Morpho-histological characterization of immature of the bioindicator midge Chironomus sancticaroli Strixino and Strixino (Diptera, Chironomidae)

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REVISTA

BRASILEIRA

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Entomologia

AJournalonInsectDiversityandEvolution

Medical

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Veterinary

Entomology

Morpho-histological

characterization

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immature

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the

bioindicator

midge

Chironomus

sancticaroli

Strixino

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Strixino

(Diptera,

Chironomidae)

Vinicius

Sobrinho

Richardi

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_Maiara

_Vicentini

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_Débora

_Rebechi

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_Luis

_Fernando

_Fávaro

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Mário

Antônio

Navarro-Silva

a,∗

a_Laboratório_de_Entomologia_Médica_e_{Veterinária,}_Departamento_de_Zoologia,_Universidade_Federal_do_Paraná,_Curitiba,_PR,_Brazil b_Laboratório_de_Reproduc¸_ão_e_Comunidade_de_Peixes,_Departamento_de_Biologia_Celular,_Universidade_Federal_do_Paraná,_Curitiba,_PR,_Brazil

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Articlehistory:

Received12February2015 Accepted18April2015 Availableonline18July2015 AssociateEditor:GustavoGraciolli Keywords:

Ecotoxicology Histologicalbiomarker Sediment

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Chironomidaeimmatureareusedasbioindicatorsofsedimentqualityinaquaticecosystemsand eco-toxicologicalassays.Histologicaldescriptionsforthisfamilyareoutdatedandlimitedandthereareno studieswithNeotropicalspecies.Theaimofthisstudywastodescribethetissuearchitectureofseveral organsofthelarvaofChironomussancticaroli.Forthedescriptionofthehistologicalpattern,thelarvae werefixedinDuboscqsolutionforinsectsat56◦_C,_followed_by_routine_histologic_processing,_{infiltration} inparaffin,andthesectionswerestainedwithHematoxylin–Eosin.Afterexaminingtheslides,thetube digestive,salivarygland,excretory,nervous,endocrine,circulatory,andintegumentarysystemsandfat bodywerehistologicallycharacterized.Thehistologyallowsevaluationofcellmorphology,andforbeing notexpensiveandeasilyaccessiblecanberoutinelyusedinbiomonitoring.Inaddition,isausefultool inecotoxicologicalassaysandallowtoevaluatebiomarkersattissueandcelllevels.

Introduction

TheimmatureformsofChironomidaeareabundantandwidely distributed in water bodies, having theirhabits closely related tothesedimenttheyareinsertedin.Oneoftheircharacteristics isthepresenceofhemoglobininthehemolymph,whichallows these organisms to tolerate low oxygen concentrations in the water(Trivinho-Strixino,2011a).Anotheraspectisthe possibil-itythatthispigmentisabletometabolizexenobiotics(Weberand Vinogradov,2001;HaandChoi,2008),makingpossiblethe pres-enceoftheseinsectsforalongertimeindegradedenvironments. Thus,sublethaleffectscanbeassessedbybiomarkersof environ-mentalcontamination.Asaresultofthesecharacteristics,these organismsareconsideredbiondicatorsofenvironmentalquality.

Several studies that applied biochemical and molecular biomarkerswereperformedusingchironomids(Callaghanetal., 2001; Callaghan et al., 2002; Crane et al., 2002; Lee and Choi, 2006; Printes et al., 2007). However, there is a lack of

∗ Correspondingauthor.

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

applicationofcellandtissuebiomarkersthatdemonstratechronic effects.

Microscopicanalysescanidentifyalterationsincellsandtissues, andhelpinunderstandingthephysiologyoforgansandsystems. Themicroscopictechniqueiswidelyusedtostudyﬁshinaquatic ecotoxicology(Bernetetal.,1999),beingsensitivetodetectsubtle effectsofpollutants,determinationoftargetorgansandpossible toxicmechanisms.Becausetherearenosufﬁcientlydetailed histo-logicaldescriptionsoftheorgansandsystemsofchironomidsand otherspeciesalsousedintoxicitytests,thehistologicaldescription ofrepresentativesofthistaxonbecomesrelevant.

Chironomus sancticaroli Strixino and Strixino, 1981 was considered synonymy to Chironomus xanthus, Rempel, 1939 (Trivinho-Strixino,2011b), beingeasilyrearedin thelaboratory (Fonseca and Rocha, 2004). Theywereused in ecotoxicological testsfortheevaluationofenvironmentalqualityinBrazil( Moreira-Santosetal.,2005;Silvérioetal.,2005;Dornfeldetal.,2006;Printes etal.,2007;Santosetal.,2007;Sotero-Santosetal.,2007;Janke etal.,2011;Printesetal.,2011;Novellietal.,2012).Toproperly understandtheeffectsof xenobioticsondifferentreducedscale bodytissuessuchasChironomidaelarvae,thereisneedforprior knowledgeofanatomicalstandardofthetissuebeforeindication ofanychange.Thisstudy aimstodescribethestructure ofthe http://dx.doi.org/10.1016/j.rbe.2015.07.003

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Retrocerebral complex

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Digestory tube Aorta Fat body Nerve cord Malpighian tubules Heart Oesophagus Aorta Oesophagus Brain Nerve Trachea Anterior gland Corpora allata Corpora cardiaca Prothoracic gland Foregut or estomodeum Midgut or mesenteron Hindgut or proctodeum Gastric caeca Midgut (region I) Midgut (region II) Midgut (region III) Valvulae region Ileum Colon e rectum Anus Salivary gland Brain Frontal ganglion

Fig.1. SchematicrepresentationsoforgansandtissuesofimmatureChironomussancticaroli.(A)Internalmorphologyofthelarva;(B)morphologyofthedigestivetract;(C) aspectofthesalivarygland;(D)dispositionoftheendocrineglandsintheretrocerebralcomplex.

standardtissueofdifferentorgansandsystemsofthelarvaeofC. sancticaroliandtooffersubsidiesfortheuseofmicroscopic tech-niquesintheseorganismsforenvironmentalanalysis.

Materialandmethods

Larvae ofC.sancticaroli werefroma colonymaintainedat a breeding room of the Department of Zoology at the Universi-dadeFederaldoParanáundercontrolledconditionsoftemperature (25±2◦C),humidity(80±10%)andphotoperiod(12hlight/12h dark),constantaerationandTetraMin®crushedfood,attherate of0.1gperliterofdechlorinatedwater.Weused30initialfourth instarlarvaefor histologicaldescriptionofthelarvalstage.The

breedingofthespecieswasadaptedfromMaieretal.(1990).The specimensweredepositedinthePadreJesusSantiagoMoure Ento-mologicalCollectionattheUniversidadeFederaldoParaná(DZUP), numbers249269–249276.

LarvaewerefixedinDuboscqsolutionforinsects(Barth,1958), for 4hin an incubator at 56◦C. After fixation,the larvae were washed in70% ethanol for theremoval ofthefixative solution andthentheproceduresofroutinehistologicaltechniqueswere followed:thematerialwasdehydratedinalcoholseries, diapha-nizationinxyleneandinfiltrationinparaffin.

Weobtainedtransversal,lateral,longitudinalanddorsoventral sectionsofthelarvae,inthethicknessof7␮m,andstainedwith Hematoxylin–Eosin.To performtheanalysisofthematerial, all

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Fig.2. MicrographsoftheforegutandgastriccaecaregionofimmatureChironomussancticaroli.(A)Cross-sectionoftheesophaguswithlongitudinalfoldsformedbythe epithelium(arrow)andthelongitudinalmusclelayer;(B)longitudinalsectionshowingtheoesophagealliningepithelium(arrow);(C)cross-sectionalofthecaecumofthe larva,notetheCuénotcells(arrow),cellsofthecaecum(arrowhead)andesophagus;(D)theCuénotcellsaredetailed;(E)indetail,cellsofgastriccaeca;(F)longitudinal sectiontheregionofthecaecumshowingtheestomodealvalve.bb:brushborder;cae:gastriccaeca;cuc:Cuénotcells;ml:musclelayer;oe:esophagus;oec:esophagus cells;oei:esophagusinvagination.Stain:Harrishematoxylinandeosin.Scalebar=20␮m.

histologicalsectionswereusedtoallowabetterdescriptionofthe standardtissueofdifferentorgansandsystems.

Results

Asinitialparameter,weelaboratedthedescriptionofthetissue patternoforgansandstructuresofthedigestivesystem,salivary gland,excretory,nervousandendocrinesystemorretrocerebral complex,fatbodyandthecirculatoryandintegumentarysystems oflarvaeofC.sancticaroli(Fig.1A).

Digestivesystem

Thedigestivetractof theC. sancticarolilarvae extendsfrom mouthtoanus,beingdividedintothreeregions:foregut,midgut

andhindgut(Fig.1B).Theesophagusisthefirstregionofthe diges-tivetract,whichcorrespondstotheregionoftheforegut.Itisa shortorganthatshows nodifferentiated region,as cropor giz-zard.Thisorganextendsfromthemouthtothemetathorax.In cross-section,itispossibletoobservefivelongitudinalfoldsofthe epithelium,inadditiontothethickmuscularlayer(Fig.2A).Inits initialportion,itissurroundedbyamuscularstructureknownas periesophagealcollar(Fig.2B).Theepitheliumofdifficult observa-tionconsistsinpavementcellsthatdonotexceed6mmthick,with basophiliccytoplasmandlargenucleus(Fig.2B).

Cuénotcellsthatareassociatedwiththeoesophageal projec-tionaregroupedandexhibitareducedsizecomparedtocellsthat arenotassociatedtotheprojection.Thecellshavelargespherical nuclei,stronglybasophilicandgranularcytoplasmandcellapex withbrushborder(Fig.2CandD).

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Fig.3. MicrographsofthemidgutofimmatureChironomussancticaroli.(A)Cross-sectionofthemidgutregionI;(B)longitudinalsectionofthemidgutregionI;(C)cellsof theepitheliumofthemidgutregionI,showingthelittlebrushborderarea(arrow)andapicalandbasaleosinophiliaofthecell(arrowhead);(D)cross-sectionofthemidgut regionII;(E)Cross-sectionoftheregionIIIofthemidgut;(F)Brushborder(arrow)andperitrophicmatrix(arrowhead)inregionIIofthemidgutand(G)Brushborder(arrow) andcellsintheprocessofsecretion(arrowhead)inregionIIIofmidgut.cae:gastriccaeca;ep:gutepithelia;fd:food;lu:lumen.Stain:Harrishematoxylinandeosin.Scale bar=20␮m.

FollowingtheCuénotcells isthe gastriccaeca zone,formed bythree or fourcrowns of ﬁfteendiverticula, being the poste-riordiverticula signiﬁcantlylarger.Theyare formedbyglobose cellsthatareslightlysmallerthantheCuénotcells,withgranular eosinophiliccytoplasm,largesphericalnucleiwherethechromatin canbeobserved,andcellswithextensivebrushborder(Fig.2E).

Inthetransitionbetweentheforegutandthemidgutisthe esto-modealvalve.Thisvalveis characterizedby aprojectionofthe epitheliumandmuscleoftheesophagus intothemidgut,being composedbyCuénotcellsandregionofgastriccaeca(Fig.2F).

