Do males and females gammarids defend themselves differently during chemical stress ?

ContentslistsavailableatScienceDirect

Aquatic

Toxicology

j o ur na l h o me p a g e : w w w . e l s e v i e r . c o m / l o c a t e / a q u a t o x

Do

male

and

female

gammarids

defend

themselves

differently

during

chemical

stress?

E.

Gismondi

,

C.

Cossu-Leguille,

J.-N.

Beisel

UniversitédeLorraine,LaboratoireInterdisciplinairedesEnvironnementsContinentaux(LIEC),CNRSUMR7360,MetzF-57070,France

a

r

t

i

c

l

e

i

n

f

o

Received18April2013

Receivedinrevisedform4July2013 Accepted13July2013 Keywords: Gammarussp. Gender Cadmium Detoxificationmechanisms Energyreserves Confoundingfactor

a

b

s

t

r

a

c

t

Toinvestigatexenobioticimpactsonorganismphysiology,severalstudiesinvolvebiomarker assess-ment.However,moststudiesdonottakeintoaccountthetoxiceffectonbothmalesandfemales.Here, wehaveinvestigatedtheinfluenceofgenderonthedetoxificationresponse(reducedglutathione, metal-lothionein,␥-glutamylcysteinligaseandcarotenoid),energyreserves(protein,lipidsandglycogen)and biomarkeroftoxiceffects(malondialdehyde)inGammarusroeseliexposedtocadmium.Aprincipal com-ponentanalysisrevealedthatG.roeselimalesandfemalesweredifferentlyimpactedbycadmium.We observedlowermalondialdehydelevelsinfemalesthaninmales,whatevertheconditiontested(i.e. control,2and8␮gCdL−1_{),althoughthepatternofresponsesofcontrolandexposuresto2or8␮gL}−1

wasthesameforbothgenders.Resultscouldbelinkedtoapparentlymoreeffectivedetoxification dis-playedbyfemalesthanbymales.Proteinconcentrationswereunchangedinbothgenders,lipidscontents werealwayssignificantlydecreasedandglycogencontentsdecreasedonlyinfemales.Thisstudy sup-portstheimportanceoftakingintoaccountthegenderinecotoxicologicalstudiestohaveanoverview ofxenobioticseffectsonapopulation.

© 2013 Published by Elsevier B.V.

1. Introduction

Arisinganthropogeniccontaminationofaquaticenvironments

isstillinprogress,andthisdisturbanceiswellknowntoinduce

dys-functionsinorganisms,whichcouldleadtopopulationdeclinesin

sensitivespecies.Toevaluatetheseenvironmentalrisks,

ecotoxic-ologicalstudiesusebiomarkers(Peakall,1994)whicharemainly

antitoxiccompounds(e.g.metallothionein,reducedglutathione)

thatrespondtovariouscontaminations,butalsovarious

detoxifi-cationenzymes,asforexamplecatalaseorglutathioneperoxidase

(Allanetal.,2006;VasseurandCossu-Leguille,2003).Ifthesetools

allowtoevaluatetheimpactofenvironmentalcontaminationson

organisms,oneproblemtheyposeisthattheirresponsesarenot

onlyinfluencedbypollutants,butcanalsovaryaccordingto

con-foundingfactorssuchasbioticfactors(e.g.age,size)(Srodaand

Cossu-Leguille,2011a;Xuereb,2009)orabioticfactors(e.g.

tem-perature)(Geffardetal.,2005,2007;Gismondietal.,2012a;Serafim

etal.,2002).

Amongfreshwaterspecies,thosethatbelongtothecrustacean

genusGammarussp.arepopularandsuitableorganismsfor

eco-toxicologicalassessment of environmentalpollutants ata large

scale,mostlybecausethisgenusiswidespreadthroughoutalarge

∗ Correspondingauthor.Tel.:+33387378429;fax:+33387378512. E-mailaddress:gismondi.eric@gmail.com(E.Gismondi).

rangeofmarineandfreshwaterhabitats.Gammaridsareknown

tobeeasytouseinbothlaboratoryandfieldstudies(seeKunz

et al.,2010 for a review).Hence manyecotoxicological studies

havebeencarriedoutusingtheseaquaticorganismstoevaluate

toxicimpactofxenobiotics(Adametal.,2010;Khanetal.,2011;

Krappetal.,2009;Sornometal.,2010;SrodaandCossu-Leguille, 2011b;Vellingeretal.,2012;Zubrodetal.,2010).Nevertheless,to

ourknowledge,onlyafewstudieshavebeendevotedto

investi-gatetheeffectofxenobioticsonbothmaleandfemalegammarids.

Inspiteoftheimportanceofthegenderasapotential

confound-ingfactor,moststudieshaveusedonlymalesorfemales,toavoid

thissourceofvariation,orhavenottakenintoaccountthe

gen-derandusedindividualsregardlessoftheirgender.Mixingboth

gendersisincontradictionwiththefactthatthefewstudiesin

whichbothgendershavebeentestedseparatelyhavehighlighted

significant differences between the responses of both genders.

Forinstance,Sornometal.(2010)observedthatGammarus

roe-selifemales were moresensitivethan maleswhen exposed for

72hto salinitystress. Indeed,females LC50 (concentration that

causedthedeathof50%ofindividuals)valuewasonaverageof

9.28gNaClL−1infemalesascomparedtomaleLC50whichwason

average12.24gNaClL−1.Inaddition,theauthorsobservedhigher

ventilationactivityinfemalesthaninmales.Undercopper

expo-sure,Sroda and Cossu-Leguille(2011b) have underlined higher

sensitivity in females as compared to malesin two gammarid

species,i.e.G.roeseliandtheinvasiveamphipodDikerogammarus

0166-445X/$–seefrontmatter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.aquatox.2013.07.006

villosus,althoughfemaleshadhigherantioxidantenzymeactivity

(e.g.glutathioneperoxidase). Infact,theLC50 valueofG.roeseli

femaleswas2.5-foldlowerthanthatofmales(22and53␮gCuL−1,

respectively),whiletheLC50valueofD.villosusfemaleswas

1.2-foldlowerthanmales(230and275␮gCuL−1,respectively).Inthe

sameway,McCahonandPascoe(1988)havefoundthatfemalesof

Gammaruspulexweretwiceassensitiveasmaleswhenexposedto

cadmiumfor48h.Thesefewstudiesultimatelyshowthat

under-standingofxenobioticeffects onbothgenders isofimportance

toprovideearlywarningsignalsofpopulationeffectsandprotect

them.

Theaimofthepresentstudywastoexaminetheimportanceof

genderontheresponsesofantitoxicdefencesandenergy

biomark-ersintheamphipodG.roeseliexposedtocadmiumfor 96h.Cd

accumulatesintheanimalprobablymostlythroughthegills,where

metalscansignificantlyinfluenceosmoregulationandrespiration

(Vellingeretal.,2012).Usually,theinternalconcentrationofmetals

in gammarids increases with exposure concentration and this

bioaccumulationcouldinduceoxidativestressduetothe

overpro-ductionofreactiveoxygenspecies(Vellingeretal.,2013).Antitoxic

defencestoprotectcellswereestimatedbymeasuringthereduced

glutathione(GSH)concentrationsandthe_{␥-glutamylcystein}ligase

(GCL)activity,aswellasmetallothionein(MT)andcarotenoid

con-centrations.GSHis an essential tripeptide inthe detoxification

system:itsthiolgroupsscavengeorganicormetallicxenobiotics

(Griffith,1999;Vasseurand Leguille,2004), butitalsoplaysan

important role as a substrate for several antioxidant enzymes

likeselenium-dependantglutathioneperoxidaseor

glutathione-S-transferases.The␥-glutamylcysteinligaseistheenzymethatlimits

thedenovoGSHsynthesis.Metallothioneins(MT)areinvolvedin

thebindingofmetallic compoundsthankstotheirthiolgroups

andcontributetoprotecttissuesagainstoxidativedamages(Bigot

etal.,2011;Roesijadi,1992).Carotenoids,whichareusedin

antiox-idantdefences(Palozzaand Krinsky,1992)andinvolved inthe

reproduction(Gilchristand Lee, 1972), werealsomeasured. To

evaluatecellulardamagesinorganisms,wemeasuredthe

malon-dialdehydelevel(MDA).Thiscompoundisanend-productofthe

lipidmembranedegradationandisconsideredasabiomarkerof

toxiceffects.Finally,energyreservesofgammaridswereestimated

by measuringprotein concentrations as well as total lipid and

glycogencontents.Wehypothesized(H1)thatG.roeselifemales

are more sensitive tocadmium than males, according tomost

resultsobtainedinpreviousstudies.Asecondhypothesisisthat

adifferenceexistsbetweenmalesandfemales intermsof

anti-toxic defence mechanisms and capacities (H2), which explains

thedifferenceofsensitivity.Ourthirdhypothesis(H3)isthatthe

energeticcost of physiologicalresponsestothe chemicalstress

increasestheuseofbodyreservesingammaridsbutwitha

dif-ferentpatternbetweenmalesandfemalesforglycogen,lipidsand

proteins.

2. Materialsandmethods

2.1. Cadmiumexposure

Malesand non-ovigerousfemalesofG.roeseliwerecollected

usingpondnetsandartificialtrapsduringthespringof2012,in

theFrenchNied River(Rémilly, North-easternFrance,49◦00 N

and6◦23 E)where cadmiumconcentrationsin thewaterwere

lower than 0.2␮gL−1 (LADROME Laboratory, Valence, France).

Non-ovigerousfemaleswerechosentoavoidtheinfluenceofthe

eggs(McCahonandPascoe,1988).Malesandfemalesweresorted

outonthespotbyobservinggnathopods(smallerinfemalesthan

inmales).Gammaridsweretransferredtothelaboratoryinlarge

containers filledwith river water. In thelaboratory, theywere

acclimated for5days at15◦Cin anEDTA-free ElendtM4

solu-tion (Elendt and Bias, 1990), and fed ad libitum with alder

leaves.TestsolutionswerepreparedusingEDTA-freeElendtM4

solution with CdCl2 added to obtain two cadmium

concentra-tions:2and8␮gCdL−1(measuredconcentrations:1.95±0.03and

7.93±0.04␮gCdL−1).ControlsconsistedofEDTA-freeElendtM4

solutiononly.Cadmiumconcentrationswerechosenaccordingto

(i)the96hLC50obtainedinourpreviousstudywhichwere107

and 50_␮gCdL−1 for femalesand males,respectively (Gismondi

etal.,2012b);andto(ii)themaximumadmissiblecadmium

con-centrationindrinkingwater,whichis5␮gCdL−1 (CD98/83/EC,

1998).Foreachexposurecondition,threereplicatesof12G.roeseli

malesandthreereplicatesof24G.roeselifemaleswereexposed

at15◦Cfor96hinaquariapreviouslysaturatedwiththe

corre-spondingcadmiumsolutionsfor5days.Duringcadmiumexposure,

animalswerenotfed.Morefemaleswereusedbecauseabout50%

areinfectedbyvertically-transmittedmicrosporidia,whichcould

biastheinterpretationofthebiomarkerresponsesandincreasethe

femalesensitivity(Gismondietal.,2012c).Asonlyfemaleswere

infected by the vertically-transmitted microsporidia (Gismondi

et al.,2012c), at theend of the experiment, gonadal tissuesof

femalesweredissectedandthemicrosporidianstatusoffemales

wasverifiedusing themethodfromGismondiet al.(2012c) to

keeponlyuninfectedfemales.Then,6poolsof6individuals(i.e.

malesandfemalesseparated)wereconstitutedforeachcondition,

frozeninliquidnitrogenandstoredat−80◦_{Cawaitingbiomarker}

analyses.

