ww w . r b e n t o m o l o g i a . c o m
REVISTA
BRASILEIRA
DE
Entomologia
AJournalonInsectDiversityandEvolution
Short
Communication
Clearing
and
dissecting
insects
for
internal
skeletal
morphological
research
with
particular
reference
to
bees
Diego
Sasso
Porto
a,∗,
Gabriel
A.R.
Melo
b,
Eduardo
A.B.
Almeida
aaLaboratóriodeBiologiaComparadaeAbelhas,DepartamentodeBiologia,FaculdadedeFilosofia,CiênciaseLetrasdeRibeirãoPreto,UniversidadedeSãoPaulo,RibeirãoPreto,SP,
Brazil
bLaboratóriodeBiologiaComparadadeHymenoptera,DepartamentodeZoologia,UniversidadeFederaldoParaná,Curitiba,PR,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received20July2015 Accepted4November2015 Availableonline1December2015 AssociateEditor:RodrigoB.Gonc¸alves Keywords: Apidae Apoidea Comparativemorphology Hymenoptera Internalmorphology
a
b
s
t
r
a
c
t
Adetailedprotocolforchemicalclearingofbeespecimensispresented.Dryspecimensaswellasthose
preservedinliquidmediacanbeclearedusingthisprotocol.Theprocedureconsistsofacombineduse
ofalkalinesolution(KOHorNaOH)andhydrogenperoxide(H2O2),followedbytheboilingofthecleared
specimensin60–70%EtOH.Clearingisparticularlyusefulforinternalskeletalmorphologicalresearch.
Thisprocedureallowsforefficientstudyofinternalprojectionsoftheexoskeleton(e.g.,apodemes,furcae,
phragmata,tentoria,internalridgesandsulci),butthisprocessmakesexternalfeaturesoftheintegument,
assomesuturesandsulci,readilyavailableforobservationaswell.Uponcompletionofthechemical
clearingprocessthespecimenscanbestoredinglycerin.Thisprocedurewasdevelopedandevaluated
forthepreparationofbeesandotherApoidea,butmodificationsforusewithotherinsecttaxashouldbe
straightforwardaftersomeexperimentationonvariationsoftimingofsteps,concentrationofsolutions,
temperatures,andthenecessityofagivenstep.Commentsonthelong-termstorage,morphological
examination,andphotodocumentationofclearedspecimensarealsoprovided.
©2015SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen
accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
The study of the morphology of different kinds of orga-nisms,especiallyanimals,certainlyisoneoftheeasiest,cheapest, andmostreadilyaccessiblewaysof obtaininglargeamountsof informationandunderstandingoftheplanet’sbiodiversity. Mor-phologicalcomparativestudieshavehadalonglife,asexemplified bytheclassictreatiseDePartibusAnimaliumbyAristotlemorethan 2300yearsago.InthecaseofEntomology,externalandinternal morphologicalstudieshavealwayshadgreatappealbecauseofthe enormousdiversityofinsects,butalsobecauseoflargeamountof variationobservableintheirexoskeleton.Despitetheemergence ofnewtechniquesforimaging complexinsectanatomy suchas CT-Scan,MRI,CLSM(e.g.,BeutelandFriedrich,2008;Deansetal., 2012), traditionallight optical microscopes remainasthemost commoninstrumentsofassessingmorphologicaldiversity. Speci-mendissectionandpreparationsformorphologicalresearchusing opticalmicroscopyisbenefitedbyclearingthecuticleusing alka-linesolutions,suchasNaOHorKOH.Clearingisparticularlyuseful wheninternalprojectionsoftheexoskeleton(e.g.,apodemes, fur-cae,phragmata,tentoria,internalridgesandsulci)areinvestigated, but this process makesexternal features ofthe integument,as
∗ Correspondingauthor.
E-mail:diegosporto@gmail.com(D.SassoPorto).
somesuturesandsulci,morereadilyavailableforobservation. Fur-thermore,clearedmaterialis appropriatefor linedrawingsand photography,inadditiontoremainavailableforposterior prepara-tionforSEMexecutionafterbeingcriticalpointeddriedifdesired
(Portoetal.,2015).
Althoughalkalinesolutions(e.g.,NaOHorKOH)havebeen com-monlyusedinentomologicalclearingproceduresbyresearchers workingwithvariousinsecttaxa,protocolscombiningtreatments withthesekindsofbasesandhydrogenperoxide(H2O2)arenot
usuallydone.Melo(1999)reportedthecombinedapplicationof analkalinecompound(KOH)andasolutionintendedtomakethe insectintegumentmoretranslucent(H2O2).Theclearingprocedure
presentedbyMelo(1999)containedthegeneralstepsbuta proto-colcomprisingdetailedapproximatetimesandalternativeroutes forthesestepsintoamorereadilyapplicableprotocolappearsto belacking.AmodifiedversionoftheMelo’s(1999)clearingmethod isprovidedinTable1.Ourprotocolwasdevelopedandevaluated forthepreparationofbees(andotherApoidea,asdonebyMelo
(1999)),butmodificationsforotherinsecttaxashouldbe
straight-forward aftersome experimentationon variations of timingof steps,concentrationofsolutions,temperatures,andthenecessity ofagivenstep.Theunderstandingoftherelationshipsbetween themainlineagesofbeeshasbeengreatlyimprovedduringthe XXcenturybymanymorphologicalworks,particularlythoseby
http://dx.doi.org/10.1016/j.rbe.2015.11.007
0085-5626/© 2015 Sociedade Brasileira de Entomologia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Table1
Protocolcontainingthesuggestedstepsforclearinganddissectingspecimensofbees(andotherrelativelylargeandwellsclerotizedhymenopteranrepresentatives)forthe studyofexternalandinternalskeletalmorphology(modifiedfromMelo,1999).
(1)Placethespecimenintoa10%solutionofKOH/NaOHtodissolvesofttissues.Thespecimenmayremainsoakedinsolutionfor10–45h,inroomtemperature.This stepoftheprocedurecanbespeedupbyheatingtheKOH/NaOHsolutionupto∼90–100◦C,preferablyinawaterbath,inwhichcasethetimeneededfortheinitial clearingcanbereducedto10–40min.ThenecessarytimeforheatedKOH/NaOHtobeeffectivedependsonvarioustraitsofthespecimen,suchasbodysize,color, thicknessoftheintegument,regionofthebody.
DrypinnedspecimensdonotneedtoberelaxedbeforebeingplacedintotheKOHsolution,althoughthismightbenecessarytosafelyremovethespecimenfrom thepin.Incasethespecimenispreservedinethanol(70–80%),boilitin100%EtOHforapproximately10min,thentransferittoheatedKOH/NaOHandproceedas indicatedinthestandardprotocol.Overall,nosignificantdifferencesbetweenethanolpreservedspecimensanddry-pinnedcanbeperceivedattheendofthe clearingprocess.
Delicatepartsofthespecimen,suchasmouthpartsandterminalia,maybedissected/disarticulatedatthisstagetopreventoverclearingthem.Additionally,the headandpropectus(i.e.,propleuronandprosternum),mesosomaandmetasomacanbeseparatedpriortothenextsteps.Thisallowsthefinetuneoftheclearing processfordistinctportionsofthebody,astheynormallyhavedifferentproperties(e.g.,cuticlethickness,colors,etc.)and,thus,willprobablydifferintheirideal timesinheatedsolution.
(2)Transferthespecimentoaroom-temperaturesolutionof3–5%H2O2,whereitshallremainforapproximately10–20min,whenitwillbeclearedenough.Once again,thenecessarytimefortheclearingtobeeffectivedependsonvarioustraitsofthespecimen,suchassize,color,integumentthickness,regionofthebody,etc. Alternatively,aheated(∼70–90◦C)solutionofH2O2maybeused,butthismustmedonewithcautionbecausetheclearingactivitymightbeoveraccelerated leadingtoareducedcontroloverthedegreeoftheoverallclearingprocess.Forproperclearing,itisimportanttoemphasizethatthespecimenneedstobe transferreddirectlyfromthealkalinesolutiontotheperoxidesolution.Donotwashitinwaterbetweenthetwosteps.
Incertaincases,treatmentwithKOH/NaOHwillbeeffectiveenoughsothatitwouldbeadvisabletoskiptheH2O2step(insuchcases,thechemicalactionof KOH/NaOHmustbestopped,andthiscanbeaccomplishedwithabathindilutedaceticacidorlacticacidforafewminutes,followedbywashinginpurewater. Goingtostep3directlyfrom1willalsowork).
(3)Transferthespecimenandallitspreviouslydisarticulatedparts(ifany)toa60–70%EtOHboilingsolution,leavingthemtherefor5–10min.Therewillbebubbles poppingoutofthebody,butthisisnormalandnecessarytoremovesmallairbubblesfromwithintheexoskeletonandremnantsofmaceratedsofttissuesaswell astointerruptthechemicaleffectsofKOH/NaOHandH2O2.
(4)Waitforthesolutiontocooldowntonaturallyreachroomtemperature.
(5)Transferthespecimentoaglycerol:60%EtOHmixture(1:1).
(6)Afterapproximately24h,removepartofthesupernatantEtOHwithapipetanddoubletheamountofglycerol.Ethanolandglyceroldonotmixperfectlywell becausetheypossessdistinctdensities.Aftersometime,itiseasilynoticeablethattherewillbemoreEtOHontheupperlayerofthissolution.
(7)Waitforthespecimentobecompletelyimmersedintheglycerol(thismayrequireafewdays),andthentransferthespecimentoapureglycerolsolution.
(5–7,alternative)Thetimerequiredforthesestepscanbesignificantlyshortenedusingaseriesofdehydratingalcoholicsolutions.Afterstep4,thedissectedpieces canbesoakedintoaseriesofEtOHsolutionswithincreasingconcentrations(70–100%)for20–30mineach.Fourbathsaresufficient(70%,80%,90%andthen100%) forasuccessfuldehydrationofthespecimensorspecimen-parts.Afterthat,storethepiecesintopureglycerol.
Roig-Alsina and Michener(1993) and Alexander and Michener
(1995),whichinvestigatedexternalandinternalskeletal
morphol-ogyofApoideainacomparativemanner.
TheprotocoladoptedbyMelo(1999)wasincurrentuseatthe EntomologyDivisionoftheUniversityofKansasNaturalHistory Museuminthe1990s.GARMrecallsnotlearningitfroma writ-tendocument,butmostlyfromtalkingtoCharlesMichenerand thelate ByronAlexander.Healsorecallslearning fromthelate SteveAshethestepofboilingspecimensindilutedethanolafter peroxideclearingtoremovetheairbubblesfromwithinthem.A searchinAshe’spublicationsatthattime(e.g.,Ashe,1992;Ahn
andAshe,1996)revealsthatuseofhydrogenperoxidetoclear
darkspecimensisindicated,butsubsequentboilinginethanolis notmentioned.Adetailedclearinganddissectingprotocolfor ale-ocharinestaphylinidbeetleswaslaterpublishedbyHanley and
Ashe(2003)inwhichthisstepismentionedanddiscussed.
Dif-ferentlyfromHanleyandAshe(2003),theprotocoldetailedinthe presentpaper focusesmostly onpreparing specimensthat will bekeptdisassembledpermanentlyinglycerininindividualwells ofplasticcultureplates,insteadofbeingmountedinpermanent slides,asitisdoneintheworkoftheformerauthors.
Protocol
Priortostartingthechemicalclearingprocessofaspecimen, itisadvisabletoremoveitswings.Thereisnoharmin leaving themattachedtothespecimen,especiallywhenitisexpectedthat astudyofitsmicroscopicstructurewillbecarriedout,buttheywill becompletelydeformedbytheproceduredescribedinTable1.It isrecommendedthatonepairispermanentlymountedinglass
slidesforstudyunderlightmicroscopyandtheotherpairisglued inapieceofpaperandpinnedwiththespecimenlabels,whichthen canbeproperlystoredforfurtherrecords.
Usefultechniquesforpreparation offinedissectingtoolsare givenbyHanleyand Ashe(2003).Thesepintoolsarealsoquite handfulwhendealingwiththewholestorageplates(seebelow) underdissectingmicroscopes.Theyallowproperpositioningofthe insectpartswithoutrequiringthattheexaminedpartbetakenout oftheplatewells.
Step1 (Table1)—Two alternativesarepossible at this step:
eitherthespecimencanbesoakedintoa10%KOH/NaOHsolutionin roomtemperatureovernight(Melo,1999:step1),orthissolution canbeheatedat90–100◦C.Theimmersionofspecimensin solu-tionbyaperiodof12–24h(generallydoneovernight),although guarantee more control over the clearing/softening process of the integument, showed inefficient results in many instances. On theone hand, for very small specimens (<3mm), this time proved tobe toolong and resulted into excessive softening of theintegument,makingthespecimensstructurallyveryweakto manipulationsduringdissections. On theotherhand,for larger specimens (>15mm),sclerites and articulations are kept struc-turallyintactoronlyslightlyaffected,evenaftermorethan24h inasolutioninroomtemperature.
Heatingacceleratesthereactionofthealkalinecompoundswith theintegument,membranes,andsofttissues,thereforefacilitating thebreakingofchemicalbondsbetweenthelinearchainsofchitin andtheproteinsthatareresponsiblefortherigidity/pigmentation ofexocuticle.Forspecimenswithbodysizebetween10and15mm (withmoderatelysclerotizedcuticle)heatingthe10%KOH/NaOH solution for about half an hour at 90–100◦C suffices in most
cases—theseconditionsalsocreateabalancebetweenthe struc-tural integrity ofthesclerites and theintention ofclearingthe cuticle.Smallerormorefragilespecimenswereclearedefficiently inca.15–20min;whereasverylargespecimens(>25mm)can gen-erallybesafelyheatedfor45–60minwithoutevidentdamage.
Step2(Table1)—Thesecondstepconsistsofsoakingthe
dissec-tedpiecesofinterestinto3–5%hydrogenperoxide(H2O2).Thisstep
isimportantbecauseitallowsadditionalclearingoftheintegument withouttheundesirableexcessivesofteningresultingfromthe con-tinuedactionofanalkalinesolution.TheuseofH2O2isindicatedfor
thosespecimenswithdarkintegumentand/orwhenthereis partic-ularinterestonthestudyofinternalprojectionsoftheexoskeleton. Extendingthetimethematerialissoakedintothehydrogen per-oxidecanimprovetheclearingofthecuticlemakingittranslucent andallowingtheobservationofinternalhardstructures(e.g., ten-torium,phragmata,furcae,apodemes,internalridgesandsulci). Thisprocessismoreefficientlyexecutedwithwarmhydrogen per-oxide.Resultscanalsobeobservedatlowertemperatures,butthe clearingwillbenoticeablyslower.
This can be the most challenging step of the protocol and requirescarefulstandardizationtopreventexcessiveclearing,as somespecimensorpartsofspecimenscanbemadealmost trans-parentafterthisstep. Asuccessfulclearingprocess isgenerally indicatedbyalightreddish/brownishcolorationoftheintegument (Fig.1),althoughthis canvarydependingonthespecimen.The
integumentcolor(i.e.,dark,pale-brown,yellowish,metallic),the overalldegreeofcuticlesclerotization,andthesizeofthespecimen werethemostimportantfactorsinfluencingtheresultsof clear-ingusingH2O2.Distinctregionsofthebodycanbedifferentially
cleared,whichoftentimesrequiresdisarticulationofhead, meso-soma,metasoma,andofcertainotherstructures(e.g.,mouthparts, legs,propleura,prosternum,malegenitalia,andstingapparatus).
Mouthparts and genitaliashould beseparatedfirst fromthe remainderof thebody and clearedindividually. Timing for the clearing for thesestructures variedfrom 30 to150s. The time neededfortheH2O2stagewasconsiderablyshorterforthehead
capsuleplusthepropectus(1–5min),anintermediateamountof timeisoptimalforthemetasoma(2–7min),andlongesttimesfor themesosoma(3–10min).Forsomestructures,asthemesosoma ofdark,heavilysclerotizedlargespecimens,thetimespentinthe warmH2O2solutioncanbeaslongas15–20min.
Step3 (Table1)—Boilingthespecimens in 60–70%EtOHfor
5–10min(depending onthesize ofthe specimenorstructure) allows theremovalof air bubblesconfinedinto thesclerotized structuresorwithinremnantsofpartiallymaceratedsofttissues. Thesecondpurposeofthisstepistheneutralizationoftheactions ofKOH/NaOHandH2O2.SpecimensboiledatEtOHforlessthan
3mintendtohaveresidualclearingresultantfromtheexcessive actionofalkalinecompounds(KOHorNaOH)afterthecompletion oftheentireprocess.
HEAD CAPSULE
PROSTERNUM & PROPLEURA
MESOFURCA & METAFURCA
MESONOTUM, METANOTUM, METAPOSTNOTUM & MESOPHRAGMA
GENERAL BODY METASOMA
MESOSOMA HEAD
CAPSULE
External: epistomal sulcus, hypostoma, occiput/postocciput
External: basisternum, furcasternum, ventral/lateral propleural areas
Internal: tentorium, epistomal ridge, mandibular apodemes
Internal: profurca, propleural arm, cervical apodeme
External: pronotum, mesoscutum, mesoscutellum, metanotum, propodeum, mesepisternum, metepisternum, episternal sulcus,
scrobe
Internal: internal ridges of mesepisternum/metepisternum, paracoxal ridge, coxal articulations
Internal: horizontal plate, mesofurcal/ metafurcal arms, mesofurcal bridge, dorsal
metafurcal lamella
Internal: prophragma, mesophragmal arched ridges, mesophragmal longitudinal ridge
Fig.1.SpecimensofScaptotrigonadepilis(Moure,1942)clearedusingtheprotocolpresentedinTable1:lateralhabitus(centralcolumn,top);dorsalhabitus(centralcolumn, middle);boxesshowdifferentportionsofthebodydisarticulated,alongwithexamplesofexternal/internalstructuresmadeavailableforrefinedmorphologicalinspections aftertheclearingprocedure.
Steps4–7(Table1)—Beforethefinalstorageinglycerol,it is importanttowaitthesolutioncooldown(step4)toavoidthermal shock,whichcandamagesomefragilestructures(e.g.,mouthparts, portionsofthegenitalia,andstingapparatus).Thetransferring pro-cedure(steps5–7)topureglycerolshouldbemadeinastepwise manner,sinceethanolandglyceroldonotmixperfectlywelldue totheirdistinctdensities.Sometimeisrequiredtothe60%EtOH inthemixturegoestotheupperphaseandcanberemovedwith apipet.Theadditionofglycerolcanbemadeonceortwice,until almostallthe60%EtOHwasremoved,beforepermanentstorage intopureglycerol.Analternativetothisoriginalprocedure out-linedbyMelo(1999:steps4–7)istouseaseriesofdehydrating alcoholicsolutionsinincreasingconcentrations(e.g.,70%,80%,90%, 100%),soakingthestructuresabout20minineachbath,thenfinally storingthenintopureglycerin.Thisshortensthetimerequiredfor thestoringprocessinglycerin,insteadofusingahydratedethanol mixtureandslowadditionofglycerin.
Specimenstorage,manipulation,andphotodocumentation
Long-termstorageoftheclearedanddissectedspecimenscan beeasilyaccomplishedbykeeping eachonein individualwells ofacryliccellcultureplates (alsotermed‘tissuecultureplates’) (Fig.2AandB).Permanentstorageincultureplatesimposes cer-tainrestrictionsfortransportationofthespecimens.Whenover shortdistances,theplatescanbeeasilyhandcarriedaslongas theyarenotstronglytiltedorjoltedintheprocess.Undernormal conditions,theycanalsobetransportedbycarifnecessary.Long distancetransportation,suchasinairplanes,shouldbepreceded bysealingofthewellswithdiscsofhigh-densityplasticfoam(e.g., polyethyleneorpolyurethane).Removalofthefoamdiscsshouldbe donecarefullytomakesurethatnodissectedpartsaretakenaway adheredtoglycerindropsthatmighthavespilledonthediscs.
Specimen study under both reflected and transmitted light canalsobereadilycarriedoutdirectlyontheplatesduetotheir
transparentwalls(Fig.2C).Cultureplatesareproducedwithwells ofdistinctdiametersandtheyshouldbechosenaccordingtothe sizeof thespecimens. However, theminimumdiameter ofthe individualwellsshouldnotbelessthan15mm,evenwhenusedto storesmallspecimens.Smallerdiametersmakeitdifficulttomove thedissectedspecimenpartswhilesimultaneouslyobservingthem underadissectingmicroscope.Insomeinstances,whenthefocal distanceofthemicroscopeis veryshort,theseplatescanmake manipulationof thespecimensdifficult.In this case,theuseof manipulativetools–asawoodenstickwithapinattachedtoitstip orsomethingsimilar–canhelptoovercometheproblemofashort focaldistance.Alternatively,thespecimenscanbetransferredto ceramicor glasswell-plates,buttheformer doesnot allowthe useoftransmittedlightand thelatterisoftenexpensive.Inthe lattercase,asmallPetridishcanbeamoreeconomicalalternative. Examinationwithconcomitantmanipulationofseveralspecimens fromasingleplateorfromdifferentplatesrequirescareful atten-tiontoavoidthatobservedpartsarenotmixedupbetweenwells. Ifnot strictly observed,specimenparts canremainattachedto dropletsofglycerinatthetipsofthemanipulationtools.
The translucent property of many liquid media, particularly glycerol,makescleared specimens and theirpartsavailable for opticalinspections, linedrawings (withaidof a cameralucida), orphotodocumentation.Duringmorphologicalinvestigationand whenusingacameralucidaforlinedrawings,thecontrastcanbe greatlyenhancedbyalteringthetechniqueoflighttransmission, varyingbetweenbrightfieldanddarkfield.
Specimens can be photographed immersed in pure glycerol insidethewellsofthecultureplate,butthisprocedureisnot rec-ommended. Although theoptical propertiesof the glycerolare generally good enough for photography due to its translucent natureanditsflatsurfacewithoutundulations(whichmakesno reflectionstothelight),itsdensityisnotashighassomeother colloidalmedia,suchaswater-solublecommercialpersonal lubri-catingjellyora1:1mixtureofglycerolandgelatin.Thechosen
Fig.2. Accessoriesforspecimenstorage,studyandphotodocumentation:(A)acryliccellcultureplatewithsixroundwellsusedforstoringspecimensinglycerolorotherliquid mediaaftertheclearingprocedure;(B)plate-coverwithanexampleofalabelingsystemforspecimensstoredinthesixwells;(C)close-upviewofonewell(diameter=35mm) containingaclearedanddisarticulatedfemalespecimenofCentrisanalisLepeletier,1841;(D)modifiedglassslideusedforaccommodatingspecimens,whichcanbefilled withhighdensitycolloidalmediumduringphotodocumentation.
mediumcanbeputintoanexcavatedglassslideformicroscopy withthespecimenorstructureofinterestproperlypositioned.A smalldropletofglycerolshallbeaddedontothetopofthe water-solublelubricanttosmoothentheundulatedsurfaceandreducethe lightreflectiononitssurface.Acoverslipshouldthenbeplaced abovetheimmersed specimen/structuretohaltmovement and preventdryingofthelubricating jelly.For largerspecimens,an alternativetotheusualexcavatedslidesistouseaglassoracrylic ring(ca.3–10mm)gluedwithsiliconeontoaregularglassslide formicroscopy,asdepictedinFig2D.Ifthespecimenofinterestis properlyhaltedwithintheliquidmedium,serialimagesfor poste-riormontage(i.e.,stacking)willbepossibleforalmostanystructure usingappropriateequipment.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
WearegratefultothankMariaIsabelP.Balbiforvaluable sug-gestions onthedehydratingand advertising aboutthe thermal shockduringtheproceduresoftheprotocol,andtoBryanN. Dan-forthforadviceonpreparingtheslidesshowninFig.2D.Weare
thankfultotwoanonymousreviewerswhoprovideduseful sug-gestionstothemanuscript.Thisprojectwaspartlysupportedby grant#2011/09477-9,SãoPauloResearchFoundation(FAPESP)to E.A.B.Almeida,andbythefellowshipsnumbers2012/22261-8and 2014/10090-0toD.S.PortoalsobyFAPESP.
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