Rearing Frankliniella zucchini Nakahara & Monteiro (Thysanoptera: Thripidae) on zucchini (Cucurbita pepo L. ‘Caserta’) fruits

RevistaBrasileiradeEntomologia63(2019)115–118

REVISTA

BRASILEIRA

DE

Entomologia

Short

Communication

Rearing

Frankliniella

zucchini

Nakahara

&

Monteiro

(Thysanoptera:

Thripidae)

on

zucchini

(Cucurbita

pepo

L.

‘Caserta’)

fruits

Viviana

M.

Camelo-García

_,

_Élison

_F.B.

_Lima

_,

_Jorge

_A.M.

_Rezende

a_{Dep.deFitopatologiaeNematologia,EscolaSuperiordeAgriculturaLuizdeQueiroz,UniversidadedeSãoPaulo,Piracicaba,SP,Brazil} b_{Dep.deBiologia,CampusAmílcarFerreiraSobral,UniversidadeFederaldoPiauí,Floriano,PI,Brazil}

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r

t

i

c

l

e

i

n

f

o

Articlehistory: Received21June2018 Accepted28February2019 Availableonline21March2019 AssociateEditor:RodrigoFeitosa Keywords: Insecta Tospovirus Thrips ZLCV Zucchinisquash

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Frankliniellazucchinitransmitszucchinilethalchlorosisvirus,causalagentoflethalchlorosisofzucchini squash.Thecharacteristicsofrelationshipbetweenthisviruswithitsvectorhavenotbeenstudied,one ofthereasonsbeingthelackofamethodforrearingthethripsforlaboratorystudies.Thisworkproposes asystemfortherearingofF.zucchinionfreshvirusfreezucchini‘Caserta’fruits,offeringapracticaland efficientalternativeforthesupplyofalargenumberofinsectsforlaterstudyofvirus/vectorrelationship. Inaddition,toaidintheidentificationofthisspeciesofthrips,theimmatureandadultformsobtained fromthecolonyweredescribed.

©2019PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeEntomologia.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

Thezucchinithrips,FrankliniellazucchiniNakahara&Monteiro (Thysanoptera: Thripidae) (Nakahara and Monteiro, 1999), is a speciesofthripssofarreportedonlyinBrazil.Itistheonlyspecies identifiedasa vectorofthetospoviruszucchinilethalchlorosis virus(ZLCV),causalagentoflethalchlorosisofzucchini (Cucur-bitapepoL.‘Caserta’);whereastransmissiontestswithF.schultzei TrybomandF.occidentalisPergandewerenegative(Bezerraetal., 1999;ICTV,2018;Monteiroetal.,2001).Moreiraetal.(2014) moni-toredthethripspopulationinexperimentalplantationsofzucchini andfoundthepredominanceoflarvaeandadultsofF.zucchini, indi-catingthatthisspecies,besidestransmittingZLCV,alsocolonizes ‘Caserta’.

In addition toC. pepo ‘Caserta’, ZLCVhas beenidentified as naturallyinfectingotherhosts,mainlycucurbitaceae:Cayaponia tibiricae,Cucurbitamaxima,C.moschata,C.moschata×C.maxima, Cucumisanguria,C.sativus,Citrulluslanatus,Luffaaegyptiacaand Sechiumedule;andsolanaceaeDaturastramonium(Camelo-García etal.,2015;Giampanetal.,2007;Yukietal.,2000).

Characteristicsoftherelationshipbetweenzucchinithripsand ZLCV,suchasacquisitionandtransmissionaccessperiods, incuba-tionperiod,virusreplicationinthevector,havenotbeenstudied, although necessary for a better understandingof epidemiolog-ical aspects of the disease in the field. Studies of virus/vector

∗ Correspondingauthor.

E-mail:jrezende@usp.br(J.A.Rezende).

relationships, however, require the availability of virus free coloniesofthripsforlarvaeandadulttrials.Theabsenceofa contin-uousandstandardizedsourceofinsectsinthefield,aswellasthe difficultyofcollectingandidentifyinglargenumbersofindividuals, makestherearingofthripsindispensableforthedevelopmentof thesestudies(LopesandAlves,2000).Theobjectiveofthepresent workwastodevelopasystemofmassrearingofF.zucchiniunder laboratoryconditionstosupportstudiesofitsrelationshipwith ZLCV.Thiscolonywasalsousedtofurtherdescribeimmatureand adultforms to expand thetaxonomic knowledgeof this thrips species.