2.2. Samplepreparation

EachpoolwashomogenizedwithaPotterElvejhemmanual

tis-suegrinderin50mMphosphatebufferKH2PO4/K2HPO4(pH7.6)

supplementedwith1mMphenylmethylsulphonylfluoride(PMSF)

and 1mM l-serine-borate mixture as protease inhibitors, and

5mMphenylglyoxalasa_␥-glutamyltranspeptidaseinhibitor.The

homogenizationbufferwasadjustedtoavolumetwo-foldthewet

weightofthesamplepool(e.g.200␮Lofhomogenizationbuffer

for100mgofwetweighttissue).Thetotalhomogenatewasdivided

intosevenpartstomeasurethedifferentparameters.Foreach

repli-cate,twoindependentmeasuresweremadeforeachbiomarker.

2.3. Biomarkermeasurements

All biomarker measurements were performed according to

Gismondietal.(2012d).Energyreserve(lipids,glycogen,proteins)

measurementswereconductedwithspectrophotometrical

meth-odswhileantitoxicdefences(GSH,GCL,MT)andbiomarkeroftoxic

effects(MDA)weremeasuredusingHPLCmethods.

In parallel, carotenoid concentrations were measured by a

spectrophotometricalmethodadaptedfromRauqueandSemenas

(2009).Avolumeof40␮Lofthetotalhomogenatewasdilutedin

450␮Lof96%ethanolandkeptfor6hinthedarkat4◦C,before

beingcentrifuged10minat3500×g. Theopticaldensityof the

resultingsupernatant wasmeasuredat422, 448and 476nm.A

commercialcarotenemixture(Sigma–Aldrich,France) wasused

as a standard. Carotenoid concentrationswere expressedin ng

carotenoidsmg−1lipids.

2.4. Statisticalanalyses

Alldatametnormalityandhomogeneityofvariance

assump-tions(ShapiroandBartletttests,p-values>0.05).Our datawere

analyzedbyusingANOVAtestswithrespectto“individualgender”

and“cadmiumexposure”asfixedfactors.Then,TukeyHSDposthoc

testswereusedtodescribesignificantdifferences.Alltestswere

The relationships between biomarker responses considered

altogetherand cadmiumexposureconditions wereassessed by

usingaprincipalcomponentanalysis(PCA)usingR2.9.0software.

3. Results

ANOVAtests(Table1)revealedaneffectofgender,cadmium

exposureand interaction betweengender and cadmium

expo-sure.Indeed,exceptforproteinconcentrations,cadmiumexposure

impactedall biomarker responses. Similarly, gender also

influ-encedallbiomarkers.Theinteractionbetweenthesetwofactors

influencedfourout ofeightbiomarkers:glycogencontent,GSH

concentration,GCLactivityandMDAlevels.

3.1. Energyreserves

Overall,cadmiumexposuresinfluencedtheenergyreservesofG.

roeselimalesandfemales.Proteinconcentrationswereunchanged

regardlessofthegender(Fig.1A),lipidcontentswerealways

sig-nificantly decreased (Fig. 1B) and glycogen contents were only

decreasedinfemales(Fig.1C).Indetail,lipidcontentwasdecreased

inmalesandfemalesaccordingtoaconcentration-response

rela-tionship.In bothgenders,lipidscontentswere1.3-and1.8-fold

lower at 2 and 8␮gCdL−1 respectively, compared to controls

(Fig. 1B). Contrary to lipid results, glycogen content was only

impactedin G.roeselifemales. Indeed, nosignificantdifference

wasobservedinmaleswhateverthecadmiumexposureswhilein

females,glycogencontentwasinaverage1.3-foldlowerinexposed

femalesthanincontrols(Fig.1C).

3.2. Antitoxicdefences

Likeenergyreserves,antitoxicdefenceswereinfluencedby

cad-miumexposure inboth gammaridgenders (Table1).In males,

theGCL activitywassignificantlyincreasedwhenanimalswere

exposedto8␮gCdL−1,whiletheexposureto2␮gCdL−1didnot

resultinasignificantincrease(Fig.1D).Theenzymeactivitywas

significantly higher (1.7-fold) in males exposed at 8␮gCdL−1,

as compared to unexposed ones. In females, regardless of the

cadmium concentration, GCL activity was significantly higher

in organisms exposed to 2␮gCdL−1 (1.6-fold increase) and to

8␮gCdL−1(2.2-foldincrease).

GSHconcentrationwasalsoinfluencedbycadmiumexposure

since an 1.2-fold increase was observed at 2␮gCdL−1 in both

genders,ascomparedtounexposed ones(Fig.1E).However,no

significantdifferenceswereobservedbetweentheGSH

concentra-tionsmeasuredat2␮gCdL−1andthosemeasuredat8␮gCdL−1in

G.roeselimalesandfemales.

Results of metallothionein concentrations showed a

concentration-dependent increase in both genders (Fig. 1F).

However, this increase was higher in males than in females.

