REVISTA
BRASILEIRA
DE
Entomologia
AJournalonInsectDiversityandEvolutionw w w . r b e n t o m o l o g i a . c o m
Biological
Control
and
Crop
Protection
Isolation,
morphological
and
molecular
characterization
of
Bacillus
thuringiensis
strains
against
Hypothenemus
hampei
Ferrari
(Coleoptera:
Curculionidae:
Scolytinae)
夽
Janaina
Zorzetti
a,∗,
Ana
Paula
Scaramal
Ricietto
a,
Fernanda
Aparecida
Pires
Fazion
a,
Ana
Maria
Meneghin
b,
Pedro
Manuel
Oliveira
Janeiro
Neves
a,
Laurival
Antonio
Vilas-Boas
a,
Gislayne
Trindade
Vilas-Bôas
aaUniversidadeEstadualdeLondrina,Londrina,PR,Brazil bInstitutoAgronômicodoParaná.Londrina,PR,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received9March2017 Accepted22July2018 Availableonline2August2018 Keywords:
Biologicalcontrol Entomopathogenicbacteria Coffeeberryborer Cryproteins
a
b
s
t
r
a
c
t
ThecoffeeberryborerHypothenemushampeiFerrari,1876(Coleoptera:Curculionidae:Scolytinae)is consideredthemostseriouspestofthecoffeecropandiscontrolledprimarilywiththeuseofchemical insecticides.Analternativetothiscontrolmethodistheuseoftheentomopathogenicbacterium,Bacillus thuringiensisBerliner,1911.Therefore,theobjectiveofthisworkwastoselectstrainsofB.thuringiensis virulentagainstH.hampeiandcharacterizethembymorphologicalandmolecularmethodstoidentify possiblegenesfortheproductionofgeneticallymodifiedplants.Toachievethisobjective,34strainsof B.thuringiensisunderwentaselectivebioassaytoevaluatetheirtoxicitytoH.hampeifirst-instarlarvae. Amongthestrainstested,11andthestandardB.thuringiensissubspeciesisraelensis(IPS-82)caused mor-talityabove90%.Then,themedianlethalconcentration(LC50)wasestimatedforthesestrainsfollowedby
characterizationusingmorphological,biochemicalandmolecularmethods.ThelowestLC50wasobtained
forstrainBR58,althoughthisconcentrationdidnotdiffersignificantlyfromthatofthestandardstrain IPS-82orfromthatofstrainsBR137,BR80andBR67.Themolecularcharacterizationdetectedcry4A, cry4B,cry10,cry11andcyt1genesin10ofthemostvirulentstrains(BR58,BR137,BR80,BR81,BR147, BR135,BR146,BR138,BR139,BR140).StrainBR67differedcompletelyfromtheothersandamplified onlythecry3gene.ThisstrainwasmorevirulentthanBR135,BR146,BR138,BR139andBR140,butit didnotdifferfromBR58,BR137,BR80,BR81andBR147.Theproteinprofilerevealedproteinsof28,65, 70and130kDa,andthemorphologicalanalysisidentifiedsphericalcrystallineinclusionsinallstrains. Theresultsshowedthatthe11strainsstudiedhavepotentialforuseasagenesourceforinsertioninto coffeeplantsforthecontrolH.hampei,especiallythecry3,cry4A,cry4B,cry10,cry11andcyt1genes,that wererepeatedinthemostvirulentisolates.
©2018SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisisanopen accessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Theroleofthecoffeecropisextremelysignificantinthewealth and financial resources of producing and consuming countries andisalsoofcrucialimportanceintheeconomyandpoliticsof manydevelopingcountries(USDA,2014).However,coffeecrops arefacedwithseveralphytosanitaryproblems,whichincludethe coffeeberryborerHypothenemushampeiFerrari,1867(Coleoptera:
夽 AssociateEditor:RegianeBueno. ∗ Correspondingauthor.
E-mail:zorzettijanaina@gmail.com(J.Zorzetti).
Curculionidae:Scolytinae),consideredthemajorpestthatattacks thecoffeecrop(Vegaetal.,2012a).
H.hampeioccursinalmostallcoffee-producingcountries(Vega etal.,2012b),causingdamagetocropsandsevereeconomiclosses. Adultfemalesproduceinternalgalleriesinthecoffeeberrytolay theireggs,andthelarvalfeedingcausesdamagesthatreduceyields andthequalityoftheseedandcanalsoresultintheabscissionofthe berry(Vegaetal.,2009).Researchinvestigatingdifferentcontrol methodsisunderway,buttheinternalfeedinghabitofremaining mostofthelifecycleinsidethecoffeefruithampersmanagement. Chemicalcontrolisthewidestmethodusedforcontrolofthe coffeeberryborer,butinadditiontohighcost,healthand envi-ronmentaldamage,fewefficientandsafeactiveingredientsare registeredformanagingthispest(Reis,2007;Souzaetal.,2013).
https://doi.org/10.1016/j.rbe.2018.07.002
0085-5626/©2018SociedadeBrasileiradeEntomologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http:// creativecommons.org/licenses/by-nc-nd/4.0/).
Endosulfan(C9H6Cl6O3S)isabroad-spectruminsecticideandis the mostefficient product against H. hampei.However, due to humanandenvironmentalrisksrelatedtoitsuse,endosulfanhas beenbanned,whichincreasestheimportanceofresearchonnew methodsofcontrol(Janssen,2011;Lubick,2010).
Among the control alternatives causing less environmental damage is the use of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. that is considered one potential biologi-calcontrolagentfor coffeeberryborerand hasbeenused asa safebioinsecticideduetopropertieslikenontoxicitytoworkers andlow impactonnon-target organismsincludingcoffeeberry borernatural enemies. However,there are somedisadvantages ofusingB.bassianaunderfieldconditionssincethefungus effec-tivenessdependsongoodweatherconditionslikehighhumidity and mild temperatures and other factors, including the strain, concentration,virulence and application efficiency (Vega et al., 2012a).
Theuseof bacteriumBacillusthuringiensis(Berliner,1911) is alsooneofthealternativestoreducetheuseofchemical insec-ticidesforthecoffeeberryborercontrol.Theentomopathogenic action of this species is produced by crystal proteins, which are formed by Cry proteins encoded by different cry genes (Angelo et al., 2010; Vidal-Quist et al., 2009; Vilas-Bôas et al., 2007),which canbeinsertedintotheplantgenomebygenetic manipulationofcoffeeplants,whichisconsideredanimportant strategy of control, since thepest behavior makes it more dif-ficult. Besidesthis, a genetically modified plantcan reducethe influenceof abioticfactors onentomopathogenagents. Consid-ering that many potential new strainsand theirgenes remain unknown, their contents and potential for use as an alterna-tive strategy in insect control must be identified (Sun et al., 2008).
B.thuringiensissubsp.israelensisIPS-82straincontainsthegenes cry4,cry10,cry11andcyt(Schnepfetal.,1998),andalthoughactive againstDiptera,werealsotoxictoH.hampei(Méndez-Lópezetal., 2003).Morerecently,strainswithcry1Baandcry3Aageneswere alsoidentified becauseof theirinsecticidal activityagainst this beetlepest(López-Pazosetal.,2009).
Theresultsobtainedbyvariousauthorshighlightthe impor-tanceofmosquitocidalstrainsinthecontrolofcoleopterans,such asAnthonomusgrandisBoheman,1843(Coleoptera:Curculionidae) andH.hampei,andhavestimulatedresearchontheeffectofgenes activeagainst dipteransfor thecontrol ofColeoptera ( Méndez-Lópezetal.,2003;Monneratetal.,2012).
Therefore,inadditiontoselectingstrainsofB.thuringiensistoxic totheinsect,thegeneticprofileofthesestrainsmustalsobe iden-tifiedfora thoroughstudyoftheircrygene contents.Thus,the purposeofthisworkwastoselectstrainsofB.thuringiensisvirulent againstH.hampeiandthencharacterizethembymorphologicaland molecularmethodsforpossibleuseasasourceofgenestoproduce geneