REVISTA
BRASILEIRA
DE
Entomologia
AJournalonInsectDiversityandEvolution w w w . r b e n t o m o l o g i a . c o mBiology,
Ecology
and
Diversity
Male
and
female
association
in
Trichomyia
Haliday
in
Curtis,
1839
using
a
molecular
approach
(Diptera,
Psychodidae,
Trichomyiinae),
and
description
of
new
species
from
Brazil
Maíra
Xavier
Araújo
a,∗,
Marcos
Aragão
a,
Danilo
Cordeiro
b,
Freddy
Bravo
a,
Claudio
José
Barros
de
Carvalho
c,
Sergio
R.
Andena
aaUniversidadeEstadualdeFeiradeSantana,DepartamentodeCiênciasBiológicas,FeiradeSantana,BA,Brazil
bInstitutoNacionaldePesquisadaAmazônia,Coordenac¸ãodeBiodiversidade,Manaus,AM,Brazil
cUniversidadeFederaldoParaná,DepartamentodeZoologia,ProgramadePós-graduac¸ãoemEntomologia,Curitiba,PR,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received28May2018
Accepted23August2018
Availableonline5September2018
AssociateEditor:AndrzejGrzywacz
Keywords: DNA-barcoding COI NeotropicalRegion Psychodid Trichomyiinae
a
b
s
t
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AnewspeciesofTrichomyiafromthestateofBahia,Brazil,isdescribedandillustrated,andmaleand femaleareassociatedusingDNAbarcoding.Additionally,fragmentsoftheCOIoftwootherspecies, TrichomyiacerdosaAraújo&Bravo,2016andTrichomyiaituberensisAraújo&Bravo,2016,andthefemales oftwounidentifiedspecies,aresequenced.
©2018SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
TaxonomyofTrichomyiaHalidayinCurtisismainlybasedon males.Duckhouse(1978),forexample,described30species,only two of which included females, and Araújo and Bravo (2016)
describedallof theforty-fourTrichomyiaspeciesbasedonly on males.Itispossiblethattheabsenceoffemalesintaxonomic treat-mentsisduetothefactthattheyarenotattractedtothesamebaits thatattractmales(Duckhouse,1978:202);alternatively,the mor-phologicalassociationbetweenmalesandfemalesinthisgenusis difficultwhenseveralspeciesarecollectedfromthesamelocality. MalesofninespeciesofTrichomyiaareunknown:T.barretoi Barreto,T.coutinhoi(Barretto),T.squamosa(Enderlein),T.eatoni Satchell,T.travassosi(Barretto),T.vazi(Barretto)andT.wasmanni (Holmgren)fromtheNeotropicalRegionandT.batuQuatefromthe OrientalRegion.
The DNA barcoding technique (Hebert et al.,2003), i.e.,the analysisofashortregionofthemitochondrialcytochromec oxi-dasegenesubunitI(COI)(<650pb),hasbeenusedwithrelative successin animalstodifferentiate species,including inDiptera
∗ Correspondingauthor.
E-mail:mah.biology@gmail.com(M.X.Araújo).
(Ekremetal.,2010;Kurina etal.,2011).Thetechniquehasalso beenusedextensivelytoassociatelifestagessuchasmalesand females(Willassen,2005;Zhangetal.,2013)or immaturesand adults (Contreras-Gutierrez et al., 2013; García-Robledo et al., 2013;Viveroetal.,2017).InPsychodidae,DNAbarcodinghasbeen usedextensivelyforspeciesdifferentiationinPhlebotominae(e.g.
Kumaretal.,2012;Gutiérrezetal.,2014;Nzeluetal.,2015;Pinto etal.,2015),butalsoforothersubfamiliesincludingPsychodinae (KvifteandAndersen,2012;KvifteandBoumans,2014;Kvifteand Menzel,2016),Sycoracinae(Jeˇzeketal.,2015)andBruchomyiinae (Polseelaetal.,2018).Inthispaper,sexassociationbyDNA barcod-ingisusedforthefirsttimeinTrichomyia.Additionally,maleand femaleofanewspeciesfromBrazilaredescribed.