Indeed,MTconcentrationincreasedbyafactorof1.6inmalesand

of1.3infemalesexposedto2␮gCdL−1ascomparedtorespective

controls.Moreover,at8␮gCdL−1,MTconcentrationdoubledin

malesandincreasedbyafactorof1.5infemales,ascomparedto

respectivecontrols.

Carotenoidlevels significantly decreased in males according

toa concentration-dependent relationship(Fig.1G). Carotenoid

contents were 1.2- and 1.4 lower in males exposed to 2 and

8␮gCdL−1 respectively, as compared to controls. In contrast,

carotenoid levels decreased significantly in females (1.5-fold

decrease)exposedto8␮gCdL−1comparedtounexposedfemales.

However, no significant difference was observed between

carotenoid levels in control females and females exposed to

2␮gCdL−1(onlyatendencyofdecreasewasobserved).

Fig.1. Energyreserves(protein,lipidsandglycogen),antitoxicdefence(GSH,GCL, MTandcarotenoids)andtoxiceffectsbiomarker(MDA)responsesinG.roeselimales andfemalesexposedto2and8␮gCdL−1_{for96h.Lettersabovethebarsindicate}

Table1

Univariateanalysesofvariance(ANOVA)investigatingvariationsinenergyreserves(lipid,proteins,andglycogen),defencecapacity(GSH,GCL,MT,carotenoids),andtoxicity biomarker(MDA),inGammarusroeseli,accordingtogenderandcadmiumexposure.Significantdifferencesareindicatedinbold(p<0.05).

Sumofsquare d.f. F p-Value

Proteins Cadmium 18.37 2 3.21 0.054 Gender 492.77 1 172.36 <0.001 Cadmium×gender 13.3 2 2.33 0.115 Lipids Cadmium 12.58 2 53.12 <0.001 Gender 8.09 1 68.35 <0.001 Cadmium×gender 0.43 2 2.04 0.148 Glycogen Cadmium 0.98 2 8.22 0.001 Gender 14.77 1 247.72 <0.001 Cadmium×gender 1.83 2 15.36 <0.001 GSH Cadmium 2.14 2 31.69 <0.001 Gender 10.57 1 313.84 <0.001 Cadmium×gender 0.29 2 4.36 0.022 GCL Cadmium 0.27 2 49.41 <0.001 Gender 0.05 1 17.66 <0.001 Cadmium×gender 0.02 2 3.56 0.041 MT Cadmium 3.19 2 29.31 <0.001 Gender 69.11 1 1267.38 <0.001 Cadmium×gender 2.25 2 20.64 0.78 Carotenoid Cadmium 449.12 2 51.77 <0.001 Gender 1730.56 1 398.98 <0.001 Cadmium×gender 27.01 2 3.11 0.059 MDA Cadmium 11.23 2 38.83 <0.001 Gender 19.73 1 136.48 <0.001 Cadmium×gender 1.38 2 4.77 0.016

3.3. Biomarkeroftoxiceffects

MDAlevel,reflectingcelldamage,wasinfluencedbycadmium

exposure(Table1andFig.1H).Whateverthegender,anincrease

inMDAlevelswasobserved.Inmales,MDAlevelswereincreased

according to a concentration-dependent response. Indeed, we

observeda1.3-and1.7-foldincreaseinmalesexposedto2and

8␮gCdL−1,respectively. In contrast,a significantincrease was

observedonlyinfemalesexposedtothehighestcadmium

concen-tration.Nosignificantdifferencewasobservedinfemalesneither

betweencontroland theexpositionat2_␮gCdL−1,norbetween

individualsexposedatthetwocadmiumconcentrations.

3.4. Principalcomponentanalysis

ThePCAperformedonallbiomarkermeasurementsresulted

inafirstfactorialplanwhichexplained89.9%ofthetotalinertia

(Fig.2).TheF1-axisexplained61.5%ofthetotalinertiawhereas

thesecondprincipalcomponent(F2)accountedfor28.3%.F1was

positivelycorrelatedtocarotenoids,glycogenandprotein

concen-trationsandnegativelycorrelatedtoGSH,andinlesserextentto

GCLactivityandMTconcentrations.F2waspositivelycorrelatedto

lipidscontentandnegativelycorrelatedtoMDAlevels.

F1-axisallowedtheseparation ofmalesand females

accord-ingtoantitoxicdefences.Femaleswereobservedwiththehighest

GSH and MT concentrations as well as GCL activity, whereas

malespresented highestlevelsof carotenoids.Similarly, F2has

alloweddifferentiatingbetweenexposureconditionswithinmale

andfemalegroupsaccordingaconcentration-dependentresponse

(controlonthenegativepartoftheF2-axis,C2attheoppositealong

thisaxis).Lowerintra-groupvariations(i.e.smallellipsesonthe

PCAprojection) wereobserved inmales accordingtocadmium

exposure, while in females, theintra-group variation increased

dependingonthecadmiumconcentration.

4. Discussion

4.1. Validationofourecotoxicologicalapproach:biomarker

responsesundercadmiumstress

AllbiomarkersmeasuredinG.roeselimalesandfemaleswere

influencedbytheexposuretocadmium.

MDAlevels increased in exposed gammarids according toa

concentration-dependent response,reflecting thetoxic effectof

cadmiumatthetestedconcentrations.Thisresultisinaccordance

withpreviousstudiessuchasthoseofCorreiaetal.(2002),and

SrodaandCossu-Leguille(2011a)whohaveobservedanincrease

ofMDAlevelsinG.locustaorG.roeseli,respectively,afteracopper

exposure.

