Factors that alter the biochemical biomarkers of environmental contamination in Chironomus sancticaroli (Diptera, Chironomidae)

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REVISTA

BRASILEIRA

DE

Entomologia

AJournalonInsectDiversityandEvolution w w w . r b e n t o m o l o g i a . c o m

Medical

and

Veterinary

Entomology

Factors

that

alter

the

biochemical

biomarkers

of

environmental

contamination

in

Chironomus

sancticaroli

(Diptera,

Chironomidae)

Débora

Rebechi-Baggio

a

_,

_Vinicius

_S.

_Richardi

a

_,

_Maiara

_Vicentini

a

_,

_Izonete

_C.

_Guiloski

b

_,

Helena

C. Silva

de

Assis

b

_,

_Mário

_A.

_{Navarro-Silva}

a,∗

a_Universidade_Federal_do_Paraná,_Departamento_de_Zoologia,_Curitiba,_PR,_Brazil b_Universidade_Federal_do_Paraná,_Departamento_de_{Farmacologia,}_Curitiba,_PR,_Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received17April2016 Accepted9July2016 Availableonline30July2016 Associateeditor:MariaAniceSallum Keywords: Centrifugation Freezing Foodstress Thermalstress

a

b

s

t

r

a

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t

Changesinphysiologyofthenervoussystemandmetabolismcanbedetectedthroughtheactivityof acetylcholinesterase(AChE),alphaesterase(EST-␣)andbetaesterase(EST-␤)inchironomidsexposed topollutants.However,tounderstandtherealeffectofxenobioticsonorganisms,itisimportantto investigatehowcertainfactorscaninterferewithenzymeactivity.Weinvestigatedtheeffectsof dif-ferenttemperatures,foodstressandtwostepsoftheenzymaticprotocolontheactivityofAChE,EST-␣ andEST-␤inChironomussancticaroli.Inexperimentofthermalstressindividualsfromtheeggstage tothefourthlarvalinstarwereexposedtodifferenttemperatures(20,25and30◦_C)._In_food_stress

experiment,larvaewerereareduntilIVinstarinastandardsetting(25◦_C_and_0.9_g_weekly_ration),_but

fromfourthinstarontheyweredividedintogroupsandoffereddifferentfeedingregimes(24,48and 72hwith/withoutfood).Insamplefreezingexperiment,agroupofsampleswasprocessedimmediately afterhomogenizationandanotherafterfreezingfor30days.Totesttheeffectofcentrifugationon sam-ples,enzymeactivitywasquantifiedfromcentrifugedandnon-centrifugedsamples.Theactivityofeach enzymereachedanoptimumatadifferenttemperature.Theabsenceoffoodtriggereddifferentchanges inenzymeactivitydependingontheperiodofstarvation.Freezingandcentrifugationofthesamples significantlyreducedtheactivityofthreeenzymes.Basedontheseresultsweconcludethatthefour factorsstudiedhadaninfluenceonAChE,EST-␣andEST-␤andthisinfluenceshouldbeconsideredin ecotoxicologicalapproaches.

Introduction

Biochemicalbiomarkerresponsesenabledetectionoftheﬁrst biological effects associatedwithexposureto xenobiotics,even atlow concentrations (Lionetto etal., 2003).Theenzyme AChE is widelyusedas a biomarkerofexposureto organophosphor-ateandcarbamatescompounds,whichinhibitthisenzyme,thus compromisingthenervoussystemoforganisms(FultonandKey,

2001;GallowayandHandy, 2003).Themetabolicenzymes

EST-␣andEST-␤bindtoxenobioticsandtransformthemintoamore hydrosolublecompoundsfacilitatingtheirexcretion(Hemingway

andRanson,2000).

However,priortousingtheenzymesAChE,EST-␣andEST-␤as biomarkers,itisnecessarytoinvestigatewhethercertainfactors

∗ Correspondingauthor.

E-mail:mnavarro@ufpr.br(M.A.Navarro-Silva).

canchangetheiractivity.Organismsinthenaturalenvironment face adverse situations on a daily basis, for instance fluctua-tionsintemperatureandfoodavailability.Inlaboratorystudies, acutetoxicitybioassaysareusuallyperformedintheabsenceof food,whichcanleadtometabolicstress.Studiesusingdifferent bioindicatorsorganisms(copepods,crustaceansandbivalves)have investigatedtheinfluenceofseasonalvariationsonselected bio-chemical biomarkers (AChE, glutathione S-transferase, catalase, metallothionein)andtheircorrelationwithseasonalfluctuations inabioticparameterssuchastemperature,salinity,turbidityand foodavailability(LeiniöandLehtonen,2005;Pfeiferetal.,2005;

Menezesetal.,2006;Cailleaudetal.,2007;Tuetal.,2012).

