Development and morphological characterization of the immature stages of Tetrastichus giffardianus Silvestri (Hymenoptera: Eulophidae)

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REVISTA

BRASILEIRA

DE

Entomologia

AJournalonInsectDiversityandEvolution

w w w . r b e n t o m o l o g i a . c o m

Systematics,

Morphology

and

Biogeography

Development

and

morphological

characterization

of

the

immature

stages

of

Tetrastichus

giffardianus

Silvestri

(Hymenoptera:

Eulophidae)

Elania

Fernandes,

Elton

Araujo

∗

,

Itala

Souza,

Mariana

Souza,

Glauber

Nunes

UniversidadeFederalRuraldoSemi-Árido,DepartamentodeCiênciasAgronômicaseFlorestais,Mossoró,RN,Brazil

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r

t

i

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l

e

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n

f

o

Articlehistory: Received18March2019 Accepted27May2019 Availableonline12June2019 AssociateEditor:MarcelHermes Keywords: Biology Biologicalcontrol Morphology Parasitoid Tephritidae

a

b

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ThegregariousendoparasitoidTetrastichusgiffardianusSilvestri(Hymenoptera:Eulophidae)isanatural enemyoffruitflies.ThisparasitoidwaspreviouslyusedtosuccessfullycontrolCeratitiscapitata (Wiede-mann)(Diptera:Tephritidae)inHawaii,USA.Despiteitsimportanceinthecontroloffruitflypests,little isknownaboutthedevelopmentorcharacteristicsofitspreimaginalstages.Theaimofthisstudywas toobservethedevelopmentandmorphologicallycharacterizetheimmaturestagesofTetrastichus gif-fardianus.TetrastichusgiffardianusindividualswererearedonC.capitatalarvae/pupaeunderlaboratory conditionsatatemperatureof25±2◦C,relativehumidityof60±10%,and12-hphotophase.Third-instar C.capitatalarvaewereexposedtoparasitismfor24h.Afterparasitism,thepupaeweredissectedevery 24htoevaluatethestageofdevelopmentattainedbyT.giffardianus,andtorecordtheirmorphological characteristics.AstereomicroscopewasusedtoobservealltheimmaturestagesofT.giffardianus.The completedevelopmentofT.giffardianusundertheseconditionswascompletedwithin14daysasfollows: egg(duration ∼= 1day);first(∼= 1day),second(∼= 1day),andthird(∼= 2days)larvalinstars;pre-pupa(∼= 2days);andpupa(∼= 7days).TheimmaturestagesofT.giffardianusdifferedsufficientlyintheirshape, color,andsizetoallowmorphologicalcharacterization.

Introduction

Biologicalcontroloffruitflies(Diptera:Tephritidae)with par-asitoids(Hymenoptera)hasbeenpracticedinmanyregionsofthe world(Alujaetal.,2014;Ovruskietal.,2000).IntheNeotropics, theparasitoidsmostcommonlyusedagainstthesefliesbelongto thefamiliesBraconidae,Chalcididae,Diapriidae,Eulophidae, Fig-itidae,and Pteromalidae(Alujaet al.,2009;Araujoet al.,2015; Nú ñez-Camperoetal.,2014;WhartonandYoder,2019).

Eulophidae is a large family of hymenopteran insects, with over4500describedspecies insome300genera(Noyes,2003). TetrastichinaeisthelargestsubfamilyofEulophidae,whichinclude parasitoidsofmorethan100familiesofinsectsofdifferentorders (LaSalle,1994).Tetrastichusgenusisthethirdlargestinthe subfam-ilyTetrastichinae,withcosmopolitandistribution,butthespecies (onlythree–TetrastichusgiffardianusSilvestri,Tetrastichusgiffardii Silvestriand TetrastichusoxyurusSilvestri)that havebeenmost commonlyassociatedwithfruit-infestingTephritidaeareofAfrican origin(LaSalleandWharton,2002).

