© 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,23-34doi:10.1242/jeb.089722 *Author ([email protected]) for correspondence Vancouver, BC, V6T1Z4Canada. Zoology, BritishColumbia, of 6270University Blvd,Department University of experiencing them(Angilletta andSears,2011). Anadditional environmental variationandthe characteristicsoftheorganisms depend onboththetemporalandspatialcharacteristicsof the case remainsextremelychallenging,inpartbecausetheanswers Parsons, 2005;Via etal.,1995), answeringthemforanyspecific (Auld etal.,2010;Gabriel,2005;GabrielandLynch, 1992; substantial attentiontothesequestionsfromevolutionarytheorists promote orimpedeadaptationtolocalconditions?Despite environmental change,orboth?Dohighlyvariableenvironments the abilitytomodifytheirsensitivitystressorsinresponse to individuals withbroadtolerancetostressorsorfor with environmental change?Dovariableenvironmentsselect for How doesevolutionshapethewaysinwhichanimalsrespond to Adaptation, Intraspecificvariation Temperature, Salinity, Cortisol,Phenotypicplasticity, Acclimation, KEY WORDS:Homeostasis,Allostasis,Stress,Hypoxia, evolutionary perspectiveonstressors,stressandresponses. Examination ofthisempiricalexamplehighlightstheutilityusingan integrative natureoftheresponsetoenvironmentalstressors. biochemical andmolecularlevelsinkillifish,emphasizingthe stressor sensitivityandphenotypicplasticityarereflectedatthe highly plastic.Whole-organismdifferences betweenpopulationsin the stressresponseeveninorganismsthatarebroadlytolerantand their abilitytoacclimate,suggestingthatlocaladaptationcanshape variation inthesensitivityofkillifishtoenvironmentalstressors,and temperature, salinityandoxygenation.Thereisinter-population resistance andlong-termplasticityinthefaceofchanging marsh habitats,killifishhavesubstantialcapacityforbothshort-term the extremeenvironmentalvariationthattheyexperienceintheirsalt Atlantic killifish,Fundulusheteroclitus animals thathaveevolvedinhighlyvariableenvironments,usingthe phenomenon ofstress.Iexaminethisframeworkinthecontext fitness providesthebroadestandmostusefulconceptionof environmental changesthatcausereductionsinperformanceor environmental stressors.Isuggestthatviewingstressorsas general frameworkforunderstandinghoworganismsrespondto homeostasis andallostasis,attempttounifythemdevelopa of existingdefinitionsthetermstress,andrelatedconcepts responses toenvironmentalchange.HereIprovideabriefsummary caused confusionwhenattemptingtounderstandorganismal in biology, theinherentambiguityassociatedwithitsdefinitionhas Although thetermenvironmentalstressisusedacrossmultiplefields Patricia M.Schulte* variable environment What isenvironmentalstress?Insightsfromfishlivingina REVIEW Introduction ABSTRACT , asacasestudy. Consistentwith classified aseither stressfulornon-stressful. duration ofexposurethatresults inanenvironmentalfactorbeing or whetheritispossibletoclearly definealevelofintensityand change thatcausesaresponse in anorganism representsa stressor issue inthefieldofstressbiology iswhethereveryenvironmental biological organization. Oneimportantandsomewhatunresolved behavioural andphysiological changesatmultiplelevelsof mounting astressresponsethatinvolvescomplexset of (Galhardo andOliveira,2009).Organisms respondtostressorsby Ehlert, 2012),andtheyarelikelyimportantinotheranimalsas well in humansandothermammals(Armarioetal.,2012;Campbell and stressors, whichinvolveperceivedthreats,areextremelyimportant availability andtoxicants.Inaddition,psychologicalemotional conspecifics, aswellabioticfactorssuchtemperature, water predators, infectionwithpathogenicorganisms orinteractionswith They includebioticfactorssuchasfoodavailability, thepresenceof response itself.Stressorsareenvironmentalfactorsthatcausestress. refer toboththeenvironmentalfactorcausingresponseand the and thewordstress,sostressisoftenusedsomewhatloosely to authors arecarefultomakethedistinctionbetweenwordstressor precision inthedistinctionsbetweenthem.Forexample,notall confusion surroundingtheseconceptsmayarisefromalackof the termsstressor, stressandresponse,partofthe to accommodatealloftheseperspectives. 2005). Anygeneraldefinitionoftheconceptstressshouldbeable to environmentalchangesuchasthecellularstressresponse(Kültz, (Wingfield, 2013)andthoseinterestedinotheraspectsofresponses phenomenon oftheglucocorticoid-mediatedstressresponse between thoseprimarilyinterestedinthewhole-organism Boonstra, 2013).Anotherimportantconceptualdivideoccurs stressors innaturalpopulations(BijlsmaandLoeschcke,2005; from abiomedicalperspectiveandthoseinterestedintheeffects of of biology, withsubstantialgulfsbetweenthoseinterestedinstress research hasdevelopedrelativelyindependentlyacrossseveralfields contexts. Muchofthisconfusionmaystemfromthefactthatstress ambiguous andthewordisusedratherdifferently indifferent et al.,2009).Despitetheseattempts,however, thedefinitionremains et al.,2011; LeMoal,2007;McEwenandWingfield, 2010;Romero 2009; GoldsteinandKopin,2007;Johnstoneetal.,2012;Koolhaas critiques oftheconcept,andattemptstorefineitsmeaning(Fink, response ofthebodytoanydemand’,therehavebeenmanycogent 1950) firstintroducedtheconceptofstressas‘thenon-specific meant whenweusetheword‘stress’.EversinceHansSelye(Selye, dividing linebetweenthem? change andastressresponse,ifso,howcanwedraw between anormalhomeostaticresponsetoanenvironmental defining whatismeantbytheword‘stress’.Isthereadifference environmental stressorsstemssimplyfromthedifficulty ofclearly complication whenthinkingabouttheevolutionofresponsesto There isalsoadegreeofinherentcircularityinthedefinitions Unfortunately, itisnotalways aneasytasktodecidewhatis 23
The Journal of Experimental Biology 24 require dynamic changesinavarietyofparameters. emphasize theideathatmaintaining functionalhomeostasismay functionality ofasystem(Mangum andTowle, 1977).These concepts physiological variablesarevaried inordertomaintaintheoverall term enantiostasiswascoinedto refertoasituationinwhichmultiple may varyacrossenvironmentsor seasons(Mrosovsky, 1990), and the introduced toemphasizethatthe setpointsforhomeostaticregulation exposure toastressor(Fink,2009).Thetermrheostasis was describe theprocessofachievinganewequilibriumstatefollowing mechanisms. Forexample,Selyecoinedthetermheterostasis to focused onthedynamicandchangingnatureofregulatory organismal responsetoastressor. Inparticular, thesediscussionshave on whethertheideaofdefendinghomeostasisfullycaptures the conditions. response, astheremaynotbeasharpthresholdbetweenthese two factors thatcausesstress,asopposedtoanormalhomeostatic course, istoidentifythetype,levelandintensityofenvironmental (Day, 2005).Thedifficulty withthesetypesofdefinitions, of likely tooverwhelm,selectivehomeostaticresponsemechanisms’ multi-system responsetoanychallengethatoverwhelms,oris stress. Thishasledtoattemptsredefinestressas‘thebody’s broad responsestoseriouschallengesasmeetingthedefinitionof cycle’ (McEwen andWingfield, 2010)],orhaveconsideredonly to bestressful[e.g.