REVISTA
BRASILEIRA
DE
Entomologia
AJournalonInsectDiversityandEvolutionw w w . r b e n t o m o l o g i a . c o m
Medical
and
Veterinary
Entomology
Intra-puparial
development
of
the
Cochliomyia
macellaria
and
Lucilia
cuprina
(Diptera,
Calliphoridae)
Karine
Brenda
Barros-Cordeiro
a,b,∗,
José
Roberto
Pujol-Luz
a,
Khesller
Patricia
Olazia
Name
c,
Sônia
Nair
Báo
baUniversidadedeBrasília,InstitutodeCiênciasBiológicas,DepartamentodeZoologia,Brasília,DF,Brazil bUniversidadedeBrasília,InstitutodeCiênciasBiológicas,DepartamentodeBiologiaCelular,Brasília,DF,Brazil cUniversidadePaulista,Coordenac¸ãodeCiênciasBiológicas,InstitutodeCiênciasdaSaúde,Brasília,DF,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received25February2016 Accepted23June2016 Availableonline18July2016 AssociateEditor:AndrzejGrzywacz Keywords: Insectanatomy Insectmetamorphosis Morphology Ontogeny Taxonomy
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b
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Theintra-puparialdevelopmentoftheblowfliesCochliomyiamacellaria(n=310)andLuciliacuprina (n=470),wasstudiedundercontrolledconditionsinlaboratory.The3rdinstarlarvaewerereareduntil theystoppedfeeding,andthepre-pupaewereseparatedaccordingtothesizeinlarvallengthanddegree ofpigmentationandofthecuticle.Weobserveasetoffivecontinuouseventsorphases:(1)pupariation, (2)larva-pupaapolysis,(3)cryptocephalicpupa,(4)phanerocephalicpupaand(5)pharateadult.The totaltimeoftheintra-puparialdevelopment,larva-pupaapolysistopharateadult,lastedfor120h(5 days)toC.macellariaand210h(8.75days)toL.cuprina.
©2016SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Themajorityofstudiesonintra-pupaldevelopmentinDiptera havefocusedMuscoideaandOestroidea,especiallyCalliphoridae andOestridae(Cepeda-PalaciosandScholl, 2000;Pujol-Luz and Barros-Cordeiro,2012;Richardsetal.,2012;Defilippoetal.,2013; KarabeyandSert,2014;Proenc¸aetal.,2014;Maetal.,2015),as wellasonanorthorraphousBrachyceraStratiomyoideaHermetia illucens(Linnaeus,1758)(Barros-Cordeiroetal.,2014).The infor-mationonthisphaseofpost-embryonarydevelopmentisusefulin variousbranchesofentomology,especiallyinthecontrolofpests andendemicdiseases,controlofparasiticdiseases,inforensic ento-mologyandstudiesofontogeny.
Studiesaboutpost-embryonarydevelopmentoftheoestroids dipterous may help with pest control of Calliphoridae, which includevectorspeciesofpathogenicmicroorganismsorthatcause myiasis(Zumpt,1965)suchasCochliomyiamacellaria(Fabricius, 1775)and Luciliacuprina(Wiedemann,1830). C.macellariait is oneofthespeciesthatcausesecondarymyiasis,alesioncaused byhistophagouslarvaethataggravatesapre-establishedinfection.
∗ Correspondingauthor.
E-mail:karine.brenda22@gmail.com(K.B.Barros-Cordeiro).
Theconditionaffects humansas wellasotheranimals,making itofparticularmedicalimportance(Greenberg,1973;Guimarães andPapavero,1999;Marquezetal.,2007).Thisspeciesisalsoa potentialvectorofvariousenteropathogensthatcausehuman dis-eases,andisrecordedasavectorofDermatobiahominis(Linnaeus, 1781),abotflyresponsibleforcutaneousmyiasis(Guimarãesand Papavero,1999).L.cuprinacausesprimarymyiasisinsheepin Aus-traliaandNewZealand,beingresponsibleforthelossofmillions ofdollarsannuallyinthewoolandmeatindustries(Sackettetal., 2006;Wall,2012).InBrazil,thespeciesisassociatedtosecondary myiasisinsheep(Moreira-LimaandMoya-Borja,1997).Recordsof thisspeciescausingmyiasisinhumansandotheranimalsare com-monasreportedbyFosteretal.(1985)andConchaetal.(2011).C. macellariaandL.cuprinaarealsopresentinthedecompositionof animalcarcasses(Wolffetal.,2001;Biavatietal.,2010).
The purposeof this study is to present thedescription and comparisonofintra-pupaldevelopmentaltimeinCalliphoridaeof medical-sanitaryandforensicimportanceinBrazil:thesecondary screwwormC.macellariaandtheblowflyL.cuprina.
Materialandmethods
WecollectedtheadultsofC.macellariaandL.cuprinainurban areas in the neighborhoods of Universidade de Brasília – UnB,
http://dx.doi.org/10.1016/j.rbe.2016.06.009
0085-5626/©2016SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Table1
DevelopmenttimeofCochliomyiamacellariaandLuciliacuprina,minimumhours(h),foreachstageofdevelopmentat23±1◦C.
Species Event Time(h) Mean±S.D. Range n
Cochliomyiamacellaria Larva-pupaapolysis 6 8.45±6.75 (b00–27) 55
Cryptocephalicpupa 6 12.6±10.37 (06–42) 10
Phanerocephalicpupa 3 28.2±25 (12–78) 5a
Pharateadult(ce)
Transparent 15 28.57±7.61 (15–45) 65
Yellowish 48 54.85±15.09 (30–46) 85
Pinkish 12 83.05±4.02 (78–90) 19
Reddish 30 107.32±10.72 (90–120) 44
Luciliacuprina Larva-pupaapolysis 6 8.6±8.4 (b00–36) 45
Cryptocephalicpupa 9 15.5±10.4 (06–39) 25
Phanerocephalicpupa 3 32.6±11 (15–54) 38
Pharateadult(ce)
Transparent 6 33.8±14.7 (18–72) 24
Yellowish 132 98.9±44 (24–192) 237
Pinkish 18 178.2±16 (156–204) 30
Reddish 36 196.4±11.6 (174–210) 41
Time(h),referstotheminimumdurationofeachevent;S.D.,standarddeviation;n,numberofsamples;ce,compoundeyes.
aOftotal10samples,wereonly5infanerocephalicphasetheotherand5sampleswereapolysisprocessorcryptocephalicphase. bBeginningofthestage.
usingamodifiedVanSomeren-Rydontrap.Wesetuptwocolonies of150couples,oneforeachspecies, intwocagesinsidea BOD incubator chamber (23.0±1.0◦C, 60±10% RH, 12:12 L:D). We reared the colonies ona meat substrate using the methods of
Barros-CordeiroandPujol-Luz(2010).Oncelarvaemigratedfrom themeattothevermiculatesubstrate,theyreducedinsize,changed color,andpupated.Wedefinedthestartofpupationasthemoment whenthelarvaechangedcolortobrownandadoptedabarrelshape.
Assoonaspupationstarted,weinitiatedcollectionofsamplesof10 pupaeatatimeaccordingtothefollowingschedule:time0;every 3huntil48hofdevelopment;every6huntiladultemergence.The collectedpupaewerefixedinCarnoysolutionfor48h,then trans-ferredto5%formicacidforanother48h,andthentransferredto andstoredin70%ethanol.
Thespecimens were dissectedand photographed withLeica M205-C®stereomicroscope.Atotalof310pupaeofC.macellaria
Table2
Comparisonbetweentheintervals,minimumduration,foreachintra-pupaldevelopmenteventandtotaltimeofdevelopmentofspeciesofCalliphoridae,inhoursandat differenttemperatures.
Event Cochliomyiamacellaria Luciliacuprina Luciliacuprina
23.0±1.0◦C 23.0±1.0◦C 30◦C Larva-pupaapolysis 6 6 4 Cryptocephalicpupa 6 9 4 Phanerocephalicpupa 3 3 8 Pharateadult 105 192 128 Totaltime 120 210 144
References Thisstudy Thisstudy BarrittandBirt(1971)
Totaltimeofdevelopment(egg-adultemergence) 23.0±1.0◦C 23.0±1.0◦C 22.9±1.1–29.5±1.0◦C
241 354 336
References Thisstudy Thisstudy Greenberg&Szyska(1984)
Event Luciliasericata Phormiaregina Calliphoravicina
25◦C 22◦C 23.0±0.6◦C Larva-pupaapolysis 8 9 48 Cryptocephalicpupa 4 14.5 84 Phanerocephalicpupa 12 18.5 67 Pharateadult 148 107 209 Totaltime 172 149 408
References KarabeyandSert(2014) Greenberg(1991) Defilippoetal.(2013)
Totaltimeofdevelopment(egg-adultemergence) 25◦C 22◦C 22◦C
516 350.4 465.6
References Marchenko(2001) GreenbergandKunich(2002) GreenbergandKunich(2002)
Event Chrysomyaalbiceps Chrysomyaputoria Chrysomyarufifacies
26.0±1.0◦C 25–27◦C 24◦C Larva-pupaapolysis 3 18 16 Cryptocephalicpupa 3 6 8 Phanerocephalicpupa 3 24 16 Pharateadult 81 68 96 Totaltime 90 116,00 136
References Pujol-LuzandBarros-Cordeiro(2012) Proenc¸aetal.(2014) Maetal.(2015) Totaltimeofdevelopment(egg-adultemergence) 26.0±1.0◦C 22.9±1.1–29.5±1.0◦C 15.6–35◦C
264 252 190–598
and470pupaeofL.cuprinawereexaminedanddissected.The ter-minologyadoptedtodescribethemorphologyofthepuparium andtheintra-puparialdevelopmentphasesfollowsFraenkeland Bhaskaran(1973),Cepeda-PalaciosandScholl(2000)and Barros-Cordeiroetal.(2014).
Results
We observedfive chronologicalevents, inthe intra-puparial developmentof C. macellaria and L. cuprina. The first phase is knownaspupariationandoccursafterthelarvaleavesthediet sub-strate.Graduallyitbeginstoburyintotheadjacentsubstrateandit mobilitydecreases.Duringthisprocesstherearearetractionand invaginationofthesegmentsofthebody.Thecuticleofthelarvais progressivelysclerotizedandpigmented,usuallyfromdarkyellow
todarkbrown.Theposteriorspiraclecollapsesandsinksontoanal tubercle,andthelarvaassumestheformofabarrel.Thepuparium ofC.macellariapresentedameanlengthof8.87±0.47mm,which isabout40%lessthanthemeanlengthofthethird-instarlarva,and ameanweightof48.60±5.20mg.Theminimumdurationofthis processwas12h(Table1).ThepupariumofL.cuprinapresenteda meanlengthof7.1±0.2mm,whichisabout38%lessthanthemean lengthofthethird-instarlarva,andameanweightof28.4±1.7mg. Thisphaselastedatleast46h(Tables1and2).
Inthesecondevent,orlarval-pupalapolysis,theseparationof thecuticlesoccursinitiallyinthemedianportionofthepuparium movingtotheanteriorpartofthebody,andfromtheventraltothe dorsalregionofthebody.Theextremitiesinitiallybecomestuck inthepuparium,theanteriorregionbythemaxillaandmandible, andtheposteriorpartbythespiraclesandintestine.Theprocess
A
B
C
D
E
F
G
H
asp
Fig.1.Morphologicalsequenceoftheintra-puparialdevelopmentofCochliomyiamacellaria.(A)Ventralviewand(B)dorsalview,cryptocephalicpupa;(C)ventralview, phanerocephalicpupa;(D,E)dorsaland(F)ventralview,pharateadult;(G)Pharateadultindorsalview,sequenceofbristlepigmentationandbodysutures;(H)Imagowith formationoftheptilinealsacinventralview.Arrow,anteriorspiracle(asp).Scale:1mm.
oflarval-pupalapolysisoccurslikewiseinbothspecies.The mini-mumtimetocompletetheprocessofapolysiswassixhoursinboth species(Tables1and2).
Thethirdeventisthecryptocephalicpupa.Itstartsafter com-pletionofapolysis,whenthepupariumbecomesmorepigmented andsclerotized.Themandibleandmaxillaaredetachedfromthe restofthecephalopharyngealskeletonandremainstucktothe puparium.Thepupaatthismomenthasanundefinedformandis wrappedinafinemembrane(Figs.1–3).Thiseventlastedsixhours inC.macellariaandninehoursinL.cuprina(Tables1and2).
Thefourtheventis thephanerocephalicpupa.It containsto theprocessof evaginationofthecephalic capsuleand the tho-racicappendices.Wecandistinguishthehead,thethoraxandthe abdomenoftheimago.Thepairofprothoracicspiraclesisformed byalateralprojection,similartoacylindricaltube,whichconnects tothepuparium(Figs.1–3).Thisprocesslastedanaverageofthree hoursforbothspecies(Tables1and2).
Thefiftheventisthepharateadult,whentothematurationof theadultinsecthappensinfourstepsdefinedbychangesinthe colorofthepigmentationofthecompoundeyes(Tables1and2). Withinthesesteps,otherstructuresofthebodygraduallybecame pigmentedandsclerotizedinbothspecies.
InC.macellaria(Figs.1and2)weobservedinthesequential steps:(i)transparenteyes,head,thoraxandabdomendefined,legs and wingsnotmembranous.Thiseventlastedatleast15h;(ii) yellowisheyes,suturesofthethoraxandabdomendefined, visi-bleterminalia,startofpigmentationofhairsandbristles.Inthis step,weobservedfiliformmaxillarypalps,andthestartofthe for-mationofthethreelongitudinalvitaeinthedorsalregionofthe thorax.Thisperiodlastedatleast48h;(iii)pinkisheyes,greater pigmentationofhairs,bristles,veinsofwingsandlegs;proboscis andantennaeclear.Thissteplastedatleast12h;(iv)reddisheyes, bodycompletelyformed,antennae,palpsandocelliwelldefined; wingsmembranousandveinsblackened;longitudinalstripsinthe dorsalregionofthethoraxstronglymarked;externalgenitalia vis-ible;scleritesdefinedanddelimited;ptilinealsacisformed.This periodlasted30h.
InL.cuprina(Figs.3and4)weobservedinthesequentialsteps: (i)transparenteyes,head,thorax,abdomenandlegsdefined,wings notmembranous.Thisperiodlastedatleastsixhours;(ii) yellow-isheyes,suturesofthethoraxandabdomendefined,theterminalia arevisible,startofpigmentationofsetaeandbristles.Thisperiod lasted at least 132h;(iii) pinkish eyes, strong pigmentationof setae,bristles,veinsof wingsand legs;proboscisandantennae
A
B
ce ceC
D
ce ceFig.2.ThepharateadultofCochliomyiamacellaria,accordingtothecolorofthecompoundeyes.(A)Transparenteyes;(B)yellowisheyes;(C)pinkisheyes;(D)reddisheyes. ce,compoundeyes.Scale:(A)0.5mm;(B–D)1mm.
Fig.3.Morphologicalsequenceoftheintra-puparialdevelopmentofLuciliacuprina.(A)ventralviewofthecryptocephalicpupa;(B,C)ventralviewofthephanerocephalic pupa;(D)ventralviewofthepharateadultand(E)dorsalview,anteriorspiracleofthepupa(arrow);(F)ventralviewofthepharateadult,sequenceofpigmentationofthe hairs,bristlesandbodystructures;(G)formedadultandformationoftheptilinealsacinthedorsalview,anteriorspiracleofthepupa(arrow)and(H)ventralview.Scale: 1mm.
translucent; thethree acrosticalbristles visibleand highly pig-mented.Thisperiodlasted atleast18h;(iv)reddisheyes,body completelyformed,antennae,palpsandocelliwelldefined;wings andveinsblackened;externalgenitaliavisible;bristlesofthe5th sternitestronglypigmented;scleritesdefinedanddelimited; pti-linealsacisformed.Thisperiodlasted36h.
Discussion
Intra-puparial development in C. macellaria and L. cuprina (Table 1) are similarto those observed for otherCyclorrhapha viz.Fraenkel and Bhaskaran (1973): Musca domestica Linnaeus, 1758;andSarcophagabullata(Parker,1916);Cepeda-Palaciosand Scholl(2000): Oestrus ovisLinnaeus,1758.Mostof the similar-itiesare restricted tothe sequence of thechronologicalevents
(larva-pupaapolysis;cryptocephalicpupa;phanerocephalicpupa; pharateadult)thatoccursintra-pupariallyasalreadydescribedfor otherspeciesofCyclorrhapha(Table2)(FraenkelandBhaskaran, 1973;Pujol-LuzandBarros-Cordeiro,2012).
Extrinsic factors, e.g. temperature and humidity, affect the developmental timing, as already suggested elsewhere (e.g.
Denlingerand ˇZdárek,1994;Cepeda-PalaciosandScholl,2000).C. macellariaandL.cuprinamayspendabout50%ofthetotaltimeof itsdevelopment(eggtoadult)inthepupalstage,asotherblowflies do(Table2).Thepharateadultphase(Table1)representsmore than60%of thetotaltime intra-puparial.However,itis among thepharateadultpinkisheyesandpharateadultreddisheyesthat someuniquemorphologicalchangestakeplace.InL.cuprinathe hardlypigmentationofthethoracicsetaeandtheappearanceof adensepubescenceof5thabdominaltergiteabdominaland,inC.
A
ce ce ce ceB
C
D
Fig.4.ThepharateadultofLuciliacuprina,accordingtothecolorofthecompoundeyes.(A)Transparenteyes;(B)yellowisheyes;(C)pinkisheyes;(D)reddisheyes.ce, compoundeyes.Scale:(A)0.5mm;(B–D)1mm.
macellariaappearthedistinctivethoracicpigmentation,witha con-spicuousvitaeandadensewhitepollinosityinabdominalventral surface.
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest. Acknowledgments
This research was developed with grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq, Fundac¸ãodeApoioàPesquisadoDistritoFederal–FAP-DF, Finan-ciadora deEstudos eProjetos –FINEP. We arealso gratefulto ÉricaS.Harterreiten-Sousa,AnaCarolinaFrancoPereira,Caroline DemoandAndréGardelinoSavino,forhelpindifferentpartsof thisresearch.ProfessorR.B.Cavalcantireadingthemanuscriptand makinghelpfulcommentsandsuggestions.
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