∗ Correspondingauthor.

E-mail:elton@ufersa.edu.br(E.Araujo).

Tetrastichusgiffardianusisagregariousfruitﬂyparasitoid.This specieswasintroducedfromWestAfricatotheHawaiianIslandsin 1914tocontrolpopulationsoftheMediterraneanfruitﬂy,Ceratitis capitata(Wiedemann)(Diptera:Tephritidae),andquicklybecame establishedthere(Purcelletal.,1996).Duringthe1920sand1930s, thisparasitoidwasalsointroducedtoLatinAmericancountriesin attemptstocontrolC.capitatapopulations(Ovruskietal.,2000). However,owingtothelackofstudiesonT.giffardianusatthe begin-ningofthelastcentury,practicallyalloftheselaterattemptsto controlC.capitatawiththisparasitoidfailed.

Tetrastichusgiffardianuspenetrateslarvalexitholesandother openingsinfruitstoovipositinlarvae(third-instar)oftephritid fruitﬂies(PembertonandWillard,1918)anddevelopsasa gre-gariouskoinobiontendoparasitoid.Themeandevelopmenttime fromeggtoadultis17.4daysat26◦C(Purcelletal.,1996).Sex ratiosarestronglyfemale-biasedinpopulations,andmatingoccurs betweensiblingsshortlyaftertheyemergefromthehost(Purcell etal.,1996).

Despitetheimportanceof T.giffardianus asa naturalenemy of fruit ﬂies, information on its preimaginal development and themorphologicalcharacteristicsofitsimmaturestagesisscarce. However,this information is important becauseit can beused asthebasisfor planningitsrearing,todeterminetheidealage at which to release these parasitoids during biological control https://doi.org/10.1016/j.rbe.2019.05.005

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programs,aswellastoallowtheimmaturephasesofthisspecies toberecognizedanddistinguishedfromthoseofotherparasitoids. Theaimof this studywastodescribe thedevelopmentand morphologicallycharacterizetheimmaturestagesofT.giffardianus rearedonC.capitatalarvae/pupaeunderlaboratoryconditionsto helpfuturestudiesonthisnaturalenemyoffruitﬂies.

Materialandmethods

Specimensourcesandrearingprocedures

Thespecimens usedin this studywerefromC. capitataand T. giffardianus populations reared at the Laboratory of Applied Entomology,UniversidadeFederalRuraldoSemi-Árido,Mossoró, Rio Grande do Norte, Brazil. The insects were reared in an air-conditioned room (temperature 25±2◦C, relative humidity 60±10%,and12-hphotophase).TheinitialpopulationofC.capitata wasobtainedfromBiofábricaMoscamedBrasil,Juazeiro,Bahia.The specimensfortheinitialpopulationofT.giffardianuswerecollected inchestnutfruits(TerminaliacatappaL.–Combretaceae)infested withC.capitata,inthecityofMossoró.

Ceratitiscapitatawererearedinsemitransparentplasticcages (27.6×33.1×48.7cm),withonesidecoveredwithvoilefabricon whichthefemalescouldoviposit.Foodandwaterwereavailable insidethecagesthrougha1:4mixofbrewer’syeastandsugarand two250-mlplasticwaterbottleswithabsorbenttape(Spontex®), respectively.Aplastictraycontainingwaterwasplacedintheouter frontpartofthecagetocollecteggs,justbelowthevoilefabric.Eggs werecollecteddailyandtransferredontoanartiﬁcialdiettolarval developmentandthesubsequentformationofpuparia,whichwere thenusedtoestablishnewrearingcages.Theartiﬁcialdietusedwas describedbyAlbajesandSantiago-Alvarez(1980).