‘inadditionthoseimposedbythenormallife considering onlyasubsetofmoreextremeenvironmentalfactors stress responses. draw acleardistinctionbetweennormalhomeostaticresponsesand (Goldstein andKopin,2007)thefieldcontinuestostruggle define inpractice,supportforitsexistenceismixedatbest (Fink, 2009;Selye,1950).However, theGAShasbeendifficult to conceived ofasanon-specificmultisystemresponsetostressors termed the‘generaladaptationsyndrome’ (GAS),whichhe homeostatic responsesofsinglephysiologicalsystemsandwhathe In fact,Selyedrewacleardistinctionbetweenindividual activities ofanorganism’s dailylifecould,inprinciple,causestress. one weretoapplythisdefinitionwithoutqualification,most transient physiologicaladjustmenttoanyenvironmentalchange.If as tobeoflimitedutility, asstresscouldpresumablyincludeevery However, thisdefinitionresultsinaconceptofstressthatissobroad threatened disturbanceofhomeostasis’ (Johnsonetal.,1992). often beenrestatedas‘thebody’s responsetoanyactualor response (reviewedinFink,2009).Selye’s definitionofstresshas from theearlierworkofWalter Cannondefiningthefight-or-flight stress asanon-specificresponsetochangesindemand,emerging The fieldofstressbiologyoriginatedwithSelye’s conceptionof REVIEW for understandinghoworganisms respondtoenvironmentalchange. to demonstratetheutilityofthisperspectiveonenvironmentalstress case studyofafishthatlivesinanextremelyvariableenvironment single over-arching concept.Finally, Iapplythisframeworktoa to bringtogetherthevariousdefinitionsofwordstressintoa a shortsurveyofthehistoryconceptsstress,andthenattempt and thestressresponse.To furtherexploretheseideas,hereIprovide conceiving ofstressasaninterveningprocessbetweenthestressor other hand,Iwouldargue thattheremaybesomevaluein of theorganism, andsothesetermsmaybesynonymous.Onthe hand, stresshasbeendefinedsincethetimeofSelyeasresponse stress andresponseessentiallyinterchangeably. Ontheone Stress asathreattohomeostasis Other approachestorefiningthedefinitionofstresshavefocused Attempts tonarrowthedefinitionofstressusuallyinvolve An additionalareaofimprecisionliesintheuseterms conserved features acrosstaxa. response differ, thehormonalresponse tostressorssharesmany Thus, althoughthespecific hormones involvedinthestress mobilizing glucose(Adamo, 2008; AdamoandBaker, 2011). is analogoustothefunction of theglucocorticoidhormonesin mobilizes lipidreservestofuel thefight-or-flight response,which a peptidehormonecalledadipokinetic hormoneisreleased,which response andischemicallysimilartonoradrenaline.Subsequently, (Adamo andBaker, 2011), whichisinvolvedinthefight-or-flight threat ofpredatorsbyreleasingthesignalingmoleculeoctopamine environmental challenges.Forexample,insectsrespondto the Invertebrates useanalogoussystemsforresponding to of thesteroidglucocorticoidhormones(MoyesandSchulte,2007). hypothalamic–pituitary–interrenal axis),whichcausestherelease hypothalamic–pituitary–adrenal axis(or, infishes,the (adrenaline andnoradrenaline)theactivationof the sympathetic nervoussystem,thereleaseofcatecholamines and psychologicalstressorsresultsintheactivationof the responses tostressors.Invertebrates,exposureenvironmental Much ofthefieldstressbiologyhasfocusedonneurohormonal that mayactuallycauseharm. are beneficial,thosethatmayimposeasignificantcostand change, andtheimportanceofdistinguishingbetweenresponsesthat issue ofthecostsmountingaresponsetoanenvironmental issues aside,theconceptofallostaticloadfocusesusonkey laboratory phenomenon(Boonstra,2013),butputtingthesespecific II allostasicoverloadeveroccursinnatureorwhetheritisstrictlya sufficient energy isavailable.Somehavequestionedwhether type activation oftheallostaticmechanismscausepathology, evenwhen performance. Type IIallostaticoverloadoccurswhentheprolonged allostasis exceedstheavailableenergy supply, resultinginreduced Type Iallostaticoverloadoccurswhentheenergy demandfrom activation ofallostaticmechanisms(McEwenandWingfield, 2010). serious negativephysiologicalconsequencesasaresultofthe ‘Allostatic overload’ isthepointatwhichananimalstartstosuffer dysregulation ofallostaticmechanisms(McEwenandStellar, 1993). as thewearandtearassociatedwithchronicoveractivityor allostasis istherelatedconceptof‘allostaticload’,whichdefined may notbestressfulinanother. stress, sinceanenvironmentalchangethatisstressfulinonecontext mechanisms thatmustbeconsideredwhenattemptingtodefine important inthattheypointoutthedynamicaspectsofregulatory various attemptsatarefinementoftheconcepthomeostasisare (Dallman, 2003;Day, 2005; Romeroetal.,2009).However, the conceived broadly, already encompasses alloftheseconcepts is necessaryoruseful,andhaveinsteadargued thathomeostasis,as introducing thetermallostasis,orindeedanyoftheseotherterms, environmental changetomaintainorganismal function. physiological mechanismsthatareusedtoanticipateorcopewith all cases,allostasisemphasizesthedynamicbehaviouraland Wingfield, 2003;McEwenandWingfield, 2010;Sterling,2012),in the definitionhasbeenrefinedbyvariousauthors(McEwenand proponents ofallostasishaveusedthetermslightlydifferently and through change’ (McEwenandWingfield, 2010).Althoughdifferent Wingfield, 2003).Allostasisis‘theprocessofachievingstability greatest attention(McEwenandStellar, 1993;McEwenand concept ofallostasis(SterlingandEyer, 1988)hasreceivedthe Stress asahormonallymediatedresponse Another importantideaemerging fromthediscussionsof A numberofauthorshavereasonablyquestionedwhether Among thesevariousreshapingsoftheideahomeostasis, The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089722
The Journal of Experimental Biology REVIEW natural conditions areseldomoptimal(Hoffmann andParsons, natural settingsexperiencestress muchormostofthetime,as reduces fitnessmightresultin theconclusionthatorganisms in Second, definingastressoras anyenvironmentalchangethat and thereforethisdefinitionmay besomewhatdifficult to apply. problems. First,measuringfitness ischallenginginmanyorganisms, of attractivefeatures,theysuffer fromsomepracticalandconceptual Parsons, 1991).Whiletheseevolutionarydefinitionshaveanumber biological systemwhichispotentiallyinjurious’ (Hoffmann and Calow, 1989)or‘anenvironmentalfactorcausingchangeinthe that, whenfirstapplied,impairsDarwinianfitness’ (Siblyand stressors byconsideringastressortobe‘anenvironmentalcondition Evolutionary biologistshavefocusedonthedetrimentaleffects of normal featuresofdailylife. environment, whereunpredictabilityanduncontrollability are particularly foranimalsthatareadaptedtoliveinahighlyvariable an environmentalfactormightbequitedifficult inpractice, 2011). However, usingthisdefinitiontoidentify stressfullevelsof include unpredictabilityanduncontrollability’ (Koolhaasetal., regulatory capacityofanorganism, inparticularsituationsthat conditions whereanenvironmentaldemandexceedsthenatural led tothesuggestionthat‘thetermstressshouldberestricted cannot beanticipatedorcompensatedforbytheorganism. Thishas focused onthedegreetowhichanenvironmentalchangecanor responses. A numberofattemptsto makethesedistinctionshave distinguish betweennormalhomeostaticresponsesandstress distinction betweenbenignandstressfulenvironmentsortoclearly provide anyadditionalinsightintotheappropriatewaytodrawa responses otherthanglucocorticoidreleasedoesnot,however, Broadening thedefinitionofwordstresstoaccommodate molecule totheindividualpopulation. of responsesatmultiplelevelsbiologicalorganization fromthe organism toenvironmentalchange,whichencompassesthediversity response assimplyonefacetofthecompletean broader perspectivewouldviewtheglucocorticoid-mediated arguing fortheutilityofabroaderdefinitiontermstress.This responses areconsideredtobestress(Gasch,2003), the capacitytorespondenvironmentalchallenges,andthese hormonal stressresponse.Certainlyunicellularorganisms alsohave considered tobeastressor, despitethelackofaglucocorticoid a deleteriousenvironmentalchangesuchasthiscouldreasonablybe even whenitcausesreductionsinperformance.Iwouldargue that always resultinareleaseofcortisolfish(O’Connoretal.,2011), mediated response.Forexample,environmentalhypoxiadoesnot are deleterioustovertebratesbutthatdonotinduceaglucocorticoid- addition, itseemspossibletoenvisageenvironmentalchangesthat et al.,2012),butthesechangesmaynotbetheresultofstress.In levels varywithtimeofdayandseasoninmanyanimals(Johnstone broad astoencompassallsituations.Forexample,glucocorticoid quantified, itisnotclearthatthisdefinitionofstresssufficiently simplifies thedetectionofstress,becausethesehormonesareeasily glucocorticoid-mediated stressresponse.Whilethisgreatly ‘stress response’ isoftentreatedassynonymouswiththe (Armario etal.,2012;CampbellandEhlert,2012),theterm treated asthekeyindicatorofoccurrencestressinvertebrates mechanisms hasbeensuchthatglucocorticoidreleaseisoften Stress asreductioninfitness environmental changes Stress astheresponsetounpredictableanduncontrollable The focusofthefieldstressbiologyonthesehormonal ‘mechanisms at thelevelofbrain,peripheral physiology, and an adequateresponsetothechallenges oftheenvironment,including was consideredtobetheability ofanindividualorspeciestomount ‘adaptive capacity’ isplottedonthe environmental factorareplotted onthe curves fromevolutionarybiology. Inthismodel,thevalues ofan reference totherichexistingliterature onperformanceandfitness the reactive-scopemodel(Romeroetal.,2009),butwithoutexplicit (Koolhaas etal.,2011) asanextensionofatime-basedmodelcalled curve oranycombinationoftheseshifts(Schulteetal.,2011). axis, changingtheperformancebreadth,heightof the curve hasanenvironmentalvariableonthe thus couldpotentiallybeusedtodetectstress.A typicalperformance curves displaytheeffects oftheenvironmentonaphenotype,and for fitnessinordertodetectthepresenceofastressor. Performance necessary tomeasurevariousperformancecharacteristicsasaproxy and evolutionaryhistory(BijlsmaLoeschcke,2005). to aparticularenvironmentalfactorisshapedbyitspriorexperience and theorganism experiencingit,becauseanorganism’s sensitivity experienced byanorganism isaconsequenceofboththestressor alter aspecies’ sensitivitytoastressor. Thus,thedegreeofstress adaptation mayresultingeneticchangesatthepopulationlevelthat adjust toastressor, andacrossmanygenerationsevolutionary Across afewgenerations,epigeneticchangesmayallowlineagesto effects ofastressorusingvarioustypesphenotypicplasticity. an individual’s lifetime,anorganism couldcompensateforthe mechanisms operatingatvarioustimescales.Forexample,within effect ofastressoronspeciesisnotfixed,andmayvarydueto occurred intheabsenceofresponse. resulting inanevengreaterdeclinefitnessthanwouldhave allostatic load).Alternatively, theresponsecouldbedeleterious, consequences fortheorganism (i.e.theycouldcontributetothe involve acost,whichwouldthenfactorintothefitness fitness. However, evenbeneficialresponseshavethepotentialto they compensate,inwholeorpart,forthepotentialreduction responses tothisstress.Thesemaybebeneficial,inthat would beconsideredtothesuiteofphysiologicalandbehavioural result ofexposuretoaparticularstressor, andthestressresponse would beconsideredtotheactualorpotentiallossinfitnessasa and theconceptofstressresponse.Inthisframework, allow acleardistinctiontobedrawnbetweentheconceptofstress on declinesinfitness,isthattheyprovideaquantitativemetricand different timescaleswillalwaysbeachallengingendeavour. (or anythinginbetween).Linkingprocessesoperatingatsuch transient andwellcompensated,orchronichighlydeleterious performance overanorganism’s lifetime,whilestressorsmaybe a problemoftimescale.Fitnessisanintegratedmeasure be oflimitedutilityinpractice.Finally, thesedefinitionssuffer from definition ofstressfirstproposedbySelye)maybesobroadasto 1991). Thus,thisdefinitionofstress(likethephysiological curves inavarietyofways,includingshiftingthecurvealong the plasticity andtransgenerationalplasticity)canshapeperformance phenotypic plasticity(includingacclimation,developmental stress thatistestableandquantifiable.Bothnaturalselection and decline. Performancecurvesprovideawaytovisualizeandidentify stressful atorbeyondthepointthatfitnessperformancebegins to 1).Anenvironmental changewouldthenbedefinedas (Fig. performance traitthatmaybeaproxyforfitness,onthe A similarconceptualmodelofstresshasbeendeveloped Because fitnessisdifficult tomeasureinpractice,itmaybe Taking anevolutionaryperspectiveemphasizestheideathat The appealofevolutionarydefinitionsstress,withtheirfocus The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089722 y -axis. Theadaptivecapacity x -axis, andtheso-called x -axis andfitness,ora y -axis 25 x -
The Journal of Experimental Biology From thisperspective, anenvironmentmightbe stressfulforaperiod the same,butitslocationwould change,orthepeakwouldbroaden. compensation, themaximumheight ofthefitnesspeakwouldremain fitness surface.Forexample, if theresponseresultedinperfect response, anditwouldhavethe resultofchangingtheshape This phenotypicplasticitywould beconsideredtoastress phenotypic plasticitytocompensate fortheeffects ofthe stressor. reduce performanceorfitness,itmayrespondbyusingexisting and anunstressfulenvironment. possible tomoreclearlydefinethedividinglinebetweenastressful physiological limitorthresholdisreached.Inthiscase,itmight be of environmentsandthendropsprecipitouslyonce some showed thresholdeffects, where fitnessremainshighacrossarange environment. However, onecouldalsoimagine afitnesssurfacethat is nosharpdividinglinebetweenastressfulandnon-stressful stress response. performance, andinsteadanewrangeofenvironments(S2)provokes previously experiencedasstressful(S1)nolongerresultsindeclines 26 greater theeffect onfitnessand thegreaterstress. The longertheenvironmentdeviatesfromitsoptimalcondition, the declines andtheorganism couldbesaidtoincreasinglystressed. duration. Astheenvironmentdeviatesfromoptimum,fitness dimensional spaceandconsidersbothstressorintensity performance curvesshowninFig. important indeterminingtheeffect onfitness;therefore,unlikethe and thelengthofexposuretothatenvironmentarelikelybe environmental variable.Boththelevelofvariable performance) mightbeaffected bychangesinaspecific hypothetical exampleofhowfitness(oraproxysuchas illustratesa 2 starting pointforaunifiedconceptofstress.Fig. clearly themostbroadlyapplicable,anditservesasanexcellent unified conceptofstress?Theevolutionarydefinitionstressis Considering alloftheseperspectives,isitpossibletodevelopa evolutionary definitionofstress. decline inadaptivecapacity)thatisessentiallythesameas performance breadth.Thenetresultisadefinitionofstress(as performance curve,andthattheregulatoryrangeisanalogousto practice, itisclearthattheresultingcurvewouldbeanalogoustoa adaptive capacitywouldbedifficult tomeasureorquantifyin range overwhichadaptivecapacitycanbemaintained.Although rangeisthenthe behaviour’ (Koolhaasetal.,2011). Theregulatory population) alongthe performance curveofanorganism(orthemeana set ofconditions.Phenotypicplasticityoradaptationcanshiftthe panel displaystheeffects oftheenvironmentonperformanceunderinitial those thatcauseperformancetodeclinebelowaspecifiedlevel.Theleft variable onperformanceorfitness. Fig. REVIEW A unifiedconceptofstress If anorganism encountersan environmentthathasthepotentialto The fitnesssurfaceshowninFig. Performance
1. Performancecurvesdisplayingtheeffectsofanenvironmental tesrSrso 1S2 S1 Stressor Stressor Environment x -axis (rightpanel)suchthatanenvironmentwas Stressful environmentsaredefinedas
1, thisexampleisplottedinthree-
2 isrelativelysmooth,sothere Performance Environment but chronicexposuremaybedeleterious,andthusmorestressful. the stressfulenvironments.Briefexposuresmayhavelittleeffect onfitness, decline andthedegreeofstressalsodependsondurationexposureto is lowerthantheoptimumcanbeconsideredstressful.Theextentoffitness (indicated bybluewhenfitnessdeclinestozero).Environmentswhere environment, anddecreasesastheenvironmentdeviatesfromthisvalue duration andfitness. Fig. response arespecific toaparticularspecies, andindeedto environmental factorscausinga stressresponseandthenatureof Ottaviani andFranceschi,1996), theexactnatureof taxa (Kassahnetal.,2009;Kültz, 2005;NesseandYoung, 2000; organismal andcellularlevelsaresharedacrossabroadrange of Although thegeneralfeatures of responsestostressorsatboththe ways inwhichanimalsrespondtoahighlyvariableenvironment. perspective onenvironmentalstressprovidesanyinsightinto the heteroclitus the workofmyresearchgroup,onAtlantickillifish, when itisappliedtoanempiricalexample.Here,Iutilizesome of has avarietyofattractivefeatures,itsutilitycanonlybeassessed stressor atalltimescales. individuals exposedtothestressor, and this factorwillactasa than intheoptimalenvironmentandlifetimefitnesswilldecline for is muchclearer:themaximumvalueoffitnesspeakwillbe lower stress responseiscostly, orthecompensationimperfect,situation detecting potentialstressorsincasessuchasthis.However, ifthe susceptibility andstressresponsescouldalsobeausefultoolfor challenge. Inter-individual orinter-population variabilityinstressor detectable declinesinperformanceorfitnessduringenvironmental normal stressresponsewaspreventedormanipulated,withresulting scale. Presumably, sucha‘hidden’ stressor mightberevealedifthe possible tomeasureadeclineinperformanceorfitnessatanytime response issorapidandcompensationcompletethatitnot whether tocallanenvironmentalfactorastressorifthestress to beimportant,leadingthesomewhatphilosophicalquestionof compensation. Inthiscontext,thetimescaleofresponseislikely to experiencingthestressor, evenwhenthestressresponseallows is analogoustotheconceptofallostaticloadinthatthereacost with organisms thatdidnotexperiencethisstressor. Thisdescription the resultingtransientdeclineinperformanceandfitness,compared would belowerinorganisms thathadexperiencedthestressorand value intheoriginalenvironment.Note,however, thatlifetimefitness of time,untilcompensatorymechanismsreturnperformancetoits Killifish asanempiricalexample Although thisevolutionaryframeworkforthinkingaboutstress