Rearing of F. zucchini was initiated from insects collected fromzucchiniplants‘Caserta’grownintheexperimentalfieldat theDepartmentofPhytopathologyandNematology,ESALQ/USP, Piracicaba, SP, Brazil,where highincidence of thripsand ZLCV havebeenobservedsince1996(Camelo-Garcíaetal.,2015). Peri-odicidentificationofthripsspecimenswasperformedbymeansof examinationofslidesmountedfollowingthetechniqueproposed byMoundandMarullo(1996)andusingthekeytoFrankliniella speciesinBrazilproposedbyCavalleriandMound(2012).Voucher specimens aredeposited in theEntomologicalCollection of the EntomologyandAcarologyDepartmentESALQ/USP,Piracicaba,SP, Brazil.Leavesandflowerscollectedfromtheseplantswere exam-inedunder astereomicroscope forcollectionof adultswiththe aid of a fine brush. The adults were then transferred to fresh virusfreezucchini‘Caserta’fruits(∼20cmlong)previouslywashed with0.5% sodium hypochloriteand subsequently with running https://doi.org/10.1016/j.rbe.2019.02.005

0085-5626/©2019PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeEntomologia.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).

116 V.M.Camelo-Garcíaetal./RevistaBrasileiradeEntomologia63(2019)115–118

26cm

19cm

9cm

Lid with voil fabric

Zucchini squash

Vermiculite

Fig.1. ContainersforrearingFrankliniellazucchinionfreshzucchinifruits.

waterinabundance.Thefruitswereplacedinplasticboxes(26cm long×19cmwide×9cmhigh)withalidprotectedwithvoilfabric toallowairexchangeandalayer(2cm)ofexpandedvermiculite sterilizedinthebottomofthebox.Theplasticboxeswiththefruits werekeptinaBOD(Biothec)incubator,witha12hlightand tem-peratureof24.5◦C±0.5◦Cfollowedby12hofdarkand20◦C±0, 5◦C(Fig.1).Thefruitswererenewedbetween8and10days, trans-ferringlarvaeoftheoldfruitswiththeaidofafinebrush,keeping theoldfruitsintheboxforafew moredaystomakesurethat larvaecouldbecollectedafterpossiblyhatching.Onceamonth,a completecleaningoftheplasticboxwiththevermiculiteexchange wascarriedout.

The resultsobtained during12 months of studies indicated theefficiencyandfeasibilityoftheuseoffreshzucchini‘Caserta’ fruitsforrearing F.zucchini. In general,it waspossibleto esti-mateapproximately500adultsofthripsperplasticbox.Attempts torearF.zucchini onbeanpods (Phaseolus vulgaris),and plants of zucchini ‘Caserta’, Cucurbita maxima ‘Exposic¸ão’, long neck squash(C.moschata‘MeninaBrasileira’),cucumber(Cucumissativus Safira’)andcommonbeangrown ina BOD (Biothec)incubator, witha12hlightandtemperatureof24.5◦C±0.5◦Cfollowedby 12hofdarkand20◦C±0,5◦C werenotsuccessful,duemainly to the poor development of these species under such condi-tions.

TheidentificationofimmaturesinThysanopteraisrestricted toafew species(Vierbergenetal.,2010)andislargely concen-tratedinthecomparisonofsecondinstarlarvae.InFrankliniella, DeBorbon(2007)andSkarlinskyandFunderburk(2016)provided keystosomespeciesoftheAmericas,howeverthe characteriza-tionofthisstageforF.zucchiniisstillunavailable.Secondinstar immaturesofthespeciescollectedfromthecolonywereusedto characterizethemorphologyoflarvalstageinthezucchinithrips, whichwillallowcertifyingitspossiblehost-associationin differ-entplantspecies.Slidesoftheimmatureformswerestudiedunder ZeissAxioLabA1lightmicroscope.Inaddition,adultswere exam-inedinlightandscanningelectronmicroscopyLEO435VP(Fig.2) toprovidenewinformationonthemorphologyofF.zucchini.

AdultfemalesofFrankliniellazucchini(Fig.2a)aredistinguished fromother congenericspecies bythesimple pedicel on anten-nalsegmentIII,ocellarsetaepairIIIonposition3,absenceofat leastonepostocularsetaI(describedbelow)(Fig.2b),presence of a pairof campaniformsensilla on metanotum and postero-marginalcombonabdominaltergiteVIIIcompleteandregularly spacedwithpointedteeth(Fig.2c).Thespeciesiscloselyrelated to F. distinguenda Bagnall and F. gemina. The first is regarded usuallyas a smallerspecies,withocellarsetaeIIIand pronotal anteromarginalsetaesmallerincomparison withF.gemina and F.zucchini.AlthoughF.zucchiniisregardedashostspecifictoC. pepo and F.gemina as more polyphagous, there is nocertainty thatdiscretebiologicalentitiesareinvolved(CavalleriandMound, 2012).