The midgutis divided into three regions with differentcell types.RegionIis formedbylargecellswithirregularshape. In cross-section,theepitheliumhasastarshapedcontour,whilethe lumenhasacircularshape(Fig.3A).Thebasalregionofthecell isroundedandconvex,and protrudesbetweenadjacentmuscle ﬁbers(Fig.3B).Intheapicalandbasalregionofthecytoplasmof thecellispossibletoseeamoreeosinophilicoutlinethanthe cyto-plasm,whichisgranular.Thebrushborderisnotextensive(Fig.3C). RegionIIhascubicalcellswithlargenucleiandbasophilic cyto-plasm(Fig.3D).RegionIIIisformedbycubicalcells,slightlysmaller

thantheanteriorregion(Fig.3E).ThebasalpoleofregionIIcells displaysheterogeneouslystainedregionsintheformofstriations (Fig.3F).Itispossibletoobserveaconspicuousbrushborderatthe apicalcellportion,moreextensivethanthoseofcellsintheregionI (Fig.3F).Thecytoplasmofthesecellsisstronglybasophillicandhas striationsperpendiculartothebaseofthecell,beingmoreevident thantheonesontheanteriorregion(Fig.3G).Intheregionbelow thenucleiandintheapicalcellportion,thecytoplasmisgranular andthebrushborderisconspicuous,representingaquarterofthe sizeofthecells.Also,inregionIII,thereareisolatedregenerative cellslocatedinthebasalepithelium(Fig.3G).

Theﬁrstregionofhindgutorvalveregionisformedbya cylin-dricalepitheliumwithbasophiliccytoplasm,mid-basalnucleiand peripheralchromatin(Fig.4A).Thevalveisformedbyacubical epitheliumwitheosinophiliccytoplasmandnucleiwithnot con-densedchromatin(Fig.4BandC).Theregionoftheileumissimilar instructuretotheesophagus,consistingofathickcircularmuscle layerandathinliningepitheliumcells,formedbycellswith impre-ciseboundaries,withlargeandsphericalnucleus,andchromatin arrangedinperipheralgranules.Thethinliningepitheliumforms

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Fig.4. MicrographsofhindgutimmatureofChironomussancticaroli.(A)cross-sectionbetweenthetransitionalepitheliumofthemidgutandhindgut;(B)longitudinalsection ofthetransitionregionbetweenthemidandhindgut,showingtheproctodealvalve(arrow);(C)indetail,epitheliumoftheproctodealvalve;(D)cross-sectionofileum showingextensivemusclelayerandthelongitudinalfoldsformedbytheepithelium(arrow);(E)longitudinalsectionofthecolonandrectum;(F)cross-sectionofthe epitheliumofthecolonandrectumdemonstratingbasaleosinophiliaofthecell(arrowhead).p:epithelia;fd:food;lu:lumen;ml:musclelayer;mlp:Malpighiantubule, pm:perithroﬁcmembrane;vep:valveepithelia.Stain:Harrishematoxylinandeosin.Scalebar=20␮m.

numerouslongitudinalfoldslinedbycuticle(Fig.4D).Intheﬁnal portionofthedigestivetract,thecolonandrectumcanbeobserved. Theyhavethesamecellularmorphology,composedoflargecells withelongatedbasalregioncontaininglargesphericalnucleus.The cytoplasmisslightlybasophilic,withaeosinophilicbasalandapical regions(Fig.4EandF).

Salivarygland

Thesalivaryglandsarepairs,andslightlycurvedstructures, fol-lowingthedorsalwallofthebody,locatedinthesecondandthird larvalthoracicsegment(Fig.1C).Inaddition,theglandular epithe-lialcellsarearrangedinasinglelayer,delimitingthelargecentral lumenofthesecretoryportion,whereisdepositedthesecretion thatexhibitshighafﬁnitytoeosin.Thesecellshavelargenuclei

containingpolytenechromosomes,thecytoplasmisuniformand stronglybasophilic(Fig.5AandB).Theexcretoryductopensinto theesophagus,consistingofcubicalepitheliumandsphericalnuclei withcondensedchromatinandbasophiliccytoplasm(Fig.5C). Excretorysystem

The species has four Malpighian tubules, with its proximal portionopeningintheﬁrstregionofthehindgutandacloseddistal portion.Thesetubulesarefreeinthebodycavity,beingbathedby hemolymph(Fig.5D).Theepitheliumiscomposedofasinglelayer ofpavementcells,withalargenucleusprojectedtothelumenofthe tubule.Thecytoplasmisgranuloseandeosinophilicandinthe api-calpoleofthecell,ashortbrushborderiseasilyobserved(Fig.5D andF).