Withintheantitoxicdefencesmeasured,thepatternofGSH

con-centrationsisthemostdifficulttointerpret.TheincreaseofGSHin

G.roeseliexposedto2␮gCdL−1couldresultfromtheincreaseof

theGCLactivity,whichistheenzymelimitingthedenovoGSH

syn-thesis(Volohonskyetal.,2002).Itisnowadmittedthatanincrease

ofGSHconcentrationfollowingGCLactivationistheresultofan

oxidativestress(Dickinsonetal.,2004).Inourstudy,theabsence

ofGSHconcentrationincreaseinG.roeseliexposedto8␮gCdL−1

compared with gammarids exposed to 2␮gCdL−1, while GCL

activity was increased, could be explained by the use of GSH

asascavengerofreactiveoxygenspeciesproducedbycadmium

(VasseurandLeguille,2004),andalsobythefactthatGSHisused

asasubstratebysomeantioxidantenzymessuchas

glutathione-S-transferasesorglutathioneperoxidases(Saezetal.,1990).

WiththesamepatternasthatofGSH,MTconcentrationswere

increasedincadmium-exposedG.roeseli.Thisrisecouldbelinked

withtheMTsynthesisinducedbytheexposuretoseveralmetals

includingcadmium(Bigotetal.,2011;Stillman,1995),asobserved

byMartinezetal.(1996)intheamphipodEchinogammarus

Fig.2. ResultsofthecorrelationcircleandtheprincipalcomponentanalysisonbiomarkerresponsesofGammarusroeselimalesandfemalesexposedtocadmiumfor96h. F:females,M:males,C1:2␮gCdL−1_{andC2:8␮gCdL}−1_.

alsodescribed in bivalves suchas in Mytilus edulis exposed at

400␮gCdL−1for65days(BebiannoandLangston,1991).

Carotenoidlevelswerealsoimpacted bycadmiumexposure

duetothefactthattheyplayanantitoxicantrolebyscavenging

freeradicals(KrinskyandDeneke,1982;Krinsky,1989).Indeed,

byscavengingfreeradicals, carotenoidscouldreducelipid

per-oxidationandthus,maintainthecellularintegrity(Jörgensenand

Skibsted,1993).

Concerningenergyreserves,proteinconcentrationsdidnotvary

aftercadmium exposure but lipid and glycogen contents were

reducedinexposedgammarids.Theseresultscouldbeexplainedby

themobilizationofenergyreservestoestablishantitoxicdefences.

Indeed,lipidsandglycogenareknowntobemobilizedinexposed

organisms(Durouetal.,2008),andthusrapidlyimpactedbytoxic

stress,evenduringashort-termexposuresuchasinourstudy.In

contrast,proteinconcentrationsareprincipallymobilizedinlong

termexposure,whichcouldexplainthatwedidnotobserveprotein

concentrationdecrease.However,thelipidcontentdecreasecould

belinkedtotheglycogencontentdecrease.Sanchoetal.(1998)and

Dutraetal.(2009)havesuggestedthatglycogendecrease,observed

in Anguillaanguilla exposed tofenitrothion and Hyallelacastroi

exposedtocarbofuran,respectively,couldbeduetopollutantstress

cost,whilethedecreaseinlipidcontentcouldbeexplainedbythe

useoflipidsasanenergysourcetocompensatefortheglycogen

lost.

4.2. Differencesbetweengenders

Overall,thePCAusedinourstudyhighlighteddifferencesinthe

responsesofG.roeseliaccordingtogender.

Celldamageislessimportantinfemalesthaninmalesaccording

tothelowerMDAlevelsregardlessofthecadmiumconcentration

tested,suggestingthanfemalesarelesssensitivetocadmiumthan

males(H1refuted).Thisresultcontrastswithseveralother

stud-iessuchasthoseofSornometal.(2010)aboutG.roeseliexposed

toasalinitystress,SrodaandCossu-Leguille(2011b)aboutG.

roe-seliandtheinvasiveD.villosusundercopperexposure,McCahon

andPascoe(1988)concerningG.pulexandacadmiumexposure

for48h.Thisdifferencecouldbeexplainedbythedifferenttypeof

stressused(e.g.salinity,copper)orthedifferentspeciesused(e.g.G.

pulex,G.roeseli).Nevertheless,ourresultsconfirmthoseobserved

inapreviousstudywherewefoundthatthe96hLC50ofG.

roe-selifemalesexposedtocadmiumwashigherthanthe96hLC50of

males(Gismondietal.,2012b).

Differencesofcelldamagebetweenmalesandfemalescouldbe

theconsequenceofadifferenceinantitoxiccompound

concentra-tions.OurstudyhighlightedhigherGSHandMTconcentrationsand

higherGCLactivityinfemales(H2validated),allowingthemtohave

abetterantitoxicdefencewhenexposedtopollutants.Thisisin

agreementwithotherinvestigationsonamphipodswhich

under-linedhigherantioxidantenzymesactivityinfemalesasforexample

in G.roeseli(Srodaand Cossu-Leguille,2011a), or lower

metal-lothioneinconcentrationsinmalessuchasinGammaruslocustra

(Correiaetal.,2004).