.Hypotheticalrelationshipbetweenstressorintensity, stressor 2. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089722 , todeterminewhethertakinganevolutionary Duration of exposure Fitness ishighest(indicatedbyred)intheoptimal Level ofenvironmentalfactor Fundulus
The Journal of Experimental Biology REVIEW Florida (acrossadistanceofover 2000 from theStLawrenceestuary inCanadatothenorthernpartof radiation andthedegreeofinfluencetides. depending onwaterdepth,airtemperature,theamountof solar tidal cycle.Similarly, temperaturemayvaryacrossthemarsh influenced creeksthewatermaybeclosetofreshformuchof the of freshwaterinput,whereasattheupstreamendtidally to full-strengthseawaterdependingonthetidalcycleandextent the mouthofamarsh,closetosea,salinityrangesfrombrackish changes therangeofabioticvariablesencountered.Forexample, at geographical scales.Atasmallscale,positionwithinsinglemarsh variation experiencedbykillifish,atbothsmalland large summer (Laymanetal.,2000)andarelesscommoninthewinter. result, bothextremehyperoxiaandhypoxiaaremorecommoninthe photosynthesis andrespirationincreasewithtemperature.Asa oxygen levels,andatnightrespirationdecreasesthem.Ratesofboth algae andbacteria.Duringtheday, photosynthesisincreaseswater is affected bythephotosyntheticandrespiratoryactivityofplants, seasonal patterns.Forexample,oxygenationofthewaterinmarshes with variationinotherabioticvariables,producingadditional through theyear. Thisseasonalvariation intemperatureinteracts in ashiftthemaximumandminimumtemperaturesexperienced temperature varieswithinadaydiffers betweenseasons,resulting some locations(Schulte,2007).Inaddition,therangeoverwhich example, meanmonthlytemperaturesvarybyasmuch15°Cat variation intheabioticcharacteristicsofkillifishhabitats.For time inthetidalcycle(Haasetal.,2009). strength seawaterdependingonthepositioninmarshand Able, 2004).Salinityalsovariesfromnearfreshwatertofull- complete anoxiatosupersaturation(Laymanetal.,2000;Smithand 1983). Similarly, dissolvedoxygenlevelscanvaryfromalmost change asmuch15°Cacrossasingletidalcycle(Sidelletal., temporal andspatialscales.Onadailyscale,temperaturecan including temperature,salinity, oxygenationandpHatavarietyof environments thatundergo dramaticchangesinabioticfactors Atlantic coastofNorthAmerica.Saltmarshesarehighlyvariable Populations ofF. heteroclitus stressors inkillifish,toanswerthequestionsoutlinedabove. comprehensive reviewoftheenvironmentalfactorsthatmayactas genetic variationpresentinthisspecies.Ithenprovidea emphasizing thevariablenatureofhabitatandhighlevels killifish habitatsandthepopulationgeneticstructureofthisspecies, the stressresponse,ifany, vary amongindividualsorpopulations? stressor sensitivityevolveinsuchasystem;and(3)whataspectsof a highlyvariableenvironment;(2)canintraspecificvariationin stress tounambiguouslyidentifystressors,eveninanorganism from following questions:(1)canweusetheevolutionarydefinitionof heteroclitus based onspecificexamples.Here,IuseAtlantickillifish( biology isnecessarilyempirical,andidentifiesgeneralprinciples of theirevolutionaryhistoryandpriorexperience.Thus,stress individuals withinthatspecies(Aubin-Horthetal.,2012)asaresult Killifish habitat temperature more than10°Clowermarshes atthesouthernend in thenorthernendofspecies rangehave,onaverage,a There isasteepthermalgradient alongthiscoast,suchthatmarshes to environmentalvariationat much broadergeographicalscales. Because There isalsoageographicalcomponenttotheenvironmental In additiontodailyvariation,thereisalsopronouncedseasonal I beginbybrieflydescribingtheenvironmentalfeaturesof F. heteroclitus ) asanempiricalcasestudywithwhichtoconsiderthe is foundalongmuchoftheAtlantic coast, are foundinsaltmarshesalongthe
km), theyarealsoexposed F. home rangecloserto300 be aslow40 the summerhomerangeofanadultkillifishhasbeenestimatedto remain withinarelativelysmallareasinglemarsh.Infact, season (Laymanetal.,2000;Nordlie,2006). than 40°Cinkillifishhabitatsdependingonthelocation,dayand in temperatureresultstemperaturesaslow fish. Thecombinationofdaily, seasonalandgeographicalvariation lower thantheminimumtemperatureexperiencedbyasouthern month, themaximumtemperatureexperiencedbyanorthernfishis of thespecies’ rangearesufficiently differentiated thatinanygiven in anygivenmonth(Schulte,2007).Thetemperaturesattheseends environmental change. underlying mechanisticbasis ofvariationinresponsesto variation makesthemexcellent modelswithwhichtoexaminethe both geneticallyandphenotypically inavarietyoftraits.This are lessimportantthanthefact thatthekillifishsubspeciesdiffer However, for thepurposesofthisreview, thesehistoricalarguments associated withpresent-dayenvironmental variationbychance. Alternatively, thecurrentdistributionofgeneticvariationcouldbe environmental factorssimilartothosenowpresentalongthecoast. genetic differentiation between theseformswasdrivenby glaciation haslikelyplayedarole.Itispossiblethattheoriginal between twopreviouslyisolatedpopulationsfollowingthe last Duvernell etal.,2008;Powers1991),secondarycontact variation inkillifishremainamatterofdebate(Adamsetal., 2006; reaches oftheselarge estuaries (Powersetal.,1991). coast andF. heteroclitus macrolepidotus heteroclitus heteroclitus are presentintheChesapeakeandDelawareBays,with 1990; Strandetal.,2012).Similargeneticandphysiologicalclines Vilasenor andPowers,1990;Powersetal.,1991;Ropson having clinesofdifferent steepness(Adamsetal.,2006;Gonzalez- and phenotypictraitsalongthecoast,withdifferent geneticmarkers 1983). Thisvariationtakestheformofaclineingenefrequencies subspecies centredinorjustnorthofNewJersey(MorinandAble, heteroclitus northern partofthespeciesrangeandisreplacedby recognized. populations of There isgenetic,morphologicalandphysiologicalvariationamong killifish, hasthepotentialtoresultinlocaladaptation. environmental variation,coupledwiththelowdispersalpotentialof phenotypic plasticity. Finally, thegeographiccomponentof environments indifferent seasons,mightbeexpectedtoselectfor and particularlythefactthatfishexperiencedifferent rangesof addition, theseasonalcomponentofenvironmentalvariation, individuals thatareresistanttolarge changesinabioticvariables.In variation atasinglelocationmightbeexpectedtoselectfor across multipletemporalandspatialscales.Thehighlevelof killifish experiencelarge fluctuationsintheirabioticenvironment occur (Haasetal.,2009). However, movementsofseveralkilometresalongtidalcreekscan (Halpin, 2000;McMahonetal.,2005;Teo andAble,2003). and theyusuallyreturntothetidalcreekswithhighsitefidelity salt marshsurfacetoforagebutseldommovemorethanakilometre, 2003). Duringtheday, killifish movefromtidalcreeksoutontothe Killifish genetics Atlantic killifishdonothavepelagiclarvaeandadultstendto Although thehistoricalfactorscausingthisdistributionofgenetic As aresultoftheirrestrictedmovementswithinsinglemarsh, The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089722 Fundulus heteroclitus macrolepidotus in thesouth,withtransitionbetweentwo F. heteroclitus m 2 (Lotrich, 1975),althoughmoststudiessuggesta (the southernsubspecies)dominatingatthe
m 2 (McMahon etal.,2005;Teo andAble, to thepointthattwosubspeciesare dominating thefreshwater −1.5°C orgreater F. heteroclitus occupies the 27 F.
