A spontaneous body color mutation in Drosophila nappae (Diptera, Drosophilidae)

RevistaBrasileiradeEntomologia61(2017)111–113

REVISTA

BRASILEIRA

DE

Entomologia

Short

Communication

A

spontaneous

body

color

mutation

in

Drosophila

nappae

(Diptera,

Drosophilidae)

Augusto

Santos

Rampasso,

Carlos

Ribeiro

Vilela

UniversidadedeSãoPaulo,InstitutodeBiociências,DepartamentodeGenéticaeBiologiaEvolutiva,SãoPaulo,SP,Brazil

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r

t

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c

l

e

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n

f

o

Received25November2016 Accepted17January2017 Availableonline16February2017 AssociateEditor:MarciaSoutoCouri Keywords: Recessive SãoPaulo Tripunctatagroup X-linked Yellow

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Ayellow-bodiedmaleappearedspontaneouslyinanisofemalelineofDrosophilanappaeestablishedfrom awild-caughtfemalecollectedattheForestReserveoftheInstitutodeBiociênciasdaUniversidadedeSão Paulo,CidadeUniversitária“ArmandodeSallesOliveira”,SãoPaulocity,stateofSãoPaulo,Brazil.This isthefirstmutationfoundinD.nappae,aspeciesbelongingtothetripunctatagroup.Theyellowmale wasisolatedandindividuallycrossedtotwowild-type(brown-colored)virginfemalesfromthesame generation,yieldingnumerousoffspring.AllF1individualswerewild-type,butthephenotypesyieldedin theF2generationwerewild-typefemales,andbothwild-typeandyellow-bodiedmales.Thelatteryellow malemutantsbackcrossedwithvirginwild-typeF1femalesyieldedfourphenotypes(brown-coloredand yellow-coloredfliesofbothsexes),indicatinganinheritancepatternofX-linkedrecessive.Chi-square goodnessoffittests(˛=5%)detectednosignificantdifferencesamongthenumberoffliesperphenotype. Thenewmutationisherebynamedyellow,duetoitsprobablehomologytoasimilarmutationwithan identicalinheritancepatternfoundinDrosophilamelanogaster.

©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

DrosophilanappaeVilela,Basso-da-SivaandValente,2004isa memberoftheDrosophilatripunctataspeciesgroup.Itisthesecond largestgroupwithintheDrosophilasubgenusintheNeotropical region(Vilela,1992),currentlywith86describedspecies(Bächli, 2016),onlysurpassedbytherepletagroup.Amongthosespecies, oneinparticularhasbeenextensivelystudied.Aimingtotransform DrosophilamediopunctataintoamodelorganismtostudyEcological andEvolutionaryGenetics,Dr.L.B.Klaczko’sresearchgrouphas beeninvestigatingnumerousaspectsofitsbiologyforover30years (Klaczko,2006;Batistaetal.,2014).Sofar,theywereabletoobtain ninemutations(mostofthempresentingallelicvariation),using bothX-raytreatmentsandinbreedingoffliescollectedinthefield (Marquesetal.,1989,1991;Klaczko,2006;Batistaetal.,2014).

D.nappaewasmisidentifiedasDrosophilaangustibuccaDuda, 1925fordecades.Wheeler(1963),whileanalyzingthematerial thatFrota-Pessoa(1954)usedtoredescribethespecies,suspected those fliescouldin fact belongtoan undescribed species.This suspicionwaslaterconfirmedbyVilelaandBächli(1990),who per-formedaredescriptionofD.angustibuccabasedontheonlymale paralectotype,which hadnotbeenanalyzedbyFrota-Pessoa.D. angustibuccaDuda,sensuFrota-Pessoa,1954,waslaterdescribed byVilelaetal.(2004)underthebinomialnameD.nappae,upon

∗ Correspondingauthor.

E-mail:crvilela@ib.usp.br(C.R.Vilela).

studying a multifemale strain established from specimens col-lectedbetween1994and1995atMorroSantana,inPortoAlegre (RS),byLucianoBassodaSilva.

AssumingthatallspecimensidentifiedbyFrota-Pessoa(1954)

asD.angustibuccaactuallybelongtoD.nappae,thedistributionof thelattermaybeconsideredwell-known(Vilelaetal.,2004).Thus, D. angustibuccaDuda sensuFrota-Pessoahad beeninitially col-lectedinthefollowingBrazilianstates:RiodeJaneiro(RiodeJaneiro city[thenDistrito Federal]),Rio Grandedo Sul(Felizand Porto Alegre),andSãoPaulo(CamposdoJordão,MogidasCruzes, Mon-gaguá[referredtoasVilaAtlântica],andPirassununga);locatedin southeasternandsouthernBrazil.Lateron,theoccurrenceofthis species(yetunderthenameD.angustibucca)inthestateofSanta CatarinaandatadditionalsitesofthestatesofRioGrandedoSul andSãoPaulohasbeenrecordedbydifferentauthors(Araújoand Valente,1981;Francketal.,1984;FranckandValente,1985;Valand Kaneshiro,1988;ValenteandAraújo,1991;DeToniandHofmann, 1995;Saavedraetal.,1995), whoprobablybasedtheir identifi-cationsonthekeyproposedbyFrota-Pessoa(op.cit.).Additional collectionsites areSerrado Cipó(stateof MinasGerais,Brazil) [Tidonetal.,1994,asspeciesR3]andHohenau(Paraguay)[Bächli etal.,2000,asD.paraguayensisDuda,1927].