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Fig.5.MicrographsofstructuresattachedtothedigestivetractofimmatureChironomussancticaroli.(A,B)Tangentialsectionofthesalivarygland,showingpartsofthe lumencontainingsecretion(arrow)andcells,highlightingthenucleuswithpolytenechromosomes(arrowhead);(C)longitudinalsectionofsalivaryglandduct,notethe presenceofcontentinthelumen(*);(D)longitudinalsectionshowingtheinsertionsiteofaMalpighiantubuleinthedigestivesystem;(E)cross-sectionofthetubuleswith thelumenandthebrushborder(arrow);(F)longitudinalsectionofatubulewiththebrushborder(arrow)andtheprojectionofthenucleusintothelumen(arrowhead). hg:hindgut;ir:insertionregion;lu:lumen;mg:midgut;oe:esophagus.Stain:Harrishematoxylinandeosin.Scalebar=20␮m.

Nervoussystem

Thecentral nervoussystemconsistsof brain,subesophageal ganglionandventralnervecord(withabdominalandthoracic gan-glia).Thebrainisabilobedorganlocatedabovetheesophagus, beingorganizedintocorticalandmedullarlayer.Theassociation oftheneurallamellawiththeperineuriumconstitutesthenerve sheath(Fig.6A).Theventralnervouscordisconstitutedbythree thoracicgangliaandeightabdominalgangliathatareconnected togetherandtothesubesophagealganglionbyconnectivepairs, whichare formedby theaxons oftheneuronsenclosedbythe neurallamella.Theﬁrstabdominalganglionisdislocatedtothe metathoraxofthelarvae,andthethoracicgangliaareslightlylarger

thantheabdominals.Thecellularstructureofthegangliainthe nervechainissimilartotheonedescribedforthebrain,butthe cellbodies,thatarelocatedintheventralganglion(Fig.6BandC). Thecorticalregionofbrainshowsthecellbodiesofneurons,while themedullaryregionischaracterizedbytheneuropile,aregion formedbytheaxonsofneurons(Fig.6D).Thefrontalganglionis locatedantertiortothebrainandaroundtheoralcavity(Fig.6E). Endocrinesystemorretrocerebralcomplex

Theretrocerebralcomplexiscomposedmainlybythecorpora allata, corpora cardiaca, and prothoracic gland (Fig. 1D). Cor-poraallataarepairedsphericalstructures,consistingofglandular

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Fig.6.MicrographsofnervoussystemstructuresofimmatureofChironomussancticaroli.(A)Longitudinalsectionshowingthebraincorticalandneuropile;(B)longitudinal sectionofaganglioninthenervecordandaconnectivesheafformedbyaxons;(C)longitudinalsectionshowingthebrain,thoracicgangliaandthefirstabdominalventral nervecord(numbers);(D)cross-sectionofthebrain;(E)longitudinalsectionofthecephalicregionofthelarva,indetailisthefrontalganglion,anteriortothebrain.1st: firstthoracicgangliom,2nd:secondthoracicgangliom;3rd:thirdthoracicgangliom,I–III:thoracicsegments;4th:firstabdominalgangliom;br:brain;cl:corticallayer, cn:connective;dv:diverticulum;fg:frontalgangliom;g:gangliom,ne:neuropile;nl:neurallamella,oe:esophagus;sg:salivarygland,tr:trophoblastes.Stain:Harris hematoxylinandeosin.Scalebar=20␮m.

epitheliumformedby afew cells of varyingsizes, withcentral nucleusandvacuolatedcytoplasm.Theyarelocatedclosetothe aorta,posterior tothebrain andtothesalivarygland(Fig.7A). Prothoracicglands are formed by slightly pyramidal cells with basophiliccytoplasmand largenuclei,varyingfromsphericalto oval,andwithchromatinarrangedinclumps.Theyarearranged longitudinally,involvingthecephalictracheae(Fig.7B).The ante-riorpostcerebralglandsarepairsandunicellularstructures.The largercellhasintenselystainednucleus,duetothecondensation ofchromatin,andcytoplasmwithvacuolesandgranules.Theyare locatedcloselyassociatedwiththewalloftheesophagus(Fig.7C andD). Corpora cardiaca arepairedstructures, formedbya set ofsmallirregularlyshapedcells,withvacuolatedcytoplasmand nucleiwithcondensed chromatin.Theyarelocatedclosetothe cephalicdorsaltrachea,betweenanteriorpost-cerebralglandsand prothoracicgland(Fig.7CandE).