Inadditiontoenvironmentalvariations,theeffectsoflaboratory protocolsthataimtoquantifyenzymaticactivityneedtobe stan-dardizedforthebioindicatorspecies.Somestepsoftheprotocol,for examplecentrifugationandfreezingofsamples,caninﬂuencethe enzymatic analysisof thebiochemical biomarkers(Guilhermino

etal.,1996;Muriasetal.,2005).

http://dx.doi.org/10.1016/j.rbe.2016.07.002

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ImmatureChironomidae(Diptera)inhabitthebenthic compart-mentofaquaticecosystems(Lagauzèreetal.,2009;DiVerolietal.,

2012a).Theyare importantcomponentsofthefoodchain,

rep-resenting thestrongest link betweenproducers and secondary consumers(PorinchuandMacDonald,2003).Becausetheyare sen-sitivetovariouspollutants(Preston2002),areeasytorearandhave ashortlifespan(FonsecaandRocha,2004),chironomidsarewidely usedasbioindicatorsofacuteandchronictoxicityincontaminated sedimentsandwater(Leeetal.,2006;Roulieretal.,2008;Yoshimi

etal.,2009;Al-Shamietal.,2010;DeJongeetal.,2012;DiVeroli

etal.,2012b;Ebauetal.,2012;Choungetal.,2013).Chironomus

sancticaroliStrixinoandStrixino,1981isawell-knownbioindicator ofwaterquality,andhasbeenusedinvariousbiochemicalstudies involvingbiomarkers,inanattempttoelucidateitsresponsesto environmentalcontamination(Moreira-Santosetal.,2005;Printes

etal.,2007,2011).

Theaimof this studywastoinvestigate experimentallythe potentialeffectsoffoodandthermalstressontheactivityofthe enzymesAChE,EST-␣andEST-␤ofC.sancticarolilarvae.In addi-tion,theeffectsoftwostepsoftheenzymaticprotocol(freezingand centrifugationofsamples)onenzymaticactivitywereassessedin ordertostandardizethemethodology.

Materialandmethods Biologicalmaterial

SpecimenswereobtainedfromtheLaboratoryoftheMedical andVeterinaryEntomology,FederalUniversityofParaná(UFPR). TheirbreedingcolonyismaintainedfollowingMaieretal’sprotocol

(1990),withmodiﬁcationsinthetemperature(25◦C±2)and

pho-toperiod(12hlight:12hdark).Voucherspecimensaredeposited inthePe.JesusSantiagoMoureEntomological Collectionofthe DepartmentofZoology,UFPR(DZUP),numbers249269to249276. Enzymaticassay

Larvaewerestoredina−80◦_C_freezer_and_were_subsequently homogenizedin300␮L0.1MpH7.5potassiumphosphatebuffer (for the enzyme AChE) and in 150␮L 0.2M pH 7.2 potassium phosphatebuffer(fortheenzymesEST-␣andEST-␤),followedby centrifugationat12,000×gfor1minat4◦C.

TheprotocolusedfortheenzymeAChEwasbasedonEllman

et al. (1961),modiﬁed for microplatesfollowing Silva deAssis

(1998).TheactivitiesoftheEST-␣andEST-␤wereascertained

fol-lowingthemethodologyofValleetal.(2006).Totalproteinperlarva wasmeasuredfollowingBradford(1976),usingbovineserum albu-minasstandard.BiochemicalanalyseswerecarriedoutinaBioTek microplatereader.

Temperatureeffectsonthelarvae

Fromhatchinguptothefourthinstar,differentgroupsoflarvae werekeptatthreedifferenttemperatures:20◦C,25◦Cand30◦C. Thetemperaturewascontrolledina BODconstanttemperature chamber(photoperiod12/12h).Thelarvaeweresubsequently sub-jectedtotheenzymaticquantiﬁcationprotocolsalreadydescribed. Atotalof270larvae(90larvaeforeachenzyme,30larvaeforeach temperature)wereused.Inthisexperiment,theeffectof temper-atureonlarvaldevelopmentdurationwasalsoascertained. Theeffectoffoodstressonlarvae

