Angelier,F.,Pineau,K.,Chastel,O.,&Costantini,D. (2017). Corticosterone, inflammation, immune status and telo-mere length in frigatebirdnestlings facingasevereherpesvirus in-fection.Conservation Physiology,5,cow073.

Sebastiano,M.,Eens,M.,Messina,S.,AbdElgawad,H.,Pineau,K.,Gerrit,B.,…Costantini,D.(2018).Datafrom:Resveratrolsupplementationreduces oxidative stress and modulates the immune response infree‐livinganimalsduringaviral infection.Dryad Digital Repository,https://doi.org/10.5061/dryad.r75251m

Selman,C.,McLaren,J.S.,Meyer,C.,Duncan,J.S.,Redman,P.,Collins,A.R.,…Speakman,J.R.(2006).Life‐longvitaminCsupplementationincombinationwithcoldexposuredoesnotaffectoxidativedamageorlifespaninmice,butdecreasesexpressionofantioxidantprotectiongenes.Mechanisms of Ageing and Development,127,897–904.https://doi.org/10.1016/j.mad.2006.09.008

Sinha,A.K.,AbdElgawad,H.,Giblen,T.,Zinta,G.,DeRop,M.,Asard,H.,…DeBoeck,G.(2014).Anti‐oxidativedefencesaremodulateddiffer-entiallyinthreefreshwaterteleostsinresponsetoammonia‐inducedoxidativestress.PLoS One,9,e95319.https://doi.org/10.1371/jour-nal.pone.0095319

Smith, K. F., Sax, D. F., & Lafferty, K. D. (2006). Evidence for therole of infectious disease in species extinction and endan-germent. Conservation Biology, 20, 1349–1357. https://doi.org/10.1111/j.1523‐1739.2006.00524.x

Staley,M.,&Bonneaud,C. (2015). Immune responsesofwildbirds toemerging infectious diseases. Parasite Immunology, 37, 242–254.https://doi.org/10.1111/pim.12191

vandeCrommenacker,J.,Richardson,D.S.,Koltz,A.M.,Hutchings,K.,&Komdeur,J.(2012).Parasiticinfectionandoxidativestatusareas-sociatedandvarywithbreedingactivity in theSeychelleswarbler.Proceedings of the Royal Society B: Biological Sciences,279,1466–1476.https://doi.org/10.1098/rspb.2011.1865

Vazquez‐Torres,A.,Jones‐Carson,J.,Mastroeni,P.,Ischiropoulos,H.,&Fang,F.C. (2000).Antimicrobialactionsof theNADPHphagocyteoxidaseand induciblenitricoxidesynthase inexperimental salmo-nellosis. I.Effectsonmicrobial killingbyactivatedperitonealmac-rophages in vitro. Journal of Experimental Medicine, 192, 227–236.https://doi.org/10.1084/jem.192.2.227

Wang,Y.‐J.,Chien,Y.‐H.,&Pan,C.‐H.(2006).Effectsofdietarysupple-mentationofcarotenoidsonsurvival,growth,pigmentation,andan-tioxidantcapacityofcharacins,Hyphessobrycon callistus. Aquaculture,261,641–648.https://doi.org/10.1016/j.aquaculture.2006.08.040

Xu,J.,Yin,Z.,Li,L.,Cheng,A.,Jia,R.,Song,X.,…Shi,F.(2013).Inhibitoryeffectofresveratrolagainstduckenteritisvirusinvitro.PLoS One,8,e65213.https://doi.org/10.1371/journal.pone.0065213

SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.

How to cite this article:SebastianoM,EensM,MessinaS,etal.Resveratrolsupplementationreducesoxidativestressandmodulatestheimmuneresponseinfree‐livinganimalsduringaviralinfection.Funct Ecol. 2018;32:2509–2519. https://doi.org/10.1111/1365‐2435.13195