Thedifferentantitoxicantmoleculelevelscouldbelinkedto

energyreservesandenergymetabolism.Incontrols,weobserved

that females have higher lipid but lower glycogen and protein

contentthanmales.Thedifferencesinenergyreservesbetween

genderscouldbeexplainedbydifferencesinfeedingasobserved

by Dick et al. (2005). Indeed these authors examined guts of

Echinogammarusmarinusandconcludedthat femalesconsumed

significantly more animals than males, resulting probably in a

higherenergyintake.Thisdifferenceinenergycontentcouldalso

beexplainedbythehighneedsofenergyinfemalesfor

oogene-sis(Sutcliffe,1993).Whenindividualswereexposedtocadmium,

lipidcontentswerealwayssignificantlydecreasedandglycogen

contentswereonlydecreasedinfemales(H3validated).The

differ-encebetweenmalesandfemalesisinapparentcontradictionwith

theirrespectiveinvestmentsinreproduction.Oogenesisandegg

incubationrequirehighlipidsynthesisandmobilization,in

com-parisontoprocessesofspermatogenesis,whicharelessdemanding

intermsofenergy(BuikemaandBenfield,1979).Malesmightalso

haveamoreconstantphysiologicalstatethanfemalesduetothe

asymmetryoftheenergyrequiredingamete production.Ifthis

resourceallocationbetweengendersisgenerallyassumedtolead

toabetterphysiologicalstateformales(theoppositeofourresults),

acomplementaryviewisthatfemaleshavehigherenergyreserves

ascomparedtomalesthatcanbereallocated.Thus,thedifferencein

reproductioncostscouldallowfemalestohaveabettermetabolism

toassumeantitoxicdefencemechanismswhentheyareexposed

toachemicalstressor.Twootherhypotheseshavebeenadvanced

toexplainabetterresistanceoffemalesbycomparisonwithmales

(McClellan-Greenetal.,2007).First,highfemaleresistanceas

com-paredtomalescouldbelinkedtoahighermoltingrate,theprocess

ofexoskeletonsheddingbeingapotentialremovalpathwayof

pol-lutants.Thishypothesissupports thehighintra-group variation

weobservedinfemalesexposedtothehighestconcentrationof

cadmium.Second,ovodepositioninvolvesthetransferof

contam-inantsintotheeggswhichconferstofemalesabettermetabolism

toeliminate environmental contaminants(Dipintoet al., 1993;

Oberdörsteretal.,2000).Pöckletal.(2003)showedthatG.

roe-selifemalescanreproduceduringtheirentirelife-spanof1.5–2

yearsaboutsixtoeightsuccessivebroods,whichadd-uptothe

productionofatleast200eggs.Hence,reproductioncoulddecrease

bodyburdensofcontaminantssincemanychemicalsareassociated

withlipidsandtransferredtooocytes.However,furtherstudiesare

neededtoinvestigatetheimportanceoftheseprocesses(molting

rate,levelofdetoxificationduetoexuviaandeggproduction)in

genderdifferences.

5. Conclusions

This work underlined the differential antitoxic defence

responsesingammaridsaccordingtogender.Ourresultsrevealed

thatG.roeselifemalesseemtobelesssensitivetocadmiumthan

malesprobablyduetoabettermobilizationofantitoxicdefences.

Studiesontheinfluenceofgender,especiallyfemales,understress

conditionsseemtobeimportanttohaveanoverviewofxenobiotics

effectsonthepopulation.Inanthropogeniccontextswherefemales

aremore sensitivetocertainpollutants than males,population

declinescantakeplacefasterinenvironmentscontaminatedby

thesecompounds.AsGammarussp.isanimportantcompartment

intheecosystem,thedeclineofgammaridsinthesedisturbed

envi-ronmentscanhaveseriouseffectsonthecycleoforganicmatter

andtheoverallfoodweb.

Acknowledgments

Thisstudy wassupported bythe FrenchMinistry of

Educa-tionandResearch(Ministèredel’EnseignementSupérieuretdela

Recherche),whichwesincerelythankhere.Thepresentworkwas

fundedbytheresearchprogramEC2CO(EcosphèreContinentaleet

Côtière).WearegratefultoCéliaJoaquim-Justoforimprovingthe

Englishtextandwewishtothanktheanonymousreviewersfor

theirhelpfulcommentsonapreviousdraftofthispaper.

References

Adam,O.,Degiorgi,F.,Crini,G.,Badot,P.-M.,2010.HighsensitivityofGammarus sp.juvenilestodeltamethrin:outcomesforriskassessment.Ecotoxicologyand EnvironmentSafety73,1402–1407.

Allan,I.J.,Vrana,B.,Greenwood,R.,Mills,G.A.,Roig,B.,Gonzalez,C.,2006.Atoolbox forbiologicalandchemicalmonitoringrequirementsfortheEuropeanUnion’s WaterFrameworkDirective.Talanta69,302–322.

Bebianno,M.J.,Langston,W.J.,1991.MetallothioneininductioninMytilusedulis exposedtocadmium.MarineBiology108,91–96.

Bigot,A.,Minguez,L.,Giambérini,L.,Rodius,F.,2011.Earlydefenseresponses in thefreshwater bivalveCorbicula flumineaexposedto copper and cad-mium:transcriptionalandhistochemicalstudies.EnvironmentalToxicology26, 623–632.

Buikema,A.L.,Benfield,E.F.,1979.Useofmacroinvertebratelifehistory informa-tionintoxicitytests.JournaloftheFisheriesResearchBoardofCanada36, 321–328.

Correia,A.D.,Livingstone,D.R.,Costa,M.H.,2002.Effectsofwater-bornecopper onmetallothioneinandlipidperoxidationinthemarineamphipodGammarus locusta.MarineEnvironmentResearch54,357–360.

Correia,A.,Sousa,A.,Costa,M.,Moura,I.,Livingstone,D.,2004.Quantificationof metallothioneininwholebodyGammaruslocusta(crustacea:amphipoda)using differentialpulsepolarography.ToxicologicalandEnvironmentalChemistry86, 23–36.