Materialandmethods
ThespecimensstudiedaredepositedatColec¸ãoEntomológica ProfessorJohannBeckerdoMuseudeZoologiadaUniversidade EstadualdeFeiradeSantana,FeiradeSantana,Brazil(MZFS).The specimensforDNAextractionwerecollectedwithMalaiseandlight trapsbetween2012/2013fromReservaEcológicadaMichelin,state ofBahia,Brazil(13◦5016.0S/39◦1428.9W;139m).
Thespecimenswerecollectedin70%ethanol,transferredand storedin100%ethanol,thenpackedinafreezerat−20◦C.Thehead, https://doi.org/10.1016/j.rbe.2018.08.004
0085-5626/©2018SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://
wingandgenitaliaofeachspecimenstudiedwereseparatedand mountedonpermanentslideswithCanadabalsamaftera diapha-nizationprocesswithpotassiumhydroxide(10%KOH).Thethorax and abdomenof thestudied specimens were usedin the DNA extraction.AllvouchersaredepositedattheMZFS.
Terminology
ThemorphologicalterminologyisbasedonCummingandWood (2009),exceptfortheantenna(Ibá ˜nez-Bernal,2004).Theposterior projectionofgonocoxiteisnamedhereas‘armofgonocoxite’. Moleculartechniques
ThesequenceswereobtainedintheLaboratoryofMolecular SystematicsofPlants(Lamol)oftheUniversidadeEstadualdeFeira deSantana.TheextractionwasperformedwiththeDNeasyBlood& TissueKit(Qiagen,Valencia,USA)followingtheprotocolprovided bythemanufacturerwiththefollowingmodifications:formore concentratedDNA,elutionwasperformedintwosuccessivesteps of50leachwithBufferAE.
Apartialsequence ofthemitochondrial cytochromeoxidase gene subunit I (COI) was amplified and sequenced with the primerpairs listedinTable1.The DNAprimersLCO/HCOwere usedtoamplifysomesamples.Whentheprimerpairmentioned abovefailed,asmallerfragment,obtainedwiththeprimersMtD6 and MtD9, wasamplified instead (see Table 1). All primers at 10nmol/l.
Asolutionforpolymerasechainreaction(PCR)wasprepared withthefollowingconcentrations:0.7lMilliQwater;2lof addi-tive;0.15l ofeachprimer and5l ofTop TaqMasterMix kit (Qiagen)foreach2lofconcentratedDNA.Subsequently,thePCR wasperformedwith37cyclesofthefollowingsteps:aninitiation of94◦Cfor3min,denaturationat94◦Cfor30s,annealingat48◦C for40s,extensionat72◦Cfor40sandafinalextensionof72◦Cfor 5min.
Table1
PrimerpairsusedfortheamplificationofCOIgenefragments.
Primer Sequence(5→3) References
LCO1490 GGTCAACAAATCATAAAGATATTGG Folmeretal.(1994)
HCO2198 TAAACTTCAGGGTGACCAAAAAATCA Folmeretal.(1994)
MtD6 GGAGGATTTGGAAATTGATTAGTTCC Simonetal.(1994)
MtD9 CCCGGTAAAATTAAAATATAAACTTC Simonetal.(1994)
Theresultofthisamplificationwasanalyzedona1.0%agarose gel,stainedwithethidiumbromideandvisualizedonaUV transil-luminator.Thestrongbandsweremeasuredasanindirectmeasure oftheamountofDNA,whichwasconfirmedbymeasurementin Nanodrop®using1lofthePCRreaction.Accordingtotheseresults thesampleswereconsideredgoodforthesequencingreactionand subjectedtoaPEG(polyethyleneglycol)cleaning.
Themixforthepre-reactionsofsequencingweremadewith 10l (inboth directions, forwardand reverse)using 0.75l of BigDyeTerminatorv3.1CycleSequencingKit(AppliedBiosystems), 1.75lofbufferforsequencing(SaveMoney5×)and1.0lofthe 5pmol/lprimer.TheamountofDNAandultrapurewaterused inthissolutiondependedonthevaluesobtainedinthecountin Nanodrop®.
The sequencing reaction followed this schedule in the Biocycler® MJ96Gthermalcycler:30cyclesofinitialtemperature of96◦Cfor3min,denaturationof96◦Cfor15s,annealingof50◦C for10s, extensionof60◦C for4min andfinallyfinal extension of60◦Cfor7min.Thesampleswerethen purifiedfor sequenc-ingandinsertedintoanautomaticsequencer(ABI3130XLGenetic Analyzer).