StocklarvaeofC.sancticaroliintheIVinstarweresubjectedto sixdifferenttreatments.IntreatmentsA,BandC,4mgTetraMin®

perlarvawereofferedattime0.After24h,treatmentAwas dis-continued,followedbytreatmentBafter48handtreatmentCafter 72h.Larvaeintheremainingthreetreatments,D,EandF,werenot fedattimezeroandweremaintainedwithoutfoodfor24,48and 72h,respectively.Resultsfromthefeedingandfooddeprivation treatmentswerethencomparedforthesametimeperiods(Awith D,BwithEandCwithF).Thisexperimentwascarriedoutin con-tainerswith80mLofdechlorinatedwater.Larvaewereisolated fromoneanothertopreventpredation.Thetreatmentswere per-formedinaBODchamberwithconstanttemperature(25◦C±2◦C) andphotoperiod(12hlight/12hdark).Intotal,540IVinstarlarvae (180larvaeforeachenzyme,30larvaeforeachtreatment)were processed.

Effectsoffreezingonhomogenizedsamples

Stocklarvaewerehomogenizedasdescribedaboveforenzyme activityquantiﬁcation.However,thevolumeofeachsamplewas dividedintotwoaliquots.Onewasusedimmediatelyforenzyme quantiﬁcation,whiletheotherwasfrozenin−80◦_C_for₃₀_days beforeitwasusedforthispurpose.Atotalof90IVinstarlarvae(30 larvaeforeachenzyme)wereprocessed.

Effectsofcentrifugationonhomogenizedsamples

Stocklarvaewere homogenizedasdescribed abovefor each enzyme.However,thevolumeofeachsample wasdividedinto twoaliquots.Onewascentrifuged,whiletheotherwasnot.Both aliquotsweresubjectedtoenzymequantiﬁcation.Atotalof90IV instarlarvae(30larvaeforeachenzyme)wereprocessed. Statisticalanalysis

Analyseswereperformedin Renvironment (RDevelopment

CoreTeam,2011).Theeffectsoftemperatureontheactivityofthe

enzymesAChEandEST-␤wereanalyzedwithanadjusted gen-eralizedlinearmodel(GLM)withGammadistribution,andforthe enzymeEST-␣,aninverseGaussiandistributionwasemployed.One wayANOVAwasapplied,andTukeycontrast(p≤0.05)wasused inaposterioricomparisons.MASS(VenablesandRipley,2002)and effects(Fox,2003)librarieswereusedforGLMandthemultcomp librarywasusedinposteriorianalyses(Hothornetal.,2008).To evaluatetheeffectofcentrifugationandfreezingonenzyme activ-ity,datawerelogaritmisedandthettestforpairedsampleswas used.Intheanalysisoffoodstressonenzymeactivity,datawere alsologaritmised,buta ttestforunpairedsampleswasapplied instead.

Results

Incrementsofﬁve-degreeCelsiusduringthedevelopmentofC. sancticarolishortenedthedevelopmenttimeofimmaturesfrom twelvedaysat20◦C,tosevendaysat25◦C,andtofourdaysat 30◦C.Theenzymeactivitychangedunderdifferenttemperatures (Fig.1).AChEactivitydecreasedwithincreasingtemperatures:at 20◦Cand25◦Citwas69%and59%lowerthanat30◦C,respectively. Nosigniﬁcantchangesinenzymeactivityweredetectedbetween 20◦Cand25◦C.

NochangesintheactivityofEST-␣wereobservedbetween20◦C and25◦C(Fig.1).However,at30◦Ctheenzymeactivityincreased by44%and45%whencomparedto20◦Cand25◦C,respectively.

The enzyme activityof EST-␤was highat the intermediate temperatureof25◦C.Atthistemperature,EST-_␤activitywas24% higher than at20◦C and 18% higher than at 30◦C. In contrast, enzymeactivityat20◦Cand30◦Cdidnotdiffer(Fig.1).

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AChE EST alpha EST beta 600 25 a a b a b a 15 10 5 20 15 10 5 0 0 a a b 20ºC Temperature 25ºC 30ºC 20ºC 25ºC 25ºC Temperature 30ºC 20ºC Temperature 30ºC

Enzymatic activity (mmol/mg ptn/min) Enzymatic activity (nmol/mg ptn/min) Enzymatic activity (nmol/mg ptn/min)

400

200

0

Fig.1. Effectoftemperature(20,25and30◦_C)_on_the_activity_of_{acetylcholinesterase}_(AChE),_alpha_esterase_(EST-␣),_and_beta_alpha_esterase_(EST-␤)_of_Chironomus_{sancticaroli.}

Thevaluesareexpressedasthemeanvalueofenzymeactivity±SD(n=30foreachcondition).Differentlettersindicatesigniﬁcantdifferenceswhenp<0.05(usingANOVA –onewayandTukeycontrast).