Fatbody

Thefatbodyisconstitutedbyoenocytesandtrophocytes.Inthe parietalfatbody,onlyintheabdominalsegments,ventrally,occurs singlyamodiﬁedtrophocyteprobablywithhemoglobincontents (Fig.8A).Theoenocytesarelocateddispersed,occurringin gro-pus intheparietalfatbody. Thesecells arelargewithglobular aspect,biggerthantrophocyte,butinsmallerquantities(Fig.8B). Thehemoglobincellsandoenocyteshaveastainedsimilarly,the cytoplasmisacidophilusandthenucleusiscentral,butinthe mod-iﬁedtrophocyteshaveacidophiluscytoplasmicsgranulesandthe nucleuswithchromatinclumps(Fig.8C)whileoenocyteshavea perinuclearbasophiliccytoplasmandinthenucleus,thechromatin isdistributedintheperiphery(Fig.8D).

Theparietalfatbodyislocatedbetweentheepidermisandthe intersegmentalmuscles,whilethevisceralfatbodyisdistributed

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Fig.7. MicrographsofglandsfromtheretrocerebralcomplexoftheimmatureChironomussancticaroli.(A)Longitudinalsectionofthecorporaallata,showingtheglandular epithelium,demonstratingthecellnucleus(arrowhead);(B)longitudinalsectionoftheprothoracicgland;(C)longitudinalsectionshowingtheregionofthecomplex, demonstratingtheanteriorpostcerebralglandandthesmallgroupofcellsthatmakeupthecorporacardiac;(D)detailoftheanteriorpostcerebralgland,notethegranules intheircytoplasm(arrowhead);(E)detailofthesmallgroupofcellsthatmakeupthecorporacardiaca(arrows).cc:corporacardiaca;ga:anteriorpostcerebralgland;ptg: prothoracicgland,tr:trachea.Stain:Harrishematoxylinandeosin.Scalebar=20␮m.

betweentheinternalorgansofthelarva.Theparietalfatbodyhas trophocytesandoenocytes,whilethevisceralisconstitutedonlyby trophocytes.Thetrophocytesoftheparietalfatbodyaresmallcells andformasolidmassassociatedwiththeintegumentofthelarva, whilethevisceralfatbodycellsarelargerandformcellularmasses thatﬁllthespacesbetweentheorgans(Fig.8E).Thetrophocytes presentthecytoplasmwithvacuolatedappearanceandnoafﬁnity forthehematoxylinandeosinstain,beingstainedbyhematoxilyn intheperinuclearregion.Thenucleusisroundandsmall,when comparedtothecytoplasmicvolume,andthechromatiniswell condensed(Fig.8F).

Circulatorysystem

The heart is located dorsally on the eighth abdominal seg-ment,anditpresentsachamberlinedbyathincontractilemuscle wall,whichisdifficulttoobserveinlightmicroscopy.Inthe mus-cularwall,theostia openup,whichallows theentranceofthe hemolymph(Fig.9A).Theaortaislocateddorsally,abovethe diges-tivetract.Itextendsfromtheregionofthehearttothehead,with itscephalicendsimilartoatrumpet.Thecellsthatformthewall oftheaortaarepavementcellswithlittlecytoplasmandflattened nucleus,containingcondensedchromatininitsperiphery(Fig.9B). Throughoutitslength,itwasobservedvalvesthatpreventbackflow ofhemolymph(Fig.9C).Thepresenceofpericardialcells associ-atedwiththeaortaisalsoreported(Fig.9D),asdescribedinthe

excretorysystem.Thepericardialcellsarelarge,multinucleated, witheosinophiliccytoplasmandvacuoles,andfoundattachedto thewallofthedorsalvessel(Fig.9D).