TheDrosophilidaeStockCenteroftheDepartamentodeGenética e Biologia Evolutiva, Institutode Biociências, Universidadede São PaulocurrentlykeepsanisofemaleD.nappaestrain(codedM29F5) establishedfromafertilizedfemalecollectedbyoneofus(CRV)

http://dx.doi.org/10.1016/j.rbe.2017.01.001

0085-5626/©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

112 A.S.Rampasso,C.R.Vilela/RevistaBrasileiradeEntomologia61(2017)111–113 between22.VIIand03.VIII.2007attheforestreserveonthemain

campusofthereferreduniversity,knownasCidadeUniversitária “ArmandodeSallesOliveira”,SãoPaulocity,stateofSãoPaulo,Brazil (23◦3354.6S,46◦4342.4W;766m).IntheEighties,itwasa chal-lengingtasktoestablishastrainofthisspecies,sincewildcaught fliesinitiallydonotbreedwellincornmealorbanana-agarculture media,ascommonlynotedformostspecieswithinthetripunctata group.Asposteriorlydetermined,theuseofpowderedmilk-agar culturemediumproducesbetterresults(Bächlietal.,2000). How-ever,oneimportantdetailwasunintentionallyomittedinthecited paper:consideringthesexualmaturationofthespeciesbelonging tothetripunctatagroupisaslowprocess,duringthisphaseitis essentialtokeepfliesinamodifiedbanana-agarculturemedium (Go ˜niandVilela,2016)andonlytwoweeksafteremergencethey shouldbeplacedinthatspecialculturemedium.However,itshould bestressedthataftersuccessfullyestablishingthestrain,itmaybe keptexclusivelyinmodifiedbanana-agarculturemedium indefi-nitely.

Currently,theD.nappaestockismaintainedatconstant tem-perature(18±1◦C)andphotoperiod(13h:11h,L:D).According toGo ˜ni and Vilela (2016),after emergence,the fliesare trans-ferredweekly(forthreeweeks)tonewvialscontainingamodified banana-agarculturemedium,withasmallballoffreshbakers’yeast (Saccharomycescerevisiae).ItisworthnotingthatspecimensofD. nappaecandisplaycatalepsy(feigningdeath)(Frota-Pessoa,1954), meaningadultsgetparalyzedaftersufferingamechanicalimpact. Therefore,thetransferencetonewvialsandtheconsequent col-lisionwiththeculturemediumoftenmakesfliesgetstuckinto itssurfacewhileparalyzed,andfrequentlyarenotabletodetach themselvesafterrecoveringtheirmovements.Toovercomethe highmortalityrate,thevialswithadultfliesarekeptupsidedown forthefirstthreeweeks,whenadultsarediscarded.After discard-ingthematureflies,thevialsfromthethirdweek,withnumerous eggsandfirstinstarlarvae,arestoredintheregularposition.Ifthe upperlayeroftheculturemediumistoowet,V-shapedstripsof filterpaper(usually4pervial)areinsertedintotheculturemedium. On20.V.2015,whileoneofus(ASR)wastransferringthe1–7 daysoldwild-type(brown-colored)straintonewvials,noticeda conspicuouslycontrastingyellowspecimen(Figs.1and2).After determiningitwasamale,itwasleftisolateduntilreaching sex-ualmaturity(circa14days),buttransferredtonewvialsoncea week.On03.VI.2015itwastransferredonceagaintoanewvial togetherwithawild-typevirginfemale.Eventhoughtheywere kepttogetherunderobservationfor2h,andthemalehaddisplayed courtshipbehavior,thefemalewasnotreceptive,sothefliesdidnot mate.Afterthisperiodoftime,thefemalewasremovedfromthe vial.Onthenextdayweaddedadifferentwildvirginfemale,and crossingwasobserved.Insequence,themalewastransferredagain toanothervial,withadifferentwildvirginfemale,andthecouple waskepttogetheruntil08.VI.2015.Theoriginalmutantmaledied on13.VI.2015,wasdouble-mountedand,togetherwithsamples fromthestrainsM29F5andM29F5F1(seebelow),willbehoused inthecollectionsoftheMuseudeZoologiadaUniversidadedeSão Paulo.