400 25 20 a a a a 15 15 10 5 0 15 10 5 0 10 5 0 a a b a a b b a AChE

A

B

C

EST alpha EST beta

AChE EST alpha EST beta

Enzymatic activity (mmol/mg ptn/min) Enzymatic activity (nmol/mg ptn/min)

Enzymatic activity (nmol/mg ptn/min) Enzymatic activity (nmol/mg ptn/min) Enzymatic activity (nmol/mg ptn/min)

300

200

100

0

400

Enzymatic activity (mmol/mg ptn/min)

300 200 100 0 30 20 10 0 Without Food - 24h With Without Food - 24h With Without Food - 24h With Without Food - 48h With Without Food - 48h With Without Food - 48h With 20 15 10 5 0 a a a a b a

Enzymatic activity (nmol/mg ptn/min) Enzymatic activity (nmol/mg ptn/min)

400

Enzymatic activity (mmol/mg ptn/min)

300 200 100 0 25 20 15 10 5 0 Without Food - 72h With Without Food - 72h With Without Food - 72h With

Fig.2.Effectoffastingfor24h(A);48h(B)and72h(C)ontheactivityofacetylcholinesterase(AChE),alphaesterase(EST-␣),andbetaalphaesterase(EST-␤)ofChironomus sancticaroli.Thevaluesareexpressedasthemeanvalueofenzymeactivity±SD(n=30foreachcondition).Differentlettersindicatesigniﬁcantdifferenceswhenp<0.05 (usingunpairedt-test).

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Enzymatic activity (nm ol/mg ptn/min) Enzymatic activity (nm ol/mg ptn/min) Enzymatic activity (nm ol/mg ptn/min)

Without 600 ₆₀ ₁₅ 10 5 0 40 20 0 a b b a b a 400 200 0 30 days Freezing Without 30 days Freezing Without 30 days Freezing

Fig.3.Effectofsamplefreezingontheactivityofacetylcholinesterase(AChE),alphaesterase(EST-␣),andbetaalphaesterase(EST-␤)ofChironomussancticaroli.Thevalues areexpressedasthemeanvalueofenzymeactivity±SD(n=30foreachcondition).Differentlettersindicatesigniﬁcantdifferenceswhenp<0.05(usingpairedt-test).

After24h of fooddeprivation, activityof the AChE enzyme increasedsignificantly(39%),whileactivityoftheEST-␣and EST-␤did not (Fig. 2A). AChE activitydid not changeafter 48h of fooddeprivation.Activity oftheEST-␣andEST-␤enzymeswas significantlylowerafter48hoffooddeprivation(34%and 41%, respectively)(Fig.2B).After72h,AChEandEST-_␣activityremained constant,whereasEST-␤activitywassignificantlyreducedby47% (Fig.2C).

The resultsof the freezingand centrifugation tests indicate thatthesetwofactorsmaynegativelyinﬂuencetheactivityofthe threeenzymesevaluated.Freezingsamplesfor30daysat−80◦_C decreasedenzymaticactivityoftheAChE,EST-␣andEST-␤by12%, 32%and25%,respectively(Fig.3).Centrifugationofsamplesalso affectedtheactivityoftheAChE,EST-␣andEST-␤:whensamples werecentrifuged,enzymeactivitydecreasedby18%,10%and10%, respectively(Fig.4).

Discussion

Itisimportanttoinvestigatehowcertainfactorssuchas tem-perature and food resources affect the activity of biochemical biomarkers used toassess theeffect of pollutants. A tempera-tureincreaseduringlarvaldevelopmentshortensthedevelopment periodofinsects,thusimpactingtheﬁnalsizeoftheadults(Vogt

etal.,2007;Oetkenetal.,2009;Zillietal.,2009).Thisisanindication

thattemperaturecanbeametabolicstressor.ParkandKwak(2014)

investigatedtheeffectsofthermalstressonthedevelopmentof Chi-ronomusripariusMeigen,1804,showingthatitaltersthebiology (larvalsurvivalrate,sexratio,successfulpupationandadult emer-gence),metabolism(increasedexpressiongenerelatedtooxidative stressenzymes(catalase,peroxidase,superoxidedismutaseand glutathioneperoxidase)andendocrinesignaling(ecdysone recep-tor)oftheorganisms.