Tetrastichus giffardianus were reared following the method-ology described by Fernandes (2018). Adult parasitoids were maintainedin plasticcages(30×30×30cm)witha voilefabric opening(10×10cm)atthetopforventilationandfedwith gran-ulatedsugar(3g)providedinaPetridish(5cmdiameter×0.5cm high)andpurehoney(1ml)spreadwithbrushinapaperﬁlter.

Waterwas supplied through a 50-mlplastic water bottle with absorbent tape (Spontex®) attachedfor capillarity.To multiply theparasitoids,chestnutfruitsweremanuallyinfesteddailywith laboratory-rearedthird instarlarvae ofC. capitata.Theinfested fruitswereplacedinsidethecageandexposedtoparasitismfor 24h.Posteriorly,thelarvaeweretransferredintoacontainerwith artiﬁcialdiet(AlbajesandSantiago-Alvarez,1980)untilthe forma-tionofpuparium.ThepupaewerethentransferredintoPetridishes (8.5_×8.5_×2.5cm)containingmoistvermiculite,wheretheywere keptuntiltheadultparasitoidsemerged.

DevelopmentandcharacterizationofTetrastichusgiffardianus Third-instarC.capitatalarvaewereexposedtoT.giffardianus adults in rearing cages for a period of 24h for parasitism as describedabove.TheflylarvaewerethentransferredtoPetridishes (9.0×1.5cm)containingartificialdietandcoveredwithplasticfilm untiltheypupated.ThenthepupaeweretransferredtoPetridishes containingmoistvermiculitethatwereclosedwithplasticfilmand keptinanair-conditionedroom(temperature25±2◦C,relative humidity60±10%,and12-hphotophase).

Sixhoursaftertheendoftheparasitismperiod,tenﬂypupae wereremovedevery24hfromthePetridishesanddissectedunder astereomicroscopeusingtweezersandprobestoobservedthetime durationofeachdevelopmentalstageoftheparasitoid(egg, lar-valinstars,pre-pupaandpupa).Atotalof140C.capitatapupae weredissectedandtheimmatureparasitoidswerecountedand analyzed.Fromeachdissectedpuparium,tenimmatureparasitoids wereanalyzed,totaling 1400immaturespecimens, which were thenmeasuredandusedfor themorphologicalcharacterization ofeach developmentalstage,includingshape,color,andsizeof individualssampledateachofthedistinctphases.

TodeterminethenumberoflarvalinstarsofT.giffardianus,a multivariateanalysiswascarriedoutusingtheK-Meansmethod, basedonthewidthofthecephaliccapsule,lengthandwidthofthe larvabody,usingtheGENESsoftwarepackage(Cruz,2013).

Measurementsandphotographsoftheimmaturestageswere obtainedwithastereomicroscope(LeicaS8APO,Singapore,

Repub-Fig.1.Tetrastichusgiffardianuslarvae(A)andpupae(B)insideaCeratitiscapitatapuparium;(C)emergenceofadults;(D)C.capitatapupariumwithT.giffardianusemergence hole.

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Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 1.6392 3.2654 4.9526 5.7001 CP 1 (96,38%) -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 CP 2 (2 ,59 %) 1st_Instar 2nd Instar 3th Instar

Fig.2.NumberoflarvalinstarsofTetrastichusgiffardianusdeterminedbymultivariateanalysisbasedofthecephaliccapsulewidth,lengthandwidthofthelarvalbody, usingtheK-Meansmethod.

licofSingapore)equippedwithaDMC2900digitalcamerausing LASversion4.6(LeicaMicrosystemsImagingSolutions)software at50×magniﬁcation.

Results

Developmentofimmaturestages

The development of T. giffardianus began with the oviposi-tionofagroupofeggs(average of13.7)insidea third-instarC. capitatalarva.Afterembryonic development,oneT.giffardianus larvahatched fromeach viableegg anddeveloped gregariously withother larvae (Fig.1A) inside thehost. Three instarswere foundtooccur,whichwerecharacterizedbasedonwidthofthe cephalic capsule, length and width of the larval body (Fig. 2). Afterlarvaldevelopment,thepre-pupalandlaterthepupalstages wereobserved(Fig.1B).Theadultparasitoidsemergedusingtheir mandiblestomake a singlecircular holein thepupariumwall (Fig.1C),throughwhichtheparasitoidspassed(Fig.1D).Malesand femalesemergedwithinashorttimeintervalofeachother.

Thecompletepreimaginaldevelopmentalperiod(eggtoadult) was14dayslongundertheconditionsusedinthisstudy (temper-ature25±2◦C,relativehumidity60±10%,and12-hphotophase). Thedurationofeachdevelopmentalstagewasasfollows:egg ∼_{= 1} day),ﬁrstlarvalinstar ∼_{= 1}day,secondlarvalinstar ∼_{= 1}day,third larvalinstar ∼_{= 2}days,pre-pupa ∼_{= 2}days,andpupa ∼_{= 7}days. Morphologicalcharacterizationofimmaturestages

Theeggsareelongated,slightlycylindricalinshapewithathin, whitechorion.Acentralopaqueareaisvisible,showingthe grow-inglarvainsidetheegg(Fig.3A).Thisspeciesisgregarious,andan averageof13.7eggsperhostpupawasobserved.Themeanegg lengthwas0.20mm(Table1).

Theﬁrstlarvalinstarishymenopteriformandhas13segments plusacephaliccapsulewithoutapparentmandibles.Thebodyis translucent,andadigestivecanalgraduallybecomesﬁlledwith fatglobulesfromfeedingonthehost’sbody(Fig.3B,C).This lar-valinstar haslittlemobility, and the onlyvisible movement is

thecontractionandexpansionofthebody.Meanlarvallengthis 0.27mm,meanwidthis0.15andmeancephaliccapsulewidthis 0.04(Table1).

Thesecondlarvalinstarhasaglabrousandelongatedbody,and thesegmentsaremoredistinctthaninthepreviousinstar(Fig.3D). Thecephaliccapsulelittledifferentiatedfromtheothersegments ofthebody.Meanlarvallengthis0.59mm,meanwidthis0.26and meanheadcapsulewidthis0.08.

The third and last larval instar is white/yellowish in color. Mature(third-instar)larvaeincreaseinsizeandhaveanelongated shape(Fig.3E),buttheyarenotverydifferentfrompreviousinstars. In this instar,larvaeoccupytheentire puparium,withnoneof thehost’sbodytissuesremaining.Adarkmeconiumcanbeseen throughthecuticleatthecaudalend.Meanlarvallengthis1.19mm, meanwidthis0.50andmeanheadcapsulewidthis0.12.

Thepre-pupaisstillwhite/yellowishincolor;duringthisstage, theinsectstopsgrowingandbeginsthedeﬁnitionoftheadultbody shape(Fig.3F).Theﬁrstpartofthebodytotakeshapeisthe meta-soma.Adarkovalpileofmeconiumcanbeseenthroughthecuticle atthecaudalend.Meanpre-pupallengthis1.36mm.

Thepupaeareoftheexaratetype,shapedlikeadults,donot movewhentouchedandpresentreddish-browneyes.Thepupae areofatthebeginningofthepupalstage,theimmaturebodyis white/yellowishincolor,butovertimethepigmentationbecomes darker until thestandard adult coloris attained. Pupae have a well-deﬁnedhead,mesosoma,andmetasoma(Fig.3G,H,I).The antennae,ocelli,andmouthpartsarefullyvisible.Pupaeareformed withtheheadfacingthecephalicendofthehostpuparium.Sexual dimorphismisnotveryapparent,althoughfemalesdohaveamuch largermetasomathanthemales.Meanpupallengthis1.33mm.