Dick,J.T.A.,Johnson,M.P.,McCambridge,S.,Johnson,J.,Carson,V.E.E.,Kelly,D.W., MacNeil,C.,2005.PredatorynatureofthelittoralamphipodEchinogammarus marinus:gutcontentanalysisandeffectsofalternativefoodandsubstrate het-erogeneity.MarineEcologyProgressSeries291,151–158.

Dickinson,D.A.,Levonen,A.-L.,Moellering,D.R.,Arnold,E.K.,Zhang,H., Darley-Usmar, V.M., Forman, H.J., 2004. Human glutamate cysteine ligase gene regulationthroughtheelectrophileresponseelement.FreeRadicalBiologyand Medicine37,1152–1159.

Dipinto,L.M.,Coull,B.C.,Chandler,G.T.,1993.Lethalandsublethaleffectsofthe sediment-associatedPCBaroclor1254onameiobenthiccopepod. Environmen-talToxicologyandChemistry12,1909–1918.

Durou,C.,Mouneyrac,C.,Pellerin,J.,Péry,A.,2008.Conséquencesdesperturbations dumétabolismeénergétique.In:Lesbiomarqueursdansl’évaluationdel’état écologiquedesmilieuxaquatiques.Lavoisier,Paris,pp.273–289.

Dutra,B.K.,Fernandes,F.A.,Lauffer,A.L.,Oliveira,G.T.,2009.Carbofuran-induced alterationsintheenergymetabolismandreproductivebehaviorsofHyalella cas-troi(Crustacea,Amphipoda).ComparativeBiochemistryandPhysiology–Part C:ComparativePharmacology149,640–646.

Elendt,B.-P.,Bias,W.-R.,1990.TracenutrientdeficiencyinDaphniamagna cul-turedinstandardmediumfortoxicitytesting:effectsoftheoptimizationof cultureconditionsonlifehistoryparametersofD.magna.WaterResearch24, 1157–1167.

Geffard,A.,Amiard-Triquet,C.,Amiard,J.C.,2005.Doseasonalchangesaffect metal-lothioneininductionbymetalsinmussels,Mytilusedulis?Ecotoxicologyand EnvironmentSafety61,209–220.

Geffard,A.,Quéau,H.,Dedourge,O.,Biagianti-Risboug,S.,Geffard,O.,2007. Influ-enceofbioticandabioticfactorsonmetallothioneinlevelinGammaruspulex. ComparativeBiochemistryandPhysiology–PartC:ComparativePharmacology 145,632–640.

Gilchrist,B.M.,Lee,W.L.,1972. Carotenoidpigmentsandtheirpossiblerolein reproductioninthesandcrab,Emeritaanaloga(Stimpson,1857). Compar-ativeBiochemistryandPhysiology–PartB:ComparativeBiochemistry42, 263–294.

Gismondi,E.,Beisel,J.N.,Cossu-Leguille,C.,2012a.Influenceofgenderandseason onreducedglutathioneconcentrationandenergyreservesofGammarusroeseli. EnvironmentalResearch118,47–52.

Gismondi,E.,Cossu-Leguille,C.,Beisel,J.-N.,2012b.Acanthocephalanparasites:help orburdeningammaridamphipodsexposedtocadmium?Ecotoxicology21, 1188–1193.

Gismondi,E.,Rigaud,T.,Beisel,J.-N.,Cossu-Leguille,C.,2012c.Microsporidia para-sitesdisrupttheresponsestocadmiumexposureinagammarid.Environmental Pollution160,17–23.

Gismondi,E.,Cossu-Leguille,C.,Beisel,J.-N.,2012d.Doestheacanthocephalan par-asitePolymorphusminutusmodifytheenergyreservesandantitoxicdefencesof itsintermediatehostGammarusroeseli?Parasitology139,1–8.

Griffith,O.W.,1999.Biologicandpharmacologicregulationofmammalian glu-tathionesynthesis.FreeRadicalBiologyandMedicine27,922–935.

Jörgensen,K.,Skibsted,L.H.,1993.Carotenoidscavengingofradicals.Zeitschriftfur Lebensmittel-Untersuchungund-Forschung196,423–429.

Khan,F.R.,Irving,J.R.,Bury,N.R.,Hogstrand,C.,2011.Differentialtoleranceoftwo Gammaruspulexpopulationstransplantedfromdifferentmetallogenicregions toapolymetalgradient.AquaticToxicology102,95–103.

Krapp,R.H.,Bassinet,T.,Berge,J.,Pampanin,D.M.,Camus,L.,2009.Antioxidant responsesinthepolarmarinesea-iceamphipodGammaruswilkitzkiitonatural andexperimentallyincreasedUVlevels.AquaticToxicology94,1–7. Krinsky,N.I.,1989.Antioxidantfunctionsofcarotenoids.FreeRadicalBiologyand

Medicine7,617–635.

Krinsky, N., Deneke, S., 1982. Interaction of oxygen and oxy-radicals with carotenoids.JournaloftheNationalCancerInstitute69,205–210.

Kunz,P.Y.,Kienle,C.,Gerhardt,A.,2010.Gammarusspp.inaquaticecotoxicology andwaterqualityassessment:towardintegratedmultileveltests.In:Whitacre, D.M.(Ed.),ReviewsofEnvironmentalContaminationandToxicology,vol205. SpringerNewYork,NewYork,pp.1–76.

Martinez,M.,Ramo,J.D.,Torreblanca,A.,Pastor,A.,Diaz-Mayans,J.,1996.Cadmium toxicity,accumulationandmetallothioneininductioninEchinogammarus echi-nosetosus.JournalofEnvironmentScienceandHealth,PartA:Environmental Science31,1605–1617.