The Journal of Experimental Biology 28 expected, given thehighlyvariablesalinityin theirenvironment, Changes inenvironmentalsalinity canbestressorsinfish,butas mediated stressresponseinavariety ofways. clear thatnorthernandsouthern killifishdiffer intheircortisol- adaptive significance(ifany)of thisvariationarenotknown,itis for atleastsomeofthisvariation.Althoughtheconsequences and handling stressbetweenthesubspecies,suggestingagenetic basis differences resultindifferences inthemetabolicresponseto dehydrogenase-B (Schulteetal.,2000),andthesegenetic element withinthepromoterofgeneencodinglactate subspecies havebeendetectedinaglucocorticoid-responsive northern, fish.Inaddition,geneticdifferences betweenthe and serinethreoninekinasesintheliversofsouthern,butnot in example, handlingstressupregulatesgenesencodingMAP kinases are activatedbycortisolrelease(PicardandSchulte,2004). For (DeKoning etal.,2004).Thesubspeciesalsodiffer inthegenesthat patterns occurwhenfisharegivenaslow-releasecortisolimplant be mediatedbytherateofcortisolclearance,becausesimilar southern fishthaninnorthernfish.Interestingly, thisdifference may the courseofaweek.Bothstressorselevatecortisolmorein single acuteepisodeofhandlingorchronicrepeatedover a standardizedhandlingstressor. The protocolinvolvedeithera showstheplasmalevelsofcortisolfollowing 3 handling stress.Fig. southern subspeciesofkillifishdiffer intheirsusceptibilityto glucocorticoid infish(LeachandTaylor, 1980).Thenorthernand al., 2006),andtoincreaseplasmacortisol,whichistheprimary increased expressionofc-fosinvariousbrainregions(Saliernoet to alterneuronalactivationinthekillifishbrain,asdemonstratedby killifish hasexaminedhandlingstress.Thisstressorbeenshown stress response(LeachandTaylor, 1980).Themajorityofworkin a varietyofbehaviouralstressorsprovokeglucocorticoid-mediated and otherbioticstressorsinkillifish.Inkillifish,asmostfishes, There hasbeenlimitedinvestigationoftheeffects ofbehavioural responses inkillifishoutlinedabovearefulfilled. the predictionsofevolutionarymodelsaboutpatternsstress evolutionary definitionofthetermstress,andtodeterminewhether versus thosethatwouldnotbeconsideredstressful,underthe environmental factorsthatwouldbeconsideredtostressful determine whetheritispossibletoclearlydifferentiate between responses ofkillifishtoavarietyenvironmentalstressors Hendry, 2011). Thenextsectionexaminestheexistingdataon stressors acrosslocations(Fitzpatrick,2012;Thibert-Planteand constrain differentiation intheabilitytocopewithenvironmental across dailyandseasonalscaleswithinasinglelocationmight is possiblethathavingtocopewithawiderangeofconditions geographic variationinconditionsalongthecoast.Alternatively, it expect toobservesomeevidenceofdifferentiation relatedtothe addition, giventhelowdispersalpotentialofkillifish,wemightalso in therangeofenvironmentsthattheyexperienceasstressful.In and (3)havethecapacitytomakeadjustmentsseasonalvariation occur rarelyorthatrepresentextremesatagivenlocationseason, environmental conditions(orcombinationsofconditions)thatonly at anacutetimescale,(2)bemoststressedwhenexposedto be abletocopewithlarge changesinarangeofabioticconditions Plante andHendry, 2011) onemightpredictthatkillifishwould:(1) (Fitzpatrick, 2012;Gabriel,2005;Ghalamboretal.,2007;Thibert- Based ontheoreticalmodelsofevolutioninvariableenvironments REVIEW Salinity asastressor Behavioural andbioticstressors inkillifish Stressors deiodinase 1in thegillinresponsetofreshwater challenge.This addition, therearedifferences intheupregulationofiodothyronine in thetransitiontowardsafreshwater-type gillmorphology. In regulated bythetranscriptionfactor HNF4a(Whiteheadetal.,2012) southern subspeciestolowsalinity suggestaroleforpathways available. have atleastsomecapacitytogillremodelingifnootheroption is use behaviouralstrategiestoavoidfreshwaterinnature,but they seawater (Buckingetal.,2012),suggestingthattheywouldtypically have astrongbehaviouralpreferenceforbrackishtofull-strength are foundintidallyinfluencedpartsofestuarinemarshes,and they subspecies makesacompletetransition.Ingeneral, tidal cycle(Whiteheadetal.,2012),andonlythenorthern morphology onlyiftheyremaininfreshwaterlongerthanasingle very closetofreshwater, andmakethetransition toafreshwater-type killifish maintainaseawater-type gillmorphologydown tosalinities (Scott etal.,2004;Whitehead2012).Unlikeother fish, undergo atransitionbetween seawaterandfreshwatermorphologies investigated andinvolvedifferences intheabilityofgillto ability oftheadultstwosubspecieshavebeenintensively action ofnaturalselectiononthistrait(Whiteheadetal.,2012). salinity ofthehabitatsinwhichtheyarefoundisconsistentwith range ofthesubspeciesthatistolerantlowsalinitiesand The observationofarepeatedassociationbetweenthegeographic end andthesouthernsubspeciesatsaltwatermouthofbothbays. Delaware Bays,withthenorthernsubspeciesfoundinfreshwater observed alongthecoastarealsoseeninChesapeakeand (Whitehead etal.,2012).Interestingly, clinessimilartothose of 0.4 subspecies areabletomaintainionichomeostasisdownasalinity of thesouthernsubspecies(AbleandPalmer, 1988).Asadults,both northern subspeciescandevelopinlowersalinitiesthanthose they havepoorfertilizationandhatchingsuccess,butembryosofthe et al.,2004).Neithersubspeciesreproduceswellinfreshwater, and levels andexperiencehighermortalityundertheseconditions(Scott fish ofthesouthernsubspeciesarelessabletoregulateplasmaion the northernsubspeciesabletotolerateverysoftfreshwater, while differ intheirabilitytotolerateextremelylowsalinity, withfishof full-strength seawater(Griffith, 1974).However, thetwosubspecies killifish cantoleratesalinitiesfromnearfreshwatertogreaterthan DeKoning etal.(DeKoningal.,2004). handling threetimesdailyfor7 handling stressorofeitherasingleevent(handled)orrepeated levels morethanthenorthernsubspecieswhenexposedtoastandardized Fundulus heteroclitus. Fig. Microarray studiescomparing the responsesofnorthernand The mechanismsunderlyingdifferences intheionoregulatory
3. Elevationinplasmacortisolfollowinghandlingstress
ppt, buttheybegintodiverge atsalinitiesbelow0.1 Plasma cortisol (ng ml–1) 200 400 600 800 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089722 0 Northern fish The southernsubspeciesincreasesplasmacortisol Chronic stress Handled Control
days (chronicstress).Dataarefrom Southern fish * F. heteroclitus *
ppt