Shortlyafter mating,it waspossibletoobserveeggsonthe surfaceoftheculturemediumand3dayslaterthefirsthatched larvae werenoticed. This fact suggested the mutant male was fertile.All emerged adultswere phenotypically wild, indicating thismutation,ifhereditary,wasrecessive.FliesfromtheF1

gen-eration wereallowed tofreely mate. Some pupae from theF2

generationwere isolated and imagoeswere sexed upon

emer-gence,sincethisspeciesdoesnotexhibitareliablepupalsecondary sexualdimorphism.Onlyfemaleswereselectedandkeptisolated and,therefore,werevirgins.IntheF2 generation,emergedflies

wereanalyzedonadailybasis,andthreephenotypicclasseswere observed:wild-typefemales,wild-typemales,andmutantmales

Table1

TotalnumberofF2offspringoftwovirginwild-type(brown-colored)females(♀1 and♀2)fromisofemalelineM29F5ofDrosophilanappaecrossedtoonespontaneous yellowmutantmalefromthesamestrain.Emergedimagoeswereanalyzeddaily, between10.VIIIand16.X.2015.AlladultmalesandfemalesofF1generationwere phenotypicallywildtype.

F2phenotype ♀1 ♀2 Wild(brown-colored)♀ 585 815 Wild(brown-colored)♂ 255 377 Mutant(yellow-colored)♂ 274 345 Total 1114 1537 Table2

Total offspring of backcross [F2 (yellow-colored males)×F1 (brown-colored

females)]fromDrosophilanappaeisofemalelineM29F5.Fouroutoftheeight per-formedcrossesyieldedoffspring:threeofthem(femalesa,dande)involving descendantsofthe♀1parentalfemaleandone(femaleg)descendantsofthe♀ 2parentalfemale.Emergedadultswereanalyzedonadailybasis,between20.IX and31.X.2015. Phenotypes ♀1 ♀2 Pooled offspring a d e g Wild(brown-colored)♀ 30 24 12 21 87 Wild(brown-colored)♂ 29 27 15 16 87 Mutant(yellow-colored)♀ 26 30 9 17 82 Mutant(yellow-colored)♂ 22 21 9 12 64 Total 107 102 45 66 320

(Table 1).Theabsenceof mutant females suggestedthis muta-tionwasX-linked.Chi-squaregoodnessof fittest(Zar,1996)at 5%significancelevel(withtheuseofYatescorrectionfor conti-nuity),acceptedthenullhypothesisthattherewerenosignificant differencesbetweenthenumber ofadultwild-typeandmutant yellowmalesintheF2generation(calculated2=0.61;critical2

value=3.84;0.30<p<0.50).In sequence,F2 mutantmaleswere

backcrossed with F1 virgin wild females. Emerged flies of the

next generation were analyzed daily as well,and four pheno-typicclasseswereobserved:wildandmutantfemalesand wild andmutantmales(Table2).Eventhoughthereweremore wild-type than mutant individuals in both sexes, a chi-square test acceptedthenullhypothesis thattherewerenosignificant dif-ferencesamongthenumber ofwild-typeandmutantyellowF2

fliespooledaccordingtophenotype(calculated2_{=4.48;critical}

2_{value=7.82;0.20<p<0.30).Additionalstudiesarenecessaryto}

clarifythisquestion,sinceithasbeendemonstratedthatDrosophila melanogasteryellowmutantmalespresentreducedlevelof loco-motionandabnormalcourtship,whichcausesmatingdisadvantage whenpairedwithwild-typefemales(Bastock,1956;Wilsonetal., 1976;BurnetandWilson,1980).However,itshouldbepointedout that,inourexperiments,thebackcrossesofF2yellowmalestoF1

wildmaleswereperformedwithnochoiceforthefemales. Aftercrossing mutant virginfemales and mutant males,we successfullyestablishedtwo yellowmutantisofemalestrainsof D.nappae,coded M29F5F1andM29F5F2,butjust theformer is currentlybeingmaintainedinourlaboratory.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest. Acknowledgements

We would like to thank our technicians, C.E. Lopes and F. Flauzino,forvariousreasons,andF.P.PradofromLeica,for tak-ingandprovidinguspicturesoftheflies,Dr.MaxiP.Richmond, fromtheUniversityofCalifornia,SanDiegoforgentlyreviewing

A.S.Rampasso,C.R.Vilela/RevistaBrasileiradeEntomologia61(2017)111–113 113

Figs.1,2.Drosophilanappae,habitusofmalesinrightlateralviews;tothesamescale.1,wild-type(brown-colored),fromisofemalelineM29F5.2,mutant(yellow-colored), fromisofemalelineM29F5F1.Notethesomewhatreticulatecloudsofthewingcrossveinsr-manddm-cuoftheyellowmutant(2)contrastingwiththeconspicuousclouds ofthebrown-coloredwild-type(1).Scalebar=1mm.

theEnglishversion,andtwoanonymousrefereesforsuggestions toimprovethemanuscript.

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