Theeffectsoftemperatureonbiochemicalbiomarkerssuchas theAChE enzyme of invertebrates have been investigated, and theresultsofvariousstudiesvariedaccordingtothespecies.For instance,activityofthisenzymemayincreaseordecreaseas tem-peratureincreases(ScapsandBorot,2000;Callaghanetal.,2002;

Pfeiferetal.,2005;Menezesetal.,2006;Cailleaudetal.,2007;Tu

etal.,2012).Inthisstudy,AChEactivitydecreasedathigher tem-peratures,corroboratingtheresultsofDominguesetal.(2007),who observedthattheactivityoftheAChEofC.ripariusMeigen,1804is higherat6◦Cand16◦Cthanat26◦C.

Thefactthateachenzymebehavesdifferentlyundervarious temperature regimes highlights the fact that each enzyme has an optimum temperature activity (Callaghan et al., 2002).This abioticfactoraltersthephysicalstructureofenzymes,and mod-iﬁestheircatalytic efﬁcacyorbindingcapacity (Hochachka and

Somero,1984).Therefore,whenenzymesareusedasbiomarkersof

environmentalcontaminationinaquaticecosystems,temperature mustbetakenintoconsiderationandenzymaticactivitycanonly becomparedamongspecimensfromsimilartemperatureranges. Additionally,seasonalvariationsintemperaturemustalsobe con-sideredinanalyses.

Theeffectsoffoodstressonbiochemicalmarkers,whichhave beenstudiedonlysporadically,arenotwellunderstood.Inourdata, thelackoffoodaffectedtheactivityofthethreetestedenzymes (AChE,EST-␣andEST-␤)differently,accordingtotheperiodof star-vation.AfterastudyusingC.riparius,Craneetal.(2002)foundno differencesinAChEactivityafter48and96hoffooddeprivation, althoughthedryweightofindividuals decreased.Studiesusing otherbiomarkers,suchasﬁsh,indicatedthatfoodstressis associ-atedwithchangesinenzymaticbiomarkers,andcausedoxidative stressinindividuals(Pascualetal.,2003).

InanexperimentusingC.riparius,individualsthatweregiven enoughfoodwerelesssusceptibletopollutantsthanthosethat

Enzymatic activity (mmol/mg ptn/min) Enzymatic activity (nmol/mg ptn/min) Enzymatic activity (nmol/mg ptn/min)

Without 350 15 15 10 5 0 10 5 0 a b b a b a 300 250 200 150 100 50 0 With Centrifugation Without With Centrifugation Without With Centrifugation

Fig.4.Effectofsamplecentrifugationontheactivityofacetylcholinesterase(AChE),alphaesterase(EST-␣),andbetaalphaesterase(EST-␤)ofChironomussancticaroli.The valuesareexpressedasthemeanvalueofenzymeactivity±SD(n=30foreachcondition).Differentlettersindicatesigniﬁcantdifferenceswhenp<0.05(usingpairedt-test).

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werenot(Postmaetal.,1994).However,anoppositeeffectcanbe achievedwhenfoodisusedintoxicologicalexperiments, increas-ingthetoxicityofcertaincompounds,forinstancecadmium,which quickly binds to organicmaterials, suchas carbon-based com-poundsderivedfromfooddegradationintheexperiment(Postma

etal.,1994).

Sample freezing after homogenization has been previously investigated and can be part of laboratorial routine when the numberofsamplesislarge.Inourdata,itwasevidentthat freez-inglowersenzymeactivity.Thiswasexpected,asatendencyto decreasedenzymeactivityaftereachcycleoffreeze-thawhadbeen previouslydocumented(Muriasetal.,2005).Consequently,in lab-oratorialroutine,itisbesttohomogenizesamplesandperform enzymaticquantiﬁcationonthesamedayasameanstoachieve maximumenzymeactivityresponse.Thiscanbedifﬁcult some-times,particularlywhenthenumberofsamplesislarge,andthe onlyalternativeisfreezing.Whenfreezingbecomesnecessary,we emphasizethatsamplesthatwillbeanalyzedtogethershouldbe processedonthesamedayandbesubjectedtothesamenumberof freeze-thawcycles,thusminimizingthevariationsintroducedby thisstep.

Anotherprotocol stepanalyzedin thisworkwas centrifuga-tion,whichalsoreducedenzymaticactivityofthesamples.This happensbecauseaportionoftheenzymescanberemovedfrom thesupernatantduringcentrifugation,asenzymesremainattached tolargerfragmentsthatdepositduringthisprocess(Guilhermino

etal.,1996).

