Standard method for detecting Bombyx mori nucleopolyhedrovirus disease-resistant silkworm varieties

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RevistaBrasileiradeEntomologia62(2018)19–22

REVISTA

BRASILEIRA

DE

Entomologia

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

Biological

Control

and

Crop

Protection

Standard

method

for

detecting

Bombyx

mori

nucleopolyhedrovirus

disease-resistant

silkworm

varieties

Yang

Qiong

∗

,

Xing

Dong

Xu,

Li

Qing

Rong,

Xiao

Yang,

Ye

Ming

Qiang

Sericulture&Agri-foodResearchInstitute,GuangdongAcademyofAgriculturalSciences,Guangzhou510610,China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received24March2017 Accepted30November2017 Availableonline8December2017 AssociateEditor:MarianeA.Nickele Keywords:

Bombyxmorinucleopolyhedrovirus Groupinoculationmethod Individualinoculationmethod Resistancedetection Silkworm

a

b

s

t

r

a

c

t

Bombyxmorinucleopolyhedrovirus(BmNPV)diseaseisoneofthemostserioussilkwormdiseases,andit hascausedgreateconomiclossestothesericultureindustry.Sofar,thediseasehasnotbeencontrolled effectivelybytherapeuticagents.Breedingresistantsilkwormvarietiesbreedingmaybeaneffectiveway toimproveresistancetoBmNPVandreduceeconomiclosses.Apreciseresistance-detectionmethodwill helptoacceleratethebreedingprocess.Forthispurpose,herewedescribedtheindividualinoculation method(IIM).DetailsoftheIIMincludepathogenBmNPVpreparation,mulberryleafsize,pathogen vol-ume,rearingconditions,courseofinfection,andbreedingconditions.Finally,aresistancecomparison experimentwasperformedusingtheIIMandthetraditionalgroupinoculationmethod(GIM).The inci-denceofBmNPVinfectionandthewithin-groupvarianceresultsshowedthattheIIMwasmoreprecise andreliablethantheGIM.

Introduction

The domesticated silkworm, Bombyx mori, is an important economic insect for silkproduction. Thereare severaltypes of silkwormdiseases,and theycausegreat economiclossestothe sericultureindustry.Amongthem,B.morinucleopolyhedrovirus (BmNPV)diseaseisthemostserious(JiangandXia,2014;Xuetal., 2015).BmNPVdiseaseisacute,andthedurationfrominfectionto illnessisonlyapproximately96h.Thebodywallofinfected silk-wormsbreakseasilyand,thus,thepathogenpollutesthemulberry leavesonthesilkwormbed,whichleadstotherapid spreadof thedisease.Sofar,BmNPVhasnotbeencontrolledeffectivelyby therapeuticagents(Baoetal.,2009).

BmNPV,amemberoftheBaculoviridaefamily,hastwodifferent virionphenotypes: anocclusion-derived virusthat is transmit-tedamong hosts, and a budded virus that spreads throughout thehost.Occlusion-derivedvirusesarepackagedinpolyhedrons andformocclusionbodies(OBs)(Chenetal.,2010;Chengetal., 2014).BmNPV-resistanceisrelatedmainlytosilkwormstrains(Bao etal.,2009;Luetal.,2007).Theheredityofsilkwormresistance toBmNPViscomplicatedbecauseitiscontrolledbothbymajor dominantgenes and multiplemicro-effective genes (Yaoet al., 2003).Investigationshaveshownthatmostsilkwormstrainsare

∗ Correspondingauthor.

E-mail:[email protected](Q.Yang).

sensitivetoBmNPV infection and onlya few strainshave high resistance(Baoetal.,2009;JiangandXia,2014).Breeding resis-tantvarieties,whichimprovessilkwormresistancetoBmNPV,may helptoreduceeconomic losses(Jiang etal.,2012b).Combining a multi-generationpathogenattackwithclassicalcrossbreeding techniquescan improvethe resistanceofsilkworm varieties to someextent(Wuetal.,2010).However,traditional crossbreed-ing methodshave somelimitations, suchaspooraccuracy and lowefﬁciency.Since2000,increasingmoleculartechniqueshave beenappliedwidelytobreedingdisease-resistantsilkworms(Isobe et al., 2004;Kanginakudru et al.,2007; Huang et al., 2009; Xu etal.,2013;Zhangetal.,2014;Jiangetal.,2014).Comparedwith traditional breedingtechnologies, molecularmarkers and trans-genictechnologyarefasterandmoreeffectivewaystoselectand improvediseaseresistance(JiangandXia,2014;Subbaiahetal., 2013;Zhangetal.,2014).AfewBmNPV-resistantsilkworm vari-etieshavebeengeneratedusingsuchtechnologies(Xuetal.,2013). Atpresent,thegroupinoculationmethod(GIM)isusedwidelyby breedingunits(Luetal.,2007;Xuetal.,2013;Yangetal.,2013). However,theGIMdoesnothaveastandardoperationprogram; thus,differentresearchersusedifferentoperationalparameters, suchdifferentmulberrysizeandpathogenvolumes.Therefore,the shortcomingsofthismethod,suchasitslargestandarddeviation and poorrepeatability,leadtounreliableresults. In additionto advancesin breedingtechnology, a more scientiﬁc andreliable resistance-detectionmethodisneededtoguidethebreedingof BmNPV-resistantsilkwormvarieties.