The Journal of Experimental Biology REVIEW ( maintain equilibriumdowntothefreezingpointofbrackishwater above 10°C;belowthistemperaturebothsubspeciesareableto tolerance differences areapparentatallacclimationtemperatures Both subspeciesshowsimilarplasticityintolerance,sothese than northernfishwithoutlosingequilibrium(Fangueetal.,2006). with southernfishtoleratingtemperaturesapproximately2°Chigher and lowthermaltolerancediffers slightlybetweenthesubspecies, 41.5°C whenacclimatedto30°C(Fangueetal.,2006).Bothhigh is 28.5°Cwhenanorthernkillifishacclimatedto2.5°C,and also extremelyplastic.Forexample,maximumtoleratedtemperature losing equilibrium(Fangueetal.,2006).Theirthermaltoleranceis subspecies cantoleratetemperaturesbetween1.7and38°Cwithout example, whenacclimatedto22°C,akillifishofthenorthern Killifish alsotoleratelarge, acutechangesintemperature.For tolerance andthecapacityforacclimation. environmental stressorcanoccureveninspecieswithbothbroad subspecies, demonstratingthatlocaladaptationintheresponsetoan is associatedwithdifferences inhabitatchoicebetweenthe their abilitytoacclimatefreshwater, andthisvariationinplasticity salinity tolerance,thetwosubspeciesofkillifishhavediverged in conditions) arehighlystressfulforkillifish.Despitetheirbroad environments thatarerarelyexperienced(i.e.extremefreshwater and molecularlevelsareconsistentwiththeideathatonly freshwater fishingeneral(McCormick,2001). examination oftherolethyroidhormonesinosmoregulation stickleback (Kitanoetal.,2010),pointingtotheneedforare- hormone pathwaysmayalsoplayaroleinfreshwateradaptation in freshwater(McNabbandPickford,1970).However, thyroid et al.,1982),andthyroidhormonelevelsarehigherinseawaterthan disturbs ionregulationinseawaterbutnotfreshwater(Knoeppel surprising becauseinhibitionofthethyroidhormoneaxisinkillifish peripheral tissues(Whiteheadetal.,2012).Thisresponseis enzyme isresponsiblefortheactivationofthyroidhormonein sensitivity ofburst (anaerobic)swimmingcapacity aresimilar, with above 33°Carestressfulfor killifish. Patternsforthethermal declines inperformancesuggest thattemperaturesbelow10°Cand unknown. However, evenwithoutadirectestimateoffitness,the in thesensethatthisacclimation responseimprovesfitness,remains acclimation’ (BerriganandHuey, 1996;Wilson andFranklin, 2002), compensation ofswimmingperformance representstrue‘beneficial are acclimatedtotheirtesttemperature(Fig. allowing performancetobemaintainedfrom1033°Cinfish that (Fangue etal.,2008).Acclimationextendsthethermalbreadth, range, suggestingthatthesetemperaturesmaybeacutelystressful 4),butperformancedeclinesbeyond this from 15to25°C(Fig. swimming performancewithacuteexposuretotemperaturesranging experience asstressful. that thesubspecieshavediverged inthetemperaturesthey 50% mortalityat36.4°Candsouthernkillifish38.2°C,suggesting to whichtheycanbeacclimated,withnorthernkillifishsuffering northern andsouthernkillifishdiffer inthemaximumtemperature exposure tohightemperatureisextremelystressful.Inaddition, substantial mortality(Fangueetal.,2006),suggestingthatlong-term acclimated totemperaturesabove~35°Cwithoutexperiencing temperatures arestressfulforkillifish?Killifishcannotbe acutely, thesetemperaturescouldcausestress.Soexactlywhat Temperature asastressor − 1.7°C) (Fangueetal.,2006). Together, thedataonsalinitytoleranceatorganismal, cellular Killifish acclimatedto20°Careablemaintainaerobic Even thoughkillifishcantoleratehigh(orlow)temperatures
4). Whetherthis in thenorthernsubspecies.DataarefromFangueetal.(Fangueal.,2008). subspecies andsignificantlyimprovesperformanceatextremetemperatures temperature. SymbolsasinA.Acclimationincreasesthermalbreadthboth acclimated tovarioustemperaturesandtestedattheiracclimation exposed totemperaturechange.(B) and southern(redlinesymbols)killifishacclimatedto15°Cacutely s.e.m. inbodylengths(BL)persecond]northern(bluelineandsymbols) these temperatures arestressfulforkillifishbased ontheeffect of killifish generallyavoidtemperatures greaterthan35°C.Again, swimming performance.Atthe highendofthethermalrange, consistent withtherangeofstressful temperaturesdefinedbasedon subspecies avoidtemperatures below approximately12°C,whichis Considering thelowerendof the thermalrange,killifishofboth 15 or25°Cobservedinathermal gradient(Fangueetal.,2009). showsthepreferredtemperatureofkillifishacclimated to5, 5 Fig. al., 2009),andoptimumperformanceisindicativeoflowstress. an organism andtheoptimal temperatureforperformance(Dillonet generally astrongcorrelationbetweenthepreferredtemperature of stressful environmentswhentheyareavailable.Infact,there is killifish prefer, asorganisms wouldbepredictedtochoosenon- stressful temperaturesmightbetoexaminethe that and below10°Cwouldbeconsideredtoseverestressors. broader thermalrange.Thus,onlytemperaturesaboveabout 33°C plasticity allowscompensationtomaintainperformanceacross a acclimated tointermediatetemperatures,butthatphysiological above 25°Candbelow15°Cmaybeacutelystressfultokillifish performance andsustainedswimmingsuggeststhattemperatures and Bennett,1995).Theeffects oftemperatureonbothburst acute exposureovertemperaturesrangingfrom5to25°C(Johnson fish acclimatedto10°Cbeingablemaintainperformancewith Fig. heteroclitus. Another approachtodistinguishingbetweenstressfulandnon-
4. Effectsoftemperatureonswimmingperformancein
–1 Ucrit (BL s ) 0 2 4 6 8 0 2 4 6 8 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089722 (A) Aerobicswimmingperformance[measuredas 102030400 102030400 B A Acclimation andtesttemperature(°C) Test temperature(°C) U crit of bothsubspecieskillifish Southern fish Northern fish Fundulus U crit ; mean± 29
The Journal of Experimental Biology 30 that fecundity may bemaximalat~30°C in bothsubspecies. example, ouranecdotalobservations offishinthelaboratorysuggest than swimmingperformanceare maximizedatthistemperature.For high? Thispatterncouldbeexplained ifperformancemetricsother why dokillifishhaveastrong thermal preference,andwhyisitso killifish wouldequallyfavourall temperatureswithinthisrange.So thermal performancecurvefor swimmingmightsuggestthat (Martin andHuey, 2008).Alternatively, theextremebreadthof a safetyfactoragainstexposuretodamaginglyhightemperatures prefer temperaturesatthelowendoftheiroptimalrangetoprovide from twoperspectives.First,theorysuggeststhatorganisms should the stressfulrange.A preferenceforhightemperaturesisunexpected for temperaturesfrom29to33°C,valuesthatareatorveryclose to depending ontheiracclimationtemperature,showaclearpreference pattern isparticularlyobviousfornorthernkillifish,which, based ontheeffects oftemperature onswimmingperformance.This enter temperaturesthatarelikelytobeacutelystressful(33–36°C), their optimalperformancerange,andtheyappeartovoluntarily depending onthesubspecies)withoutsubstantialmortality. killifish cannotbeacclimatedtohightemperatures(36–38°C, temperature onswimmingperformanceandtheobservationthat monitored inathermalgradientfor10 Symbols asinA.Thepositionofeachfish( (B) Thermalpreferenceof circles), 15°C(pinklinesandsquares)or25°C(redtriangles). macrolepidotus different temperatures. Fig. REVIEW the timespentateachtemperaturefrom14:00to17:00 recalculated fromFangueetal.(Fangueal.,2009). possible diurnalrhythmsinpreference)calculated2°Cbins.Dataare In general,killifishtendtoprefertemperaturesatthehighend of
5. Thermalpreferenceof Percent of time spent 10 15 20 25 10 15 20 25 0 5 0 5 01 02 04 45 40 30 25 20 15 10 5 0 01 02 04 45 40 30 25 20 15 10 5 0 B A (northern subspecies)acclimatedto5°C(bluelinesand 25°C 15°C 5°C (A) Thermalpreferenceof F. heteroclitus heteroclitus Fundulus heteroclitus Temperature (°C)
h. Dataareexpressedaspercentof N =9 foreachsubspecies)was F. heteroclitus (southern subspecies).