Eventhoughcentrifugationcausesanegativeeffectonenzyme activity,this procedureshouldbeusedinallprotocols,because itpuriﬁesthesamplesforenzymaticquantiﬁcation,reducingthe interferenceofresiduesinabsorbancereadings.

Conclusions

TheactivityoftheenzymesAChE,EST-␣andEST-␤decreased afterfreezingand centrifugationof samples,demonstratingthe importanceof standardizedprotocols. Additionally thermal and foodstresscausedchangesintheactivityofthethreeenzymes. Basedontheseresultswerecommendthattemperatureandfood supplyshouldbemaintainedconstantintoxicitybioassaytests. Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest. Acknowledgement

WethanktheConselhoNacionaldeDesenvolvimento Cientí-ﬁcoeTecnológico(CNPq),#305038/2009-5(DR),#305470/2012-4 (MANS).

References

Al-Shami,S.A.,Rawi,C.S.M.,Ahmad,A.H.,Nor,S.A.M.,2010.Distributionof Chi-ronomidae(Insecta:Diptera)inpollutedriversoftheJuruRiverBasin,Penang, Malaysia.J.Environ.Sci.22,1718–1727.

Bradford,M.,1976.Arapidandsensitivemethodforthequantiﬁcationof micro-gramquantitiesofproteinutilizingtheprincipleofprotein-dyebinding.Anal. Biochem.72,248–254.

Cailleaud,K.,Maillet,G.,Budzinski,H.,Souissi,S.,Forget-Leray,J.,2007.Effects ofsalinityandtemperatureontheexpressionofenzymaticbiomarkersin Eurytemoraafﬁnis(Calanoida,Copepoda).Comp.Biochem.Physiol.,PartA147, 841–849.

Callaghan,A.,Fisher,T.C.,Grosso,A.,Holloway,G.J.,Crane,M.,2002.Effectof temper-atureandpirimiphosmethylonbiochemicalbiomarkersinChironomusriparius Meigen.Ecotoxicol.Environ.Saf.52,128–133.

Choung,C.B.,Hyne,R.V.,Stevens,M.M.,Hose,G.C.,2013.Theecologicaleffectsofa herbicide-insecticidemixtureonanexperimentalfreshwaterecosystem. Envi-ron.Pollut.172,264–274.

Crane,M.,Sildanchandra,W.,Kheir,R.,Callaghan,A.,2002.Relationshipbetween biomarkeractivityanddevelopmentalendpointsinChironomusripariuMeigen exposed to an organophosphate insecticide. Ecotoxicol. Environ. Saf. 53, 361–369.

DeJonge,M.,Belpaire,C.,Geeraerts,C.,DeCooman,W.,Blust,R.,Bervoets,L.,2012. Ecologicalimpactassessmentofsedimentremediationinametal-contaminated lowlandriverusingtranslocatedzebramusselsandresident macroinverte-brates.Environ.Pollut.171,99–108.

DiVeroli,A.,Goretti,E.,Paumen,M.L.,Kraak,M.H.S.,Admiraal,W.,2012a. Induc-tionofmouthpartdeformitiesinchironomidlarvaeexposedtocontaminated sediments.Environ.Pollut.166,212–217.

DiVeroli,A.,Selvaggi,R.,Goretti,E.,2012b.Chironomidmouthpartdeformitiesas indicatorofenvironmentalquality,acasestudyinLakeTrasimeno(Italy).J. Environ.Monit.14,1473–1478.

Domingues,I.,Guilhermino,L.,Soares,A.M.V.M.,Nogueira,A.J.A.,2007.Assessing dimethoatecontaminationintemperateandtropicalclimates:Potentialuse ofbiomarkersinbioassayswithtwochironomidspecies.Chemosphere69, 145–154.

Ebau,W.,Rawi,C.S.M.,Din,Z.,Al-Shami,S.A.,2012.Toxicityofcadmiumandlead ontropicalmidgelarvae,ChironomuskiiensisTokunagaandChironomusjavanus Kieffer(Diptera:Chironomidae).AsianPac.J.Trop.Biomed.2,631–634. Ellman,G.L.,Courtney,K.D.,Andres-Jr,V.,Featherstone,R.M.,1961.Anemandrapid

colorimetricdeterminationofacetylcholinesteraseactivity.Biochem. Pharma-col.7,88–95.

Fonseca,A.L.,Rocha,O.,2004.LaboratoryculturesofthenativespeciesChironomus. ActaLimnol.Brasil.16,153–161.