https://doi.org/10.1016/j.rbe.2017.11.007

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20 Q.Yangetal./RevistaBrasileiradeEntomologia62(2018)19–22

Fig.1.BmNPVOB.

Materialsandmethods Silkwormstrainsandpathogen

SilkwormstrainsDazao,Yangshi,andshi7werepreservedin thelaboratoryoftheSericultureandAgri-foodResearchInstitute, GuangdongAcademyofAgriculturalSciences,Guangzhou,China.

Thewild-typeBmNPVGuangdongstrainwasalsomaintainedin thesamelaboratory.

Theindividualinoculationmethod(IIM)

Mulberry(Morus)leaveswerecutintodifferentsizes accord-ingtotheinstarsofthetestedsilkwormlarvaeasdescribedbelow. Differentvolumesofapathogensuspensionweretransferredwith apipetteanddroppedontothesurfaceoftheleavesandgently smearedtodry.Theappropriatevolumeofthepathogen suspen-sionisonethatdrieseasilywithin1h.Subsequently,thepathogen preparation,mulberrycutting,individualinoculationparameters, andrearingconditionsweremanipulatedasdescribedinSections ‘BmNPVpreparation’to‘IIM’.

BmNPVpreparation

Newlyexuviatedﬁfth-instarlarvaewereinoculatedonto mul-berryleavescoatedwith109_OBs/mL_for₄_h,_and_then_they_were

fed normal mulberry leaves. The OBs were obtained from the hemolymphoftheinfectedsilkworms,andtheywerepuriﬁedby repeatedcentrifugation(RahmanandGopinathan,2004).TheOB (Fig.1)concentrationwasdeterminedwithahemocytometer.The pathogensuspensionwasstoredat4◦Cbeforeuse.

Mulberryleafsize

Thefavorablesizewasthatatwhichtheleavesareeatenwithin 4hbymostofthesilkworms,becausetheleavesremained sufﬁ-cientlyfreshforthesilkwormstoeatduringthetime.Thesuitable

Fig.2.MulberryleafsizestandardsofdifferentlarvalstagesfortheIIM.(A)Leafsizeforsecond-instarlarvae.(B)Leafsizeforthird-instarlarvae.(C)Leafsizeforfourth-instar larvae.(D)Leafsizeforﬁfth-instarlarvae.

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Q.Yangetal./RevistaBrasileiradeEntomologia62(2018)19–22 21 Table1

ParametersoftheIIMandGIM.

Method Perleaf size(com) Larvae num-ber/perleaf (worm) Pathogen volume/per leaf(␮L) Pathogen dosage/(OBs/mL) GIM 3.5 5 25 2×108 IIM 1.5 1 5 2×108 Table2

TheBmNPVinfectionincidencesofthethreesilkwormstrainsusingtheIIMand GIM.

Method Silkwormstrains Numberoflarvaeinoculated Incidence(%)/¯x ±S GIM Darzhao 180 39.4±20.4 Yangshi 180 26.1±10.0 shi7 180 41.1±12.9 IIM Darzhao 180 54.4±1.9 Yangshi 180 40.6±4.7 shi7 180 50.6±2.8

mulberryleafsizesforsecond-,third-,fourth-,andﬁfth-instar silk-worms shouldbe 1.0, 1.1, 1.5, and 2.0cm in diameter(Fig.2), respectively.

Thepathogeninoculationvolume

Thefavorableinoculationpathogenvolumeis5␮Lforthe 1.1-and1.5-cmdiameterleafdisks,and10␮Lforthe2.0cmleafdisk. Larvalstage

Althoughsilkwormlarvaehavefiveinstars,youngerlarvae(first andsecondinstars)weretooeasilyinjuredduringtheexperiment. Thefifthinstarwastoolatefortheexperimentbecauseitneedtoeat toomuchpathogen,butthepathogenconcentrationwastoohigh anddifficulttosmearontheleave.Thus,third-andfourth-instar larvaewereused.

Silkwormrearingcell

Thesilkwormrearingcellshouldbemadeofplastic,whichcan bedisinfectedeasily.