Discussion

Developmentoftheimmaturestages

Owingtothelownumberofknowngregariousspeciesoffruit ﬂyparasitoidsandthegenerallackofinformationontheimmature stagesofthesenaturalenemies(Ovruskietal.,2006;Purcelletal., 1996;vanAchterbergetal.,2012;Viggianietal.,2007),the

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imma-Fig.3.ImmaturestagesofTetrastichusgiffardianus.Egg(A);ﬁrst-instarlarva(B,C);second-instarlarva(D);third-instarlarva(E);pre-pupa(F);pupaatthreedifferentstages ofmelanization(G,H,I).Scalebar=0.1mm.

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Table1

Measurements(mm)oftheimmaturestagesofTetrastichusgiffardianusinlarvae/pupaeofCeratitiscapitata.Temperature25±2◦_C,_relative_humidity₆₀_±_10%_and_photophase

of12h.Samplenumber(n=100).

Stages Bodylength Bodywidth Cephaliccapsulewidth

Mean Range Mean Range Mean Range

Egg 0.20 0.17–0.21 – – – – L1 0.27 0.19–0.36 0.15 0.09–0.19 0.04 0.03–0.05 L2 0.59 0.40–0.78 0.26 0.19–0.38 0.08 0.06–0.12 L3 1.19 0.82–1.60 0.50 0.34–0.82 0.12 0.09–0.15 Pre-pupa 1.36 0.96–1.89 – – – – Pupa 1.33 0.81–1.80 – – – –

L1,1stlarvalinstar;L2,2ndlarvalinstar;andL3,3rdlarvalinstar.

turedevelopmentofT.giffardianus(agregariousendoparasitoid)

hasbeen mainlycompared withthat of solitaryendoparasitoid

speciesinthediscussionbelow.

The development of T. giffardianus is similar to that of the

solitaryendoparasitoidDiachasmimorphalongicaudata(Ashmead)

(Hymenoptera:Braconidae),meaningthatitbeginswiththeegg

phase,goesthroughthreesimilarlarvalinstars,apre-pupaland

apupalstageafterwhichitreachestheadultstage.However,we

observedthatinT.giffardianustheemergenceofmalesandfemales

occursalmostsimultaneously,whereasin D.longicaudatamales

andfemalesemergeonseparatedays(Paladinoetal.,2010).

The duration of the preimaginal period of T. giffardianus observedinthisstudywasshorterthanthedurationreportedby PembertonandWillard(1918).Theseauthorsreportedthatthe lengthoftheperiodfromtheeggtoadultphaseinT.giffardianuswas 24–31daysincoldmonths,andabout18daysinwarmermonths. AsPembertonandWillard(1918)didnotreportdetailsaboutthe parasitoidsandtheclimaticconditionsinwhichtheywerereared, sotheshorterdevelopmentalperiodobservedinthisstudywas likelyduetothedifferencesintheparasitoidlineageorclimatic conditions(temperature, relative humidity and/orphotoperiod) usedincomparisonwiththeirstudy.Additionally,thedevelopment ofparasitoidsmaybeinﬂuencedbyotherfactors,suchassize,age, andhostquality(Lawrence,1990).

ThedurationofeachimmaturestageofT.giffardianus(egg, lar-vae,pre-pupa,andpupa)wasshorterthanthedurationreported for the equivalent immature stages of Aganaspis daci (Weld) (Hymenoptera:Figitidae)(Tormosetal.,2013),andD.longicaudata (Paladinoetal.,2010),bothofwhichwererearedonC.capitata hostsataconstanttemperatureof25◦C.

Morphologicalcharacterization

Despitethelack ofinformation onthecharacteristics ofthe preimaginalstagesofgregariousfruitﬂyparasitoids,wewereable toverifythatthecharacteristicsofnewlydepositedT.giffardianus eggsaredifferentfromthoseofotherlarval-pupalparasitoids.For example,theeggsofthesolitaryendoparasitoidA.daciarelarger thanT.giffardianus;theymeasureapproximately0.51mminsize, andeachhaveapeduncleintheanteriorextremitythatdisappears after48h(Tormosetal.,2013),whichwasnotseenineggsofT. giffardianus.

Tetrastichusgiffardianuslarvaepresentedmorphological charac-teristicsthataredifferentfromthoseofthesolitaryendoparasitoid Doryctobraconareolatus(Szépligeti)(Hymenoptera:Braconidae),in whichthelarvaearelongandcylindrical,withthelastabdominal segmentin the shape ofa tail(Murillo et al., 2016). The pres-enceofacaudalappendixallowssolitaryendoparasitoidlarvaeto travelthroughthehost’shemolymphtoﬁndanddestroy compet-ingparasitoidlarvae(Harveyetal.,2013).Theabsenceofacaudal appendixinthelarvaeofgregariousparasitoidslikeT.giffardianus isanimportantevolutionaryfeature,sinceitallowsthelarvaeto

remainpracticallyimmobileduringtheirdevelopment, facilitat-ingthegregarioushabitoftheseparasitoids(PextonandMayhew, 2004).

Thethree larval instarsof T. giffardianus were characterized byincreasingsizesand changesinthesizeofthecephalic cap-sule. Some studies onparasitoids have reported that the only differences among larval stages are apparent in size increases, mandiblechanges,andcephaliccapsuledimensionchanges(Parra andHadad,1989;Tormosetal.,2013).Thepresenceofdeveloped mandiblesinmanysolitaryendoparasitoidlarvaeisacharacteristic thatcandifferentiatethemfromT.giffardianuslarvae.Ingeneral, solitaryendoparasitoidshavewell-developedmandiblesintheﬁrst larvalinstarbecausetheyusethemandiblestopierceandkill com-petingparasitoidsinsidethesamehost(Harveyetal.,2013;Wang etal.,2008).

Thegeneralcharacteristicsofthepre-pupaeofT.giffardianusare similartothoseofotherspeciesoffruitﬂyendoparasitoids,which are characterizedbymovement deceleration, evacuationofthe hostremains(meconium),andonsetofeyepigmentation(Murillo etal.,2015;Tormosetal.,2009).However,pre-pupaeofT. giffar-dianus(1.3mm)aresmallerthanthoseofD.longicaudata(2.8mm) (Paladinoetal.,2010),D.areolatus(4.3mm)(Murilloetal.,2015), andA.daci(2.1mm)(Tormosetal.,2013).

ThepupalstageofT.giffardianuspresentsmanyofthe charac-teristics(antennae,coloration,legs,etc.)thatwillbepresentinthe adults,similartothatinthepupaeofD.longicaudata(Paladinoetal., 2010).SexualdimorphismisnotveryvisibleintheT.giffardianus pupalstage,incontrasttothepupalstageofD.longicaudata,in whichfemalespresentawell-developedovipositor,andmaleshave longerantennaeandvalvesthatcorrespondtotheexternalparts oftheirreproductivesystemintheventral-caudalregion(Paladino etal.,2010).Inaddition,T.giffardianuspupaearesmallerthanthose ofD.longicaudata(Paladinoetal.,2010).

Conclusion

Thisstudyshowsfor theﬁrst timethedevelopmentpattern and morphological characteristics of the immaturestages of T. giffardianus.Theinformationobtainedcancontributetoabetter understandingofthebiologyandmorphologyofthepreimaginal stagesofthisparasitoid,andmayhelptoimprovetherearingof coloniesofthisspeciesinthelaboratory.Inaddition,the character-izationofthisspecies’immaturephasesmayaidfuturetaxonomic studieson theimmature stages of parasitoids and research on interspeciﬁc competition.However,more detailed studiesonT. giffardianusshouldbecarriedouttoevaluatethepotentialofthis parasitoidtobeusedinthebiologicalcontrolofC.capitatainthe Braziliansemiarid.

Conﬂictsofinterest

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