Fox,J.,2003.EffectdisplaysinRforgeneralisedlinearmodels.J.Stat.Softw.8,1–27. Fulton,M.H.,Key,P.B.,2001.Acetylcholinesteraseinhibitioninestuarineﬁshand invertebratesasanindicatoroforganophosphorusinsecticideexposureand effects.Environ.Toxicol.Chem.20,37–45.

Galloway,T.,Handy,R.,2003.Immunotoxicityoforganophosphorouspesticides. Ecotoxicology12,345–363.

Guilhermino,L.,Lopes,M.C.,Carvalho,A.P.,Soares,A.M.V.M.,1996.Inhibitionof acetylcholinesteraseactivityaseffectcriterioninacutetesteswithjuvenile Daphniamagna.Chemosphere32,727–738.

Hemingway,J.,Ranson,H.,2000.Inseticideresistanceininsectvectorsofhuman disease.Annu.Rev.Entomol.45,371–391.

Hochachka,P.W.,Somero,G.N.,1984.BiochemicalAdaptation.Princenton Univer-sityPress,Princenton,NJ.

Hothorn,T.,Bretz,F.,Westfall,P.,2008.Simultaneousinferenceingeneral paramet-ricmodels.Biom.J.50,346–363.

Lagauzère,S.,Pischedda,L.,Cuny,P.,Gilbert,F.,Stora,G.,Bonzom,J.-M.,2009. InﬂuenceofChironomusriparius(Diptera,Chironomidae)andTubifextubifex (Annelida,Oligochaeta)onoxygenuptakebysediments.Consequencesof ura-niumcontamination.Environ.Pollut.157,1234–1242.

Lee,S.-M.,Lee,S.-B.,Park,C.-H.,Choi,J.,2006.Expressionofheatshockprotein andhemoglobingenesinChironomustentans(Diptera,Chironomidae)larvae exposedtovariousenvironmentalpollutants:apotentialbiomarkerof fresh-watermonitoring.Chemosphere65,1074–1081.

Leiniö,S.,Lehtonen,K.K.,2005.Seasonalvariabilityinbiomarkersinthebivalve MytilusedulisandMacomabalthicafromthenorthernBalticSea.Comp.Biochem. Physiol.,PartC140,408–421.

Lionetto,M.G.,Caricato,R.,Giordano,M.E.,Pascariello,M.F.,Marinosci,L.,Schettino, T.,2003.Integrateduseofbiomarkers(acetylcholinesteraseandantioxidant enzymesactivities)inMytilusgalloprovincialisandMullusbarbatusinanItalian coastalmarinearea.Mar.Pollut.Bull.46,324–330.

Maier,K.J.,Kosalwat,P.,Knight,A.W.,1990.CultureofChironomusdecorus(Diptera: Chironomidae)andtheeffectoftemperatureonitslifehistory.Environ. Ento-mol.19,1681–1688.

Menezes,S.,Soares,A.M.V.M.,Guilhermino,L.,Peck,M.R.,2006.Biomarkerresponses oftheestuarinebrown shrimpCrangoncrangonL.to non-toxicstressors: temperature,salinityandhandlingstresseffects.J.Exp.Mar.Biol.Ecol.335, 114–122.

Moreira-Santos,M.,Fonseca,A.L.,Moreira,S.M.,Rendón-vonOsten,J.,Silva,E.M., Soares,A.M.V.M.,Guilhermino,L.,Ribeiro,R.,2005.Short-termsublethal (sed-iment and aquatic rootsof ﬂoating macrophytes) assays with a tropical chironomidbasedonpostexposurefeedingandbiomarkers.Environ.Toxicol. Chem.24,2234–2242.

Murias,M.,Rachtan,M.,Jodynis-Liebert,J.,2005.Effectofmultiplefreeze-thaw cyclesofcytoplasmsamplesontheactivityofantioxidantenzymes.J.Pharmacol. Toxicol.Methods52,302–305.

Oetken,M.,Jagodzinski,L.,Vogt,C.,Jochum,A.,Oehlmann,J.,2009.Combinedeffects ofchemicalandtemperaturestressonChironomusripariuspopulationswith differinggeneticvariability.J.Environ.Sci.Health,PartA44,955–962. Park,K.,Kwak,I.,2014.Theeffectoftemperaturegradientsonendocrinesignaling

andantioxidantgeneexpressionduringChironomusripariusdevelopment.Sci. TotalEnviron.1003–1011,470–471.

Pascual,P.,Pedrajas,J.R.,Toribio,F.,López-Barea,J.,Peinado,J.,2003.Effectoffood deprivationonoxidativestressbiomarkersinﬁsh(Sparusaurata).Chem.Biol. Interact.145,191–199.