Silkwormbreedingconditions

First- and second-instar larvae were reared at 27–28◦C at 85–90%relativehumidity(RH).Third-instarlarvaewererearedat 26–27◦Cat85–90%RH.Fourth-andﬁfth-instarlarvaewerereared at25–26◦Cat75–85%RH.A12hlight:12hdarkphotoperiodwas usedthroughoutthelarvalstage.

IIM

Leaveswerecutaccordingtothelarvainstarasdescribedin Section‘Mulberryleafsize’.Singleandintact(withoutanycrevices orholes)leafdiskwaspickedandplacedinaplasticrearingcell. Fivemicrolitersofthepathogensuspensionwastransferredwitha

pipetteanddroppedontothesurfaceoftheleafdisk,smearedwith astainlesssteelorplasticstick,andairdried.Afterexuviationand fastingfor8–12h,silkwormswereplacedintheplasticcellandfed thepathogen-contaminatedleafdisks.Onesilkwormshould corre-spondtoonemulberryleaf.Afterfeedingfor4h,thesilkwormsthat atethemulberryleaveswereselectedandgrouped,andthenthese silkwormswererearedonfreshmulberryleavesundertherequired breedingconditions.Thenumbersofthesilkwormsthatexhibited thetypicalsymptomsofBmNPVwererecordeddaily,andthe inci-denceofBmNPVinfectionwasanalyzedusingstatisticalsoftware asdescribedbelow.

ComparisonoftheIIMandGIM

ResistancedetectionwasperformedbytheIIMandGIMusing the three silkworm strainsmentioned above. Newly exuviated fourth-instarsilkwormswereusedforthetest.Thesizesofthe mul-berryleaves,numbersoflarvaeperleaf,andpathogenvolumeper leafaregiveninTable1.Infectedsilkwormswererecordedduring theprocessofbreeding.Eachmethodwasreplicatedthreetimes andeachreplicateconsistedof60larvae.TheincidenceofBmNPV infectionandthestandarddeviationwereanalyzedusingSPSSfor Windows,version16.0(SPSSInc.,Chicago,IL,USA).

Results

ComparisonoftheincidenceofBmNPVinfectionusingtheIIMand GIM

TheincidencesofBmNPVinfectionofsilkworms(Table2)ofthe samestraindifferedusingthetwodetectionmethods(Figs.3and4). TheBmNPVinfectionincidenceusingtheIIMwashigherthanthat oftheGIM,andtheintra-groupstandarddeviationwaslower. Discussion

TheincidencesofBmNPVinfectionusingtheGIMandIIMwere compared.TheIIMgroupexhibitedahigherincidenceofinfection andalowerintra-groupstandarddeviationthantheGIM,which canbeexplainedbecausetheamountofBmNPVeatenbyeach silk-wormwasnotequalusingtheGIM,assomelarvadidnoteator ateonlyasmallamountoftheBmNPV-treatedmulberryleaves. Thus,thegreatestweaknessoftheGIMwasitsinabilityto quan-tifyhowmucheachlarvaate,whichmadeitdifﬁculttodistinguish individualdifferencesofBmNPVresistance. Incontrast,theIIM, withindividualinoculationatitscore,avoidsmanyoftheproblem causedbydifferentexternalfactors.Thus,theIIMisbetterthan theGIM,anditprovidesdetectionresultswithgoodreliabilityand repeatability.Inpreviousstudies,wedetectedandselectedsome disease-resistantsilkwormvarietiesusingtheIIM,whichwere cre-atedbytransgeneoverexpressiontechnologyorRNAinterference (Jiangetal.,2012a,2012b,2013a,2013b).

Fig.3.GIMforplacingsilkwormsonmulberryleaves.(A)TheleaveswerearrangedintheboxaftersmearingthemwithBmNPV,andthentheywereairdried.(B)Fivelarvae wereplacedoneachleaf.(C)Theleavesafterbeingeatenbythesilkworms.

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22 Q.Yangetal./RevistaBrasileiradeEntomologia62(2018)19–22

Fig.4.IIMforplacingsilkwormsonmulberryleaves.(A)Onelarvawasplacedoneachleaf.(B)Thelarvaeatingtheleaf.(C)Theleafafterbeingeatenbythesilkworm. RoseMeireCostaBrancalhão’sresearchgroupusedasimilar

inoculationmethodtostudythecytopathologyoftheB.mori tra-cheaandpylorusinfectedwithBmNPV(Baggioetal.,2014;Senem etal.,2016).Theyprovidedtheparametersformulberryleafdisk sizeand pathogen volumefor newly moltedﬁfth-instar larvae. Here,wedescribedmoreparametersthataresuitablefor differ-entinstars.Themethodcouldbeusedtostudyotherinfectious silkwormdiseasesbyoralinfection,suchasB.moricytoplasmic polyhedrosisvirusandNosemabombycis.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest. Acknowledgements

This work was supportedby Guangdong Province Research ProjectGrants2015A040404032and2015A010107009.

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