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 mBiological
Control
and
Crop
Protection
Standard
method
for
detecting
Bombyx
mori
nucleopolyhedrovirus
disease-resistant
silkworm
varieties
Yang
Qiong
∗,
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.
©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
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:serilover@126.com(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
0085-5626/©2017SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).
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-berryleavescoatedwith109OBs/mLfor4h,andthentheywere
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.
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
Thefavorableinoculationpathogenvolumeis5Lforthe 1.1-and1.5-cmdiameterleafdisks,and10Lforthe2.0cmleafdisk. 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.
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.
References
Baggio,M.P.D.,Ribeiro,L.F.C.,Vessaro-Silva,S.A.,Brancalhão,R.M.C.,2014.Bombyx moripylorusinfectionbyAlphabaculovirus.Genet.Mol.Res.13,1–8. Bao,Y.Y.,Tang,X.D.,Lv,Z.Y.,Wang,X.Y.,Tian,C.H.,Xu,Y.P.,Zhang,C.X.,2009.
Geneexpressionprofilingofresistantandsusceptible Bombyxmoristrains revealsnucleopolyhedrovirus-associatedvariationsinhostgenetranscript lev-els.Genomics94,138–145.
Chen,H.,Li,G.,Huang,G.,Chen,K.P.,Yao,Q.,Guo,Z.,2010.CharacterizationofORF29 ofBombyxmorinucleopolyhedrovirus.ActaVirol.54,275–280.
Cheng,Y.,Wang,X.Y.,Hu,H.,Killiny,N.,Xu,J.P.,2014.Ahypotheticalmodelof cross-ingBombyxmorinucleopolyhedrovirusthroughitshostmidgutphysicalbarrier. PLOSONE9,e115032.
Huang,K.,Li,C.,Gan,J.,Meng,B.,Zhi,L.,Zhou,Z.,2009.Apreliminarystudyon utilizingRNAinterferencetoconferenhancedresistancetoBmNPVintransgenic silkworm.Sci.Sericult.35,308–313.
Isobe,R.,Kojima,K.,Matsuyama,T.,Quan,G.X.,Kanda,T.,Tamura,T.,Sahara,K., Asano,S.I.,Bando,H.,2004.UseofRNAitechnologytoconferenhanced resis-tancetoBmNPVontransgenicsilkworms.Arch.Virol.149,1931–1940. Jiang,L.,Xia,Q.Y.,2014.Theprogressandfutureofenhancingantiviralcapacityby
transgenictechnologyinthesilkwormBombyxmori.InsectBiochem.Mol.Biol. 48,1–7.
Jiang,L.,Cheng,T.,Zhao,P.,Yang,Q.,Wang,G.,Jin,S.,Lin,P.,Xiao,Y.,Xia,Q.,2012a. ResistancetoBmNPVviaoverexpressionofanexogenousgenecontrolledbyan
induciblepromoterandenhancerintransgenicsilkworm,Bombyxmori.PLOS ONE7,e41838.
Jiang,L.,Wang,G.,Cheng,T.,Yang,Q.,Jin,S.,Lu,G.,Wu,F.,Xiao,Y.,Xu,H.,Xia, Q.,2012b.ResistancetoBombyxmorinucleopolyhedrovirusvia overexpress-ionofanendogenousantiviralgeneintransgenicsilkworms.Arch.Virol.157, 1323–1328.
Jiang,L.,Zhao,P.,Wang,G.,Cheng,T.,Yang,Q.,Jin,S.,Lin,P.,Xiao,Y.,Sun,Q.,Xia,Q., 2013a.Comparisonoffactorsthatmayaffecttheinhibitoryefficacyoftransgenic RNAitargetingofbaculoviralgenesinsilkworm,Bombyxmori.AntiviralRes.97, 255–263.
Jiang,L.,Peng,Z.,Dang,Y.,Zhou,P.,Xiao,Y.,Xing,D.,Xia,Q.,2013b.Constructionand resistanceassayoftransgenicsilkwormexpressingRNAitargetingnonessential immediategeneie-2ofBmNPV.Sci.Sericult.39,710–715.
Jiang,L.,Zhao,P.,Xia,Q.Y.,2014.Researchprogressandprospectofsilkworm molec-ularbreedingfordiseaseresistance.Sci.Sericult.40,571–575.
Kanginakudru,S.,Royer,C.,Edupalli,S.V.,Jalabert,A.,Mauchamp,B.,Prasad,S.V., Chavancy,G.,Couble,P.,Nagaraju,J.,2007.Targetingie-1genebyRNAiinduces baculoviralresistanceinlepidopterancelllinesandintransgenicsilkworms. InsectMol.Biol.16,635–644.
Lu,R.,Shi,M.,Bi,L.,Gu,J.,Huang,J.,2007.Apreliminaryinvestigationonthe resis-tanceofthesilkwormvarietyresourcesinGuangxitoBmNPV.Sci.Sericult.33, 117–121.
Rahman,M.M.,Gopinathan,K.P.,2004.Systemicandinvitroinfectionprocessof Bombyxmorinucleopolyhedrovirus.VirusRes.101,109–118.
Senem,J.V.,Torquato,E.F.B.,Ribeiro,L.F.C.,Brancalhão,R.M.C.,2016. Cytopatho-logyofthetracheaofBombyxmori(Lepidoptera:Bombycidae)toBombyxmori nucleopolyhedrovirus.Micron(Oxford1993)80,39–44.
Subbaiah,E.V.,Royer,C.,Kanginakudru,S.,Satyavathi,V.V.,Babu,A.S.,Sivaprasad, V.,Chavancy,G.,DaRocha,M.,Jalabert,A.,Mauchamp,B.,Basha,I.,Couble,P., Nagaraju,J.,2013.Engineeringsilkwormsforresistancetobaculovirusthrough multigeneRNAinterference.Genetics193,63–75.
Wu,F.,Yang,Q.,Xiao,Y.,Chen,L.,Liu,X.,Xing,D.,Luo,G.,Tang,C.,Wang,Z.,2010. PreliminaryreportoftheselectiveeffectofcontinuousfeedingwithBmNPV. GuangdongSericult.44,20–23.
Xu,A.Y.,Lin,C.Q.,Qian,H.Y.,Sun,P.J.,Zhang,Y.H.,Liu,M.Z.,Li,L.,2013.Breedingof anewsilkwormvariety‘Huakang2’withtolerancetoBombyxmori nucleopoly-hedrovirusdisease.Sci.Sericult.39,275–282.
Xu,K.,Li,F.,Ma,L.,Wang,B.,Zhang,H.,Ni,M.,Hong,F.,Shen,W.,Li,B.,2015. Mech-anismofenhancedBombyxmorinucleopolyhedrovirus-resistancebytitanium dioxidenanoparticlesinsilkworm.PLOSONE10,e0118222.
Yang,H.,Liu,Z.H.,Liu,S.,Li,T.,Tian,M.H.,Tang,F.F.,Li,M.,Xue,A.Y.,2013.Resistance ofsilkwormvariety871N×872NinYunnanProvince,China.Sericulture34, 42–44.
Yao,Q.,Li,M.W.,Wang,Y.,Wang,W.B.,Lu,J.,Dong,Y.,Chen,K.P.,2003.Screenof molecularmarkersforNPVresistanceinBombyxmoriL.(Lep.,Bombycidae).J. Appl.Ent.127,134–136.
Zhang,P.,Wang,J.,Lu,Y.,Hu,Y.,Xue,R.,Cao,G.,Gong,C.,2014.Resistanceof trans-genicsilkwormtoBmNPVcouldbeimprovedbysilencingie-1andlef-1genes. GeneTher.21,81–88.