Pfeifer,S.,Schiedek,D.,Dippner,J.W.,2005.Effectoftemperatureandsalinityon acetylcholinesteraseactivity,acommonpollutionbiomarker,inMytilussp.from thesouth-westernBalticSea.J.Exp.Mar.Biol.Ecol.320,93–103.

Porinchu,D.F.,MacDonald,G.M.,2003.Theuseandapplicationoffreshwatermidges (Chironomidae:Insecta:Diptera)ingeographicalresearch.Prog.Phys.Geogr.27, 378–422.

(6)

Postma,J.F.,Buckert-deJong,M.C.,Staats,N.,Davids,C.,1994.Chronictoxicityof cadmiumtoChironomusriparius(Diptera,Chironomidae)atdifferentfoodlevels. Arch.Environ.Contam.Toxicol.26,143–148.

Printes,L.B.,Espíndola,E.L.G.,Fernandes,M.N.,2007.Biochemicalbiomarkersin individuallarvaeofChironomusxanthus(Rempel,1939)(Diptera, Chironomi-dae).J.Brazil.Soc.Ecotoxicol.2,53–60.

Printes,L.B.,Fernandes,M.N.,Espíndola,E.L.G.,2011.Laboratorymeasurementsof biomarkersandindividualperformancesinChironomusxanthustoevaluate pes-ticidecontaminationofsedimentsinariverofsoutheasternBrazil.Ecotoxicol. Environ.Saf.74,424–430.

RDevelopmentCoreTeam,2011.R:ALanguageandEnvironmentforStatistical Computing.RFoundationforStatisticalComputing,Vienna,Austria,ISBN 3-900051-07-0,availableat:http://www.R-project.org(accessed17.04.15). Roulier,J.L.,Tusseau-Vuillemin,M.H.,Coquery,M.,Geffard,O.,Garric,J.,2008.

Mea-surementofdynamicmobilizationoftracemetalsinsedimentsusingDGTand comparisonwithbioaccumulationinChironomusriparius:ﬁrstresultsofan experimentalstudy.Chemosphere70,925–932.

Scaps,P.,Borot,O.,2000.AcetylcholinesteraseactivityofthepolychaeteNereis diver-sicolor:effectsoftemperatureandsalinity.Comp.Biochem.Physiol.,PartC125, 377–383.

SilvadeAssis,H.C.,1998.Dereinsatzvonbiomarkernzursummarischen erfas-sungvomgewässerverschmutzungen.Ph.D.thesis,UniversityofBerlin,Berlin, Germany.

Tu,H.T.,Silvestre,F.,DeMeulder,B.,Thome,J.-P.,Phuong,N.T.,Kestemont,P.,2012. Combinedeffectsofdeltamethrin,temperatureandsalinityonoxidativestress biomarkersandacetylcholinesteraseactivityintheblacktigershrimp(Penaeus monodon).Chemosphere86,83–91.

Valle,D.,Montella,I.R.,Ribeiro,R.A.,Medeiros,P.F.V.,Martins-Jr,A.J.,Lima,J.B.P., 2006.Quantiﬁcationmethodologyforenzymeactivityrelatedtoinsecticide resistanceinAedesaegypti.Fundac¸ãoOswaldoCruzandSecretariadeVigilância emSaúde,MinistériodaSaúde,RiodeJaneiro,DistritoFederal.

Venables,W.N.,Ripley,B.D.,2002.ModernAppliedStatisticswithS-Plus,4thed. Springer,NewYork.

Vogt,C.,Pupp,A.,Nowak,C.,Jagodzinski,L.S.,Baumann,J.,Jost,D.,Oetken,M., Oehlmann,J.,2007.Interactionbetweengeneticdiversityandtemperature stressonlife-cycleparametersandgeneticvariabilityinmidgeChironomus ripariuspopulations.Clim.Res.33,207–214.

Yoshimi,T.,Odagiri,K.,Hiroshige,Y.,Yokobori,S.,Takahashi,Y.,Sugaya,Y.,Miura, T.,2009.InductionproﬁleofHSP70-cognategenesbyenvironmentalpollutants inChironomidae.Environ.Toxicol.Pharmacol.28,294–301.

Zilli,F.,Marchese,M.,Paggi,A., 2009.LifecycleofGoeldichironomus holopras-inus goeldi (Diptera: Chironomidae) in laboratory. Neotrop. Entomol. 38, 472–476.