The only major difference found between F. zucchini and F. gemina is the absence of at least one postocular seta in the pair I in the first species (usually both setae are absent) (CavalleriandMound,2012;NakaharaandMonteiro,1999).The examination of the specimens obtained from rearing and sur-veys from other regions revealed that the absence of at least one seta of the postocular pair I is constant, as no speci-menexhibited both setae,and inmost individuals (about90%) the pair was completely absent (Fig. 2b). In addition, a more recently used characterto distinguishFrankliniella species (see Gunawardanaetal.,2017)wasexaminedamongmorethan300 specimens:F.zucchinihasusuallyonlyone(sometimeszero)small microtrichia onthe dorsal surface of the hind coxae (Fig. 2d), while F.gemina hasthree or fourmore developed microtrichia (Fig.2e).Studiesinvolvingbothmolecularandmorphologicbiology shouldbecarriedouttorevealmoreconsistentspecies delimita-tions.

AmongthespeciesclosetoF.zucchini,onlyF.geminahas sec-ondinstarlarvaedescribedbyDeBorbon(2007).Thesecondinstar morphologyofbothspeciesaresimilar,andthesetallengthsexhibit overlaps(Table1).

ThemorphologicalcharacterizationofFrankliniellazucchini lar-vae II (Fig. 2f) is as follows: head with setae weakly pointed

V.M.Camelo-Garcíaetal./RevistaBrasileiradeEntomologia63(2019)115–118 117

Fig.2. MorphologyofadultfemaleandinstarIIimmature.Adultfemale(Frankliniellazucchini):(a)habitus;(b)headandpronotum;(c)abdominaltergitesVIII–X;(d)hind coxaeuppersurface.(e)HindcoxaeuppersurfaceofFrankliniellagemina.InstarIIimmature(F.zucchini):(f)habitus;(g)headandpronotum;(h)meso-andmetanotum;(i) abdominaltergitesVIII–X.

to capitate (Fig. 2g), D1 setae 5–10␮m long, D2 15–30␮m long, D3 12.5–17.5␮m long, D4 20–25␮m long; pro-, meso-and metanotal main setae capitate, pronotum medially with-out integument plaques, meso-and metanotum with rounded andelongatedplaquesmedially,plaquesontheposteriorregion smallerand sometimesweakly dentate(Fig.2gandh); abdom-inal tergites with capitate to weakly expanded setae and rounded elongated integumentplaques; tergite IX with sclero-tizedbandextendingfromD1setae1.0–1.5timesthediameter

of a setal insertion, not reaching the campaniform sensilla, posteromarginal comb usually with 16–18 teeth pointed and regularly spaced, of which 4–6 between D1 setae, teeth not longer than D1 seta insertion, transverse line of integument plaques present medially at the level of campaniform sensilla (Fig. 2i), D1 setae 30–40␮m long, D2 35–50; tergite X with sclerotizedbandintheposteriorhalf(Fig.2i);sterniteIX postero-marginalcombwithteethsmall,aboutonethirdofdorsalteeth length.

118 V.M.Camelo-Garcíaetal./RevistaBrasileiradeEntomologia63(2019)115–118 Table1

SetallengthsinFrankliniellageminaa_{andF.zucchini(inmicrons).}

Sclerite Setae F.gemina F.zucchini

Head D1 7.5–10.0 5.0–10.0 D2 15–22.5 15.0–30.0 D3 15.0–18.8 12.5–17.5 D4 22.5–32.5 20.0–25.0 Pronotum D1 18.8–25.0 22.5–25.0 D2 20.0–31.3 27.5–30.0 D3 7.5–13.8 7.5–10.0 D4 12.5–21.3 7.5–12.5 D5 22.5–32.5 25.0–30.0 D6 27.5–40.0 35.5–35.0 D7 22.5–32.5 25.0–27.5 TergiteII D1 16.3–23.8 15.0–20.0 D2 17.5–27.5 17.5–22.5 D3 16.3–25.0 12.5–17.5 TergiteVII D1 20.0–30.0 25.0–27.5 D2 25.0–35.0 25.0–30.0 D3 27.5–37.5 30.0–35.0 TergiteVIII D1 25.0–32.5 30.0–32.5 D2 27.5–35.0 30.0–32.5 D3 30.0–40.0 30.0–35.0 TergiteIX D1 30.0–36.3 30.0–40.0 D2 40.0–55.0 35.0–50.0 a_{BasedonDeBorbon(2007).} Conflictsofinterest

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