Integument

Theepidermisconsistsofasinglelayerofcubicalcellsthatmay formfoldsinthebodyoflarvae,witheosinophiliccytoplasmand sphericalnucleiwithcondensedchromatin.Thecuticleliningthe epidermisismadeupofthreelayers,withtheepicuticlebeingthe outermostand themostdifﬁculttoseein lightmicroscopy,the exocuticlethemiddlelayerandtheendocuticlethemostintern one.Thecephaliccapsuleisastronglysclerotizedstructure,soit ispossibletoobserveathinendocuticle, athickerlayerof exo-cuticulaandanextremelythinanddifﬁculttoobserveepicuticle (Fig.9E).Inthebodyofthelarvae,thecuticleisrepresentedbya largeramountofendocuticle,incomparisonwiththethicknesses oflayersofexocuticleandepicuticle(Fig.9F).

Discussion

ThemajorityofhistologicalstudieswithChironomidae histol-ogywereheldattheendofthenineteenthcenturyandtheﬁrst halfofthetwentiethcentury.However,theseauthorsonly illus-tratedthegeneralmorphology oftissues, withoutdetailing the structureoftheircells(MiallandHammond,1900;Burtt,1938;

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Fig.8. MicrographsofthefatbodyofimmatureChironomussancticaroli.(A)Hemoglobincellinparietalfatbody;(B)oenocyte,arrangedingroupsdistributedintheparietalfat body;(C)indetail,thehemoglobincellpresentinggranulesinitscytoplasm(arrowhead);(D)groupedoenocytesshowingthebasophilicperinuclearcytoplasm(arrowhead); (E)longitudinalshowingthearrangementoftheparietalandvisceralfatbody;(F)indetailthetrophocytes,cellsthatareingreaterquantityinthistissue,notetheperinuclear cytoplasm(arrowhead)andthevacuolesﬁllingthecell(arrows).ct:cuticle;dg:digestorytube;llm:longitudinalmusclelayer;pfb:parietalfatybody,tr:trophocytes;vfb: visceralbodyfat.Stain:Harrishematoxylinandeosin.Scalebar=20␮m.

Possompès, 1948; Zeeand Pai, 1944; Gouin, 1946; Possompès, 1946; Cazal, 1948; Pierson, 1956; Credlandand Phillips, 1974; CredlandandScales,1976;Credland,1978;Panov,1979;Seidman etal.,1986;Jarial,1988;Nardietal.,2009).Furthermore,the ner-vous,endocrine,circulatorysystemsandfatbodywerenotdetailed histologicallyforChironomidae.

Inecotoxicologicalcontext,manyxenobioticscancause patho-logicalchangesinorgansandsystemsoforganisms.Histopathology isanimportantanalyticaltoolbetweenmolecular,cellularand bio-chemicalbiomarkersandisconsideredanendpointwithrelevant extrapolationsonthepopulationlevel. Histologicalapproach is usedmainlyinorganismsexposedtolowconcentrations, show-ingchroniceffects. Such a damage assistsin theelucidation of xenobioticstargetorgans(ChiangandAu,2013).Histopathology isusedasbiomarkerinstudiesinvolvingseveralanimalgroups,

predominantlyﬁsh(gill,liver,kidney,andgonads)inAquatic Tox-icology(Bernetetal.,1999).Theadvantageofworkingwiththis groupislinkedtothelargeamountsoftissueineachorgan,which allows analysis of only one organ in the slide. But with some invertebrates,due totheirbody size,it isonly possibleto per-formhistologicalstudiesevaluatingwholeorganismontheslide, requiringpriorknowledgetoidentifytargettissues.