McCahon,C.P.,Pascoe,D.,1988.Increasedsensitivitytocadmiumofthefreshwater amphipodGammaruspulex(L.)duringthereproductiveperiod.Aquatic Toxico-logy13,183–193.

McClellan-Green,P.,Romano,J.,Oberdörster,E.,2007.Doesgenderreallymatterin contaminantexposure?Acasestudyusinginvertebratemodels.Environmental Research104,183–191.

Oberdörster,E.,Brouwer,M.,Hoexum-Brouwer,T.,Manning,S.,McLachlan,J.A., 2000.Long-termpyreneexposureofgrassshrimp,Palaemonetespugio,affects moltingandreproductionofexposedmalesandoffspringofexposedfemales. EnvironmentalHealthPerspectives108,641–646.

Palozza,P.,Krinsky,N.I.,1992.Antioxidanteffectsofcarotenoidsinvivoandinvitro: anoverview.In:CarotenoidsPartA:Chemistry,Separation,Quantitation,and Antioxidation.AcademicPress,pp.403–420.

Peakall,D.B.,1994.Theroleofbiomarkersinenvironmentalassessment(1). Intro-duction.Ecotoxicology3,157–160.

Pöckl,M.,Webb,B.W.,Sutcliffe,D.W.,2003.Lifehistoryandreproductivecapacity ofGammarusfossarumandG.roeseli(Crustacea:Amphipoda)undernaturally fluctuatingwatertemperatures:asimulationstudy.FreshwaterBiology48, 53–66.

Rauque,C.A.,Semenas,L.,2009.Effectsoftwoacanthocephalanspeciesonthe reproductionofHyalellapatagonica(Amphipoda,Hyalellidae)inanAndean PatagonianLake(Argentina).JournalofInvertebratePathology100,35–39. Roesijadi,G.,1992.Metallothioneinsinmetalregulationandtoxicityinaquatic

animals.AquaticToxicology22,81–113.

Saez,G.T.,Bannister,W.H.,Bannister,J.V.,1990.FreeRadicalsandThiolCompounds. TheRoleofGlutathioneAgainstFreeRadicalToxicity.CRCPressInc.,Florida. Sancho,E.,Ferrando,M.D.,Fernandez,C.,Andreu,E.,1998.Liverenergymetabolism

ofAnguillaanguillaafterexposuretofenitrothion.Ecotoxicologyand Environ-mentSafety41,168–175.

Serafim,M.,Company,R.,Bebianno,M.,Langston,W.,2002.Effectof tempera-tureandsizeonmetallothioneinsynthesisinthegillofMytilusgalloprovincialis exposedtocadmium.MarineEnvironmentResearch54,361–365.

Sornom,P.,Felten,V.,Médoc,V.,Sroda,S.,Rousselle,P.,Beisel,J.-N.,2010.Effect ofgenderonphysiologicalandbehaviouralresponses ofGammarusroeseli

(CrustaceaAmphipoda)tosalinityandtemperature.EnvironmentalPollution 158,1288–1295.

Sroda,S.,Cossu-Leguille,C.,2011a.Seasonalvariabilityofantioxidantbiomarkers andenergyreservesinthefreshwatergammaridGammarusroeseli. Chemo-sphere83,538–544.

Sroda,S.,Cossu-Leguille,C.,2011b.Effectsofsublethalcopperexposureontwo gammaridspecies:whichisthebestcompetitor?Ecotoxicology20,264–273. Stillman, M.J., 1995. Metallothioneins. Coordination Chemistry Reviews 144,

461–511.

Sutcliffe,D.W.,1993.ReproductioninGammarus(Crustacea,Amphipoda)female strategies.FreshwaterForum,26–64.

Vasseur,P.,Cossu-Leguille,C.,2003.Biomarkersandcommunityindicesas com-plementary tools forenvironmental safety.Environment International28, 711–717.

Vasseur,P.,Leguille,C.,2004.Defensesystemsofbenthicinvertebratesinresponse toenvironmentalstressors.EnvironmentalToxicology19,433–436. Vellinger,C.,Parant,M.,Rousselle,P.,Immel,F.,Wagner,P.,Usseglio-Polatera,P.,

2012.Comparisonofarsenateandcadmiumtoxicityinafreshwateramphipod (Gammaruspulex).EnvironmentalPollution160,66–73.

Vellinger,C.,Felten,V.,Rousselle,P.,Mehennaoui,K.,Parant,M.,Usseglio-Polatera, P.,2013.SingleandcombinedeffectsofcadmiumandarsenateinGammarus pulex(Crustacea,Amphipoda):understandingthelinksbetweenphysiological andbehaviouralresponses.AquaticToxicology,inpress.

Volohonsky,G.,Tuby,C.N.Y.H.,Porat,N.,Wellman-Rousseau,M.,Visvikis,A.,Leroy, P.,Rashi,S.,Steinberg,P.,Stark,A.A.,2002.Aspectrophotometricassayof ␥-glutamylcysteinesynthetaseandglutathionesynthetaseincrudeextractsfrom tissuesandculturedmammaliancells.Chemico-BiologicalInteractions140, 49–65.

Xuereb,B.,2009.AcetylcholinesteraseactivityinGammarusfossarum(Crustacea Amphipoda):intrinsicvariability,referencelevels,andareliabletoolforfield survey.AquaticToxicology93,225–233.

Zubrod,J.P.,Bundschuh,M.,Schulz,R.,2010.Effectsofsubchronicfungicide expo-sureontheenergyprocessingofGammarusfossarum(Crustacea;Amphipoda). EcotoxicologyandEnvironmentSafety73,1674–1680.