AllsequencesaredepositedinGenBank(Table2). Alignment
Each nucleotide sequence was compared to the sequences deposited in theNCBI (National Center of Biotechnology Infor-mation)databasethroughtheBLAST(StandardNucleotideBasic LocalAlignmentSearchTool)algorithm.Thesequencesobtained were edited and aligned using the program BioEdit 7.1.9 (www.mbio.ncsu.edu/BioEdit/BioEdit.html). For each sequence, theagreementbetweenthechromatogramand thenucleotides, andbetweenthetwocomplementarystrands,wasmaximized.
Intra and interspecific genetic divergences were calculated usingthep-distancesintheMEGAX10.0.4program(Kumaretal., 2018).
Taxonomy
TrichomyiapseudoannaeAraújo&Bravosp.nov. (Figs.1.1–9and2.1–3)
Diagnosis.Headwithonerowofsupraocularalveoliandonerow ofoccipitalalveoli.Palpuswiththreesegments.Maleterminalia withhypandriumfusedtogonocoxitesandexpandedposteriorlyas aapicallyslightlybifucateplatecoveringtheaedeagus,gonocoxite
Table2
Specimensanalyzedinthiswork,includingthespeciesnames;BR,Brazil;gender(M,male;F,female);code,primer,pairbasesequence,GenBankaccessionnumbersand
locality.
Species Gender DNAextraction
code Primer Sequence (pb) GenBankaccession numbers Locality
T.cerdosaAraújo& Bravo,2016
M T15 lco/hco 657 MH042537 BR,Bahia,Igrapiuna,ReservaEcológicaMichelin,Pancada
Grande,15.VI.2013(lighttrap),M.Aragão&E.Menezesleg.
T.cerdosaAraújo&
Bravo,2016
M P4 mtd6/mtd9 480 MH042535 BR,Bahia,Igrapiuna,ReservaEcológicaMichelin,Pancada
Grande,15.VI.2013(lighttrap),M.Aragão&E.Menezesleg.
T.pseudoannaesp.nov. M P28 lco/hco 468 MH042538 BR,Bahia,Igrapiuna,ReservaEcológicaMichelin,Pancada
Grande,18.V.2013(lighttrap),M.Aragão&E.Menezesleg.
T.ituberensisAraújo&
Bravo,2016
M P21 mtd6/mtd9 480 MH042540 BR,Bahia,Igrapiuna,ReservaEcológicaMichelin,Vila5,
28.IV–19.V.2013(Malaise),M.Aragão&E.Menezesleg.
T.pseudoannaesp.nov. F P46 lco/hco 657 MH042536 BR,Bahia,Igrapiuna,ReservaEcológicaMichelin,Pancada
Grande,22.IX.2012(lighttrap),M.Aragão&E.Menezesleg.
Trichomyiasp1 F P29 mtd6/mtd9 467 MH042539 BR,Bahia,Igrapiuna,ReservaEcológicaMichelin,Pancada
Grande,18.V.2013(lighttrap),M.Aragão&E.Menezesleg.
Trichomyiasp2 F P44 lco/hco 633 MH042541 BR,Bahia,Igrapiuna,ReservaEcológicaMichelin,Pancada
Grande,24.III.2013(lighttrap),E.Mota,M.Aragão&E.
0.06 mm 0.06 mm 0.06 mm 0.06 mm 0.06 mm 0.06 mm 0.06 mm 0.06 mm 2 3 1 6 8 7 9 php 4 5 hyp cer agv aed pm agd 0.5 mm
Fig.1. 1–9Trichomyiapseudoannaesp.nov.1.Scape,pedicelandflagellomeres1and
2;2.Rightwing;3.Head,dorsalview;4.Head,ventralview;5.Palpus;6.Male
ter-minalia,lateral,arrowinprojectioninthegonocoxalapodeme;7.Cerci,epandrium,
hypoproct;8.Maleterminalia,ventral;9.Aedeagusandparameres(agv,ventral
armofgonocoxite;agd,dorsalarmofgonocoxite;cer,cercus;aed,aedeagus;hyp,
hypoproct;pm,paramere;php,post-hypandrialplate).
cer cer map spm subp 1 2 3 0.06 mm 0.06 mm 0.06 mm
Fig.2.1–3Trichomyiapseudoannaesp.nov.1.Femaleterminalia,ventral;2.Median
apodemeandspermathecae;3.Femaleterminalia,dorsal(map,medianapodeme;
cer,cercus;spm,spermathecae;subp,subgenitalplate).
withtwopairsofarms.Femalewiththesubgenitalplatetrapezoidal andbifurcatedapically,cercielongated.