The ecotoxicological studies with insects regarding Hymenoptera always refer to histological patterns under nor-mal conditions of the model organism as related in previous studies (Cruz et al., 2010; Cataeet al., 2014).But for Chirono-midaelarvae,thesestandardsarenot available.In studieswith Hymenoptera, various tissues (midgut, Malpighian tubules, fat body, brain)were sensitiveto various toxic substances, and in additiontothesetissuesotherpossibletargetsystemshavealso

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Fig.9.MicrographsofthecirculatorysystemandtheintegumentofimmatureChironomussancticaroli.(A)Longitudinalsectionoftheheart,notetheostiumregion(arrows); (B)longitudinalsectionoftheregionofthevesselwallwiththecellofthevessel(arrowhead);(C)longitudinalsectionshowingtheaorticvalves(*);(D)pericardialcells attachingtothewalloftheaorta;(E)longitudinalsectionoftheintegumentoftheheadcapsule,showingtheepitheliumandthelargeamountofexocuticle;(F)longitudinal sectionoftheintegumentofthebodyofthelarva,notethegreateramountofendocuticle.alu:aorticlumen;ao:aorta,ed:endocuticle;ep:epidermis,e.g.exocuticle;h: hemolymph,ht:heart;re:rectum.Stain:Harrishematoxylinandeosin.Scalebar=20␮m.

beendescribed in thepresent study(endocrineand circulatory system,salivarygland,integument) toalloweasyidentiﬁcation oftissuesandorgansthatmaychangeinfuturehistopathological analyzesoftheChironomidaelarvae.

Inahistopathologicalanalysis,itisconsideredofgreat impor-tancetostudythetypesofchangesthatcanoccurinthecellsof themodelanimal.Themidgutappearsasatargetorganduetoits roleinabsorptionoforallyacquiredxenobioticsubstances.In hon-eybeelarvae(Cruzetal.,2010),itwasobservedvacuolationofthe cytoplasm,dilationoftheintercellularspace,extrusionofcellular contentsandchromatincompactioninducedbyexposuretoboric acidandﬁpronil.Inworkersants(Sumidaetal.,2010),thesame changesmentionedabovewereobserved,inadditiontonarrowing thewidthoftheepithelium,andnuclearpyknosisinthepresence ofboricacid.

TheMalpighiantubulesactinmetaboliteexcretion,therefore alsoaresensitivetoxenobiotics.Workersbeeslarvaeandworkers antsshowedvacuolizationofthebasalcytoplasmofepithelialcells. Furthermore,chromatincondensation,nuclearpyknosis,irregular nucleimorphology,obstructedlumenandalterationofcellvolume

wereobservedinbeelarvae(Ferreiraetal.,2013;Rossietal.,2013a; Sumidaetal.,2010).Xenobioticscanchangetheenergymetabolism ofsomeorganisms(Serviaetal.,2006),andthefatbodyis respon-sibleforthisfunction,inadditiontoproducingtheenzymesthat participateinthebiotransformationofchemicals.Thatiswhyit maybeatargetorganforcontaminants.Inworkersbeeslarvae, itwasobservednuclearpyknosisintrophocytesexposedtoboric acidandchangesincellvolumeinoenocytesexposedtoparaquat (Cruzetal.,2010;Cousinetal.,2013).

Othertissuesunrelatedtoabsorption,metabolismand excre-tionofxenobioticsmayalsobetargetsforhistologicalchanges, suchasthebrain.Adultbeesexposedtoimidacloprid,therewere alterationsinpedunculatedbodycellsandopticlobe,suchas chro-matincondensation,changesincellvolumeandcelldeath(Rossi etal.,2013b).

Conclusions

Thisstudypresentsadescriptionofthehistologyofa biondica-torofthequalityofaquaticsedimentsintheabsenceofchemical

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contaminants.We described peculiaritiesof someorgans never detailedbeforetothegroup,whichwillbeusedasareferencefor ecotoxicologicalstudies;besidestheyassistintheunderstanding ofthetargetorganofsomexenobiotics.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest. Acknowledgements

WethanktheConselhoNacionaldeDesenvolvimentoCientíﬁco eTecnológico(CNPq,process305038/2009-5)andCamiladaCosta SenkivforEnglishreview.

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