acclimated to h (tocontrolfor 35 35 subspecies, with expressionpatternsindicative ofincreasedprotein general featuresoftheheatshock responsearesimilarbetweenthe induction acrossthegenes.Microarray analysissuggeststhatthe consistent patternindifferences betweensubspeciesinthe extentof whereas induced atlowertemperatures insouthernthannorthernfish, differences betweenthesubspecies(Fangueetal.,2006). temperatures between30and33°C indicate acellularstressresponse,arefirstexpressedinkillifish at cellular level.Forexample,variousheatshockproteins,which 6). consumption areslightlycompromisedatthistemperature(Fig. oxygen consumptionto33°C,butmaximumratesof 15°C). Incontrast,southernkillifishmaintainroutinerates of temperatures above30°Cinnorthernkillifish(whenacclimated to this expectedpatternbeginstobreakdownwithacuteexposure to expected basedonArrheniuseffects (HealyandSchulte,2012),but consumption increasesexponentiallywithtemperature,aswould be killifish. Forexample,therateofbothroutineandmaximumoxygen that temperaturesgreaterthan~33°Careacutelystressfulfor likely tobestressful. temperatures greaterthan35°C,sothesearehighly it isclearfromthethermalpreferencedatathatkillifishavoid range, andseveraldegreesbelowastressfultemperature.However, gradient is~27°C,whichclosertothecentreoftheiroptimal temperatures. Theaveragetemperatureoccupiedbykillifishinthe few minutes,andisusuallyfollowedbyentryintomuchlower high temperatures,anyindividualexposureisgenerallylessthana Although theyspendasubstantialamountoftheirtotaltimeatthese apparatus –theymakebrief,butfrequent,excursionsintothem. long periodsoftimeatthesetemperaturesinthethermalgradient killifish usethethermalgradientapparatus.Killifishdonotspend the apparentthermalpreferenceisaconsequenceofwaythat to occupytemperaturesof29–33°C.Alternatively, itispossiblethat only aslightriskofexposuretodamagingtemperaturesifitchooses at thesouthernendofspecies’ range.Thus,akillifishmayrun occur formorethanafewhoursattimeinkillifishhabitat,even stressful followingacclimation.Temperatures above35°Crarely in mostsituations,andonlytemperaturesevenhigherthanthisare temperatures greaterthan30–33°Careacutelystressfulforkillifish temperatures? Basedonthedataforswimmingperformance,only exactly howriskyisitforakillifishtopreferthesehigh northern killifishselecthigherthanoptimaltemperatures.But preference behaviourarenotstronglyselected,andasaresult high temperatures,itispossiblethattheaversiveinputsregulating ones). Ifthehabitatsofnorthernkillifishseldomreachdamagingly are attractedtowarmtemperatures,butavoiddamaginglyhigh result fromabalanceofattractiveandaversiveinputs(i.e.killifish killifish couldbemaladaptive.Thermalpreferenceisthoughtto (Fangue etal.,2009).Alternatively, thehighthermalpreferenceof occur. Thisphenomenonisanexampleofcountergradient variation them totakeadvantageofreproductiveopportunitieswheneverthey preference ofnorthernkillifishforwarmtemperaturescouldallow which couldincreasegrowthandreproductiveoutput.Thestrong gain thethermodynamicadvantagesofincreasedratesreactions, northern partofthespecies’ range).Athighertemperatures,killifish situations whenthereproductiveperiodisshorter(suchasin strong preferenceforhightemperaturescouldbebeneficialin temperatures). Onehypothesisconsistentwiththispatternisthata clearest innorthernfish(andsouthernacclimatedtolower However, suchanargument doesnotexplainwhythepreferenceis These whole-organism indicators ofstressarealsoreflectedatthe Other performancetraitsarealsoconsistentwiththehypothesis Hsp70 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089722 is inducedatsimilartemperatures. Thereisalsono Fg 7), (Fig. but withnoconsistent Hsp90 is
The Journal of Experimental Biology temperature for heat shockproteininduction. higher temperaturesmightbe expected toincreasethethreshold of whole-organism thermaltoleranceandperformance,acclimation to REVIEW yet beenexaminedin plasticity oftheheatshockresponse withthermalacclimationhasnot with thepatternsobservedfollowing heatshockinmanyspecies.The downregulation ofglobaltranscription (Healyetal.,2010),consistent ubiquitination, upregulationofapoptoticpathways and and Schulte,2012). subspecies athightemperatures.DataarefromHealyandSchulte(Healy MMR, andforMMRinsouthernfish,suchthatRMRconvergesbetween the expectations attemperaturesabove30°CfornorthernfishinbothRMR and curves fittoeachdataset.Notethedeparturesfromexponential subspecies of southern subspecies.(C)ComparisonofRMRforthenorthernand various temperatures.(B)MMRandRMRunderthesameconditionsfor the (RMR) inthenorthernsubspeciesacclimatedto15°Candacutelyexposed to heteroclitus. Fig.
6. Effectsoftemperatureonmetabolicratein Routine metabolic rate (µmol h–1 g–1) Metabolic rate (µmol h–1 g–1) Metabolic rate (µmol h–1 g–1) 10 15 20 10 20 30 10 20 30 0 5 0 0 102030400 102030400 102030400 (A) Maximummetabolicrate(MMR)androutine F. heteroclitus C B A Southern fish Northern fish F. heteroclitus RMR MMR RMR MMR . Dataaremeans±s.e.m.Linesexponential Temperature (°C) . However, based ontheplasticity Fundulus (acclimated to20°Candacutelyexposedheatshocktemperaturesfor 2 temperatures between30and33°Cinnorthernsouthernkillifish Fundulus heteroclitus. and sampledfollowing2 Fig. held at5°Cinthe laboratory, andthusareinnegative energy balance southern nornorthernkillifishare abletomaintainbodymasswhen term exposuretocoldisalso stressfulforkillifish,asneither increases incortisolsecretion(Pickford etal.,1971).Similarly, long- cold shockresultsinincreases in circulatingleukocytesbecauseof temperatures areaseverestressor forkillifish.Forexample,acute at thecellularlevelareconsistent withtheideathatlow are means±s.e.m.fromFangueetal.(Fangueal.,2006). convulsions andmortalityinthissubspeciespreliminaryexperiments. Data exposed abruptlytotemperaturesabove35°Cbecausethiscaused killifish: redlinesandsymbols.Notethatnorthernkillifishcouldnotbe elongation factor-1alpha.Northernkillifish:bluelinesandsymbols;southern (A: Hsp90 As isthecaseforwhole-organism performanceindicators,traits
.Onsettemperaturesofheatshockprotein( 7. Hsp70-2 expression Hsp70-1 expression Hsp90a expression 0.5 1.0 1.5 ; B: 0.5 1.0 1.5 0.2 0.4 0.6 0.8 0 0 0
Control The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089722 Hsp70-1 C B A Handled ; C:Hsp70-2
Southern fish Northern fish h ofrecovery).Dataarerelative Heat shockproteinmRNA levelsincrease at Treatment (heatshock,°C) 03 23 43 36 35 34 33 32 31 30 ) normalizedtotheexpressionof hsp) expressionin hsp mRNA levels
31 h
The Journal of Experimental Biology 32 been showntoexhibit greaterlevelsofinter-individual variationin Killifish acclimatedtoconstant conditionsinthelaboratoryhave this question,butonedataset does providesomeintriguingclues. environment bestressful?There arefewdataavailabletoaddress extremely unusualsituationfor akillifish.Butwouldsuchan the constantenvironmenttypically imposedinthelaboratoryisan to anever-changing, somewhat unpredictableenvironment.Thus, From theprecedingdiscussion,itisclearthatkillifishareadapted for furtherinvestigation. the potentialmechanismscausingthiseffect remainafruitfularea killifish [orindeedinanymarineorganism (Crainetal.,2008)],so stressor. Therehavebeenlimitedstudiesofinteractingstressorsin compromise theorganism’s abilitytocopewithanadditional where thecostsofmountingaresponsetoonestressor may interaction mayrepresentanexampleoftheeffects ofallostaticload, a stressorwhenexperiencedincombinationwithanotherfactor. This does notrepresentastressorwhenexperiencedalonecouldbecome stressors mayactsynergistically andanenvironmentalfactorthat temperature onmetabolicrate(McBryanetal.,2013).Thus, this declineislarger thanwouldbepredictedbasedontheeffects of Similarly, hypoxiatolerancedeclinesastemperatureincreases,but temperatures (T. M.HealyandP.M.S., unpublishedobservations). handling stressorthankillifishacclimatedtomoderateorlow temperatures above25°Caremorelikelytodiefollowingasevere For example,wehaveobservedthatkillifishacclimatedto multiple stressorsthatmayinteractincomplexanddynamicways. exposed tostressorsindividually. Insteadtheymustcopewith In theircomplex,highlyvariablehabitats,killifishareseldom so stressfulthatpopulationscannotpersist. circumstances. Thus,winterisstressfulfornorthernkillifish,butnot the environment,butratherasolutionthatisgoodenough,given Natural selectiondoesnotnecessarilyresultinaperfectmatchwith not limitedbyepistaticinteractionsortrade-offs withothertraits. available geneticvariationforthetraitsofinterest,andwhenitis and Loeschcke,2005),naturalselectioncanonlyactifthereis in theenvironmentsmostcommonlyoccupiedbyaspecies(Bijlsma Fay, 2005),whichmightbeexpectedtoresultinrelativelylowstress are normalpartsoftheirlifehistory. killifish mustregularlyexperiencestressevenunderconditionsthat subspecies, thiscompensationisfarfromperfect.Thusnorthern some evidenceoftheevolutioncompensatoryresponsesinthis stress duetolowtemperatureseachwinter, andalthoughthereis thus couldbeconsideredstressful.Theyalsoexperiencesevere the year, yetthesetemperaturesaresuboptimalforperformance,and killifish experiencetemperaturesbetween10and15°Cformuchof environmental conditions(BijlsmaandLoeschcke,2005).Northern environmental conditionsaremorestressfulthantypical not consistentwiththehypothesisthatrarelyencountered killifish, particularlywhenexperiencedchronically. Thispatternis below approximately12°Candabove30–33°Carestressfulfor while southernkillifishdonot(DhillonandSchulte,2011). northern killifishincreasemitochondrialvolumedensityinmuscle, subspecies. Similarpatternsareevidentatthecellularlevel,as performance (Fangueetal.,2008),butonlyinthenorthern evidence thatcoldacclimationcanmodestlyimproveswimming under theseconditions(HealyandSchulte,2012).Thereissome REVIEW Is aconstantenvironmentstressfulforkillifish? Interacting stressors Although stresscanactasaforcedrivingadaptation(Lexerand Consideration ofallthesedatasuggeststhattemperatures organisms arecurrentlyexperiencing. rapid, anthropogenicallymediatedenvironmentalchanges that particularly helpfulasweattempttounderstandtheeffects ofthe extremely variableenvironments.Thisperspectivemay be environmental change,andisusefulevenfororganisms livingin clarify ourunderstandingoftheresponsesorganisms to an evolutionaryperspectiveonthedefinitionofstresshelpsto the responsesofkillifishtoenvironmentalchangeshowshowtaking it hasanepigeneticcomponent,iscurrentlyunknown.Examining to bioticandabioticstressorsisgeneticallydetermined,orwhether easy tointerpret.Whetherthisintraspecificvariationinsensitivity exposed tostressors,althoughthesepatternsarenotalwaysclearor differences incortisolreleaseandgeneexpressionpatternswhen stressor sensitivityandcapacityforacclimationarereflectedin range andatthesaltwatermouthsofmajorestuaries. tolerant offreshwater, isfoundinthesouthernendofspecies’ subspecies, whichismoretolerantofhightemperaturesandless in thefreshwaterreachesofmajorestuaries,whilesouthern salinities, dominatesatthenorthernextremeofspecies’ rangeand subspecies, whichismoretolerantofcoldtemperaturesandlow matches thecurrenthabitatdistributionsofkillifish:northern observed variationinstressorsensitivityandthestressresponse evolve inorganisms livinginhighlyvariableenvironments.The plasticity showthatintraspecificvariationinstressorsensitivitycan The differences betweenkillifishsubspeciesinbothtoleranceand persist andeventhrive,theseconditionsarestressfulforthisspecies. So althoughkillifishcantoleratetheseconditions,andpopulations (for exampleduringwinterinthenorthernpartofspecies’ range). encounter stressfulenvironmentsasanaturalpartoftheirlifecycle However, despitetheirbroadtoleranceandplasticity, killifishoften They alsoshowsubstantialplasticityintheresponsetostressors. tolerant oflarge changesinawiderangeofenvironmentalvariables. identified theseenvironments,wecanseethatkillifisharebroadly environments asstressfulornotforkillifish.Having stress responses.Usingthesedefinitions,wecanclearlyidentify of takinganevolutionaryperspectivetodefinestressors,stressand discussion, thecasestudyofAtlantickillifishdemonstratesutility Returning tothequestionsthatwereposedatbeginningofthis is clearthatthisquestiondeservesfurtherattention. natural environment(OleksiakandCrawford,2012).Inanycase,it expression otherwisemaskedbytheresponsestostressful under constantnear-optimal conditionsmightallowvariationin Alternatively, thereductionin stressimposedbyholdingkillifish laboratory environmentmightbeperceivedasstressfulbykillifish. al., 2009),suggestingthatsomefactorassociatedwiththeconstant gene expressionthanfishsampleddirectlyfromthefield(Scottet Engineering ResearchCouncilofCanada (NSERC)toP.M.S. This workwassupportedbyaDiscovery grantfromtheNaturalSciencesand The authordeclaresnocompetingfinancial interests. homeostasis’ andtothereviewersofthismanuscriptforhelpfuldiscussions. Thanks totheparticipantsofJEBsymposium‘Stress:challenging be .W n amr R. E. Palmer, and K.W. Able, Conclusions References Funding Competing interests Acknowledgements larval mortalityof At physiologicalandbiochemicallevels,thesedifferences in The JournalofExperimentalBiology(2014)doi:10.1242/jeb.089722 Fundulus heteroclitus (1988). Salinityeffects onfertilizationsuccess and . Copeia 1988 , 345-350.
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