Description.
Male.Headsubcircular,eyesrounded.Supraocularsetaein sin-glerow(Fig.1.3).Occipitalsetaearrangedinsinglerow(Fig.1.4). Antennalpitsubtriangular,shortdistancebetweenantennae(less than1/3ofthewidthofthepitsandwithscleroticfold).Scape sub-cylindricalandpedicelsubespherical,basalflagellomerespyriform andeccentric;withapairofmediobasaldigitiformandS-shaped ascoids,firstandsecondflagellomereequalinlength,ascoids1.4 timeslengthofflagellomere(Fig.1.1).Palpusthree-segmented;
firstsegmentwithsensillaindepressedpitoninnerside;palpus formula:1.0:0.6:0.9(Fig.1.5).Wing(Fig.1.2).Sc-rsclerotizedbut notmicrosetose,r-mpresent,radialforkdistalofapicesofCuA2and
medialfork,baseofM2sclerotizedbutwithoutmicrosetae.Male
terminalia.Hypandriumfusedwithgonocoxitesandexpanded pos-teriorlyasanapicallyslightlybifucateplatecoveringtheaedeagus. Twopairsofarmsof gonocoxite(Fig.1.8:agd, agv),onedorsal, directedtoapicalregionandwithfinebristlesdistributed irregu-larlyandaventralpair,longerthandorsalone,directedtointernal regionofgenitaliaatanangleof60◦.Pairofdorsalarmsdigitiform, withrowofrod-likesetaeatapexandsimplebristlesdistributed irregularly.Gonostylussub-circular,slightlysclerotizedandwith finebristles,articulatedwithventralregionofgonocoxite(Fig.1.8). Gonocoxalapodemewithmedium,narrowandsclerotized projec-tiondirectedtodorsalregionofgenitalia(Fig.1.6and1.8).Aedeagus bifid;twopairsofparameres,dorsallanciformandventral digiti-form,ejaculatoryapodemelong,1.75timeslengthofparameres (Fig.1.9).Cercus cuneiformwithbristles distributedirregularly. Hypoproctwithmicropilosityandapexrounded.Epandrium trape-zoidalandpilose,withalveolidistributedintwolateralpatches (Fig.1.7).
Female.Head,antennae,mouthparts,palpiandwingsasinmale. Femaleterminalia.subgenitalplatetrapezoidalbifurcatedapically. Cercielongate,about5.2timeslongerthanwide;sclerotizedarch betweencerciacuminateandwithmicroseate,0.4aslongascerci (Fig.2.1 and 2.3).Spermathecae withducts annulated,inflated apically,apexslightlytruncated.Medianapodemewithtwo sclero-tizedprojectionsanteriorlyandthreeposteriorly;medianposterior projectionthreetimeslongerthanotherprojections(Fig.2.2).
Material examined: Voucher #m and holotype #m (MZFS) Brazil,Bahia,Igrapiuna,ReservaEcológicadaMichelin,Pancada Grande,18.V.2013,M.Aragão&E.Menezescols.;1paratype#m (MZFS)thesamelocalityandcollectorasholotype,15.VI.2013;22 paratypes#m(MZFS)Brazil,Bahia,Igrapiuna,ReservaEcológica daMichelin,Pacangê,M.Aragão&E.Menezescols.27–28.X.2012 (1 paratype); 22.IX–28.X.2012 (5 paratypes); 16.XII–20.I.2013 (11 paratypes); 24.II–31.III.2013 (1 paratype); 21–22.VII.2012 (1 paratype); 27–28.IV.2013 (2 paratypes); 30–31.III.2013 (1 paratype);1paratype#m(MZFS)Brazil,Bahia,Igrapiuna,Reserva Ecológica da Michelin, Vila 5, 24.II–31.III.2013, M.Aragão &E. Menezescols.;Voucher#f(MZFS)Brazil,Bahia,Igrapiuna,Reserva EcológicadaMichelin,PancadaGrande,22.IX.2012,M.Aragão&E. Menezescols.;1paratype#f(MZFS)thesamelocalityandcollector asallotype.
Etymology.Theepithetreferstomorphologicalsimilaritywith TrichomyiaannaeBravo,2001.
Distribution.Knownonlyfromthetypelocality.
Comments. The new species is morphologically similar to Trichomyiaannae.Thedifferencesareinthemaleterminalia,the plateexpandedposteriorlyofhypandriumandgonocoxiteshasa smallbifurcationapicallywithprojectionsonroundedapexand notlanciformasinT.annae.Bothspecies,todate,havenotbeen includedinanysubgenus.
TrichomyiaituberensisAraújo&Bravo
TrichomyiaituberensisAraújo&Bravo,2016:30–31,figs.13A–I. Comments.MalesofT.ituberensisarerecognizedbythe gen-italia,withahypandriumfusedwithgonocoxitesandexpanded posteriorly as an apically strongly bifucate plate covering the aedeagus.Therearetwopairsofparameresandrod-likesetaein thearmofgonocoxite.
Materialexamined.Brazil,Bahia,Igrapiuna,ReservaEcológica Michelin,Vila5,28.IV–19.V.2013(Malaise),M.Aragão&E.Menezes cols.,1#m(MZFS).
Table3
Matrixofp-distancesamongmalesandfemalesofspecimensofTrichomyia.Bolddenotesshortestdistances;F,female.
#P4T. cerdosa #P46T. pseudoannae(F) #T15T. cerdosa #P28T. pseudoannae #P29Trichomyia sp1(F) #P21T. ituberensis #P44Trichomyia sp2(F) #P4T.cerdosa 0.156 0.010 0.162 0.197 0.216 0.276 #P46T. pseudoannae(F) 0.156 0.166 0.002 0.188 0.139 0.287 #T15T.cerdosa 0.010 0.166 0.168 0.201 0.220 0.294 #P28T. pseudoannae 0.162 0.002 0.168 0.202 0.154 0.304 #P29Trichomyia sp.1(F) 0.197 0.188 0.201 0.202 0.218 0.388 #P21T.ituberensis 0.216 0.139 0.220 0.154 0.218 0.314 #P44Trichomyia sp.2(F) 0.276 0.287 0.294 0.304 0.388 0.314 P46 T. pseudoannae (F) P28 T. pseudoannae P21 T. ituberensis P4 T. cerdosa T15 T. cerdosa P29Trichomyia sp1(F) P44Trichomyia sp2 (F) 0.050
Fig.3.DendrogramofgeneticsimilarityamongTrichomyiaspeciesanalyzed,F, female.
TrichomyiacerdosaAraújo&Bravo
TrichomyiacerdosaAraújo&Bravo,2016:39–40,figs.20A–H. Comments.MalesofT.cerdosaarerecognizedbythegenitalia, withanelongatearmofgonocoxitewithelongateapicalbristles. Thecercushasfourapicalbristlesrod-like.
Materialexamined.Brazil,Bahia,Igrapiuna,ReservaEcológica Michelin,PancadaGrande,15.VI.2013(lighttrap),M.Aragão&E. Menezescols.,2#m(MZFS).
Distribution.Brazil(Bahia).
SexualassociationbyDNAbarcoding
FragmentsofCOIofsevenspecimensofatleastthreespecies weresequenced(Table2).
Themale–femaleassociationwaspossibleonlyinTrichomyia pseudoannae spnov. The COI sequences frommale and female divergedinonlyinnucleotideposition361(Ginthesequenced femaleand A in themale).Data from morphology,close DNA-barcoding(0.002)andthefactthatbothspecimenswerecollected fromthesamelocalitycorroborateourhypothesisthatofthatthey belongtothesamespecies(Fig.3).
Concerningtheother specimensanalyzed here,thedistance betweenthesequencesoftwomalespecimensofT.cerdosawas 0.011.Ingeneral,theintra/interspecificgeneticdistancesrange from0.002–0.010/0.390–0.314respectively(Table3).
Conflictsofinterest
Theauthorsdeclarenoconflictsofinterest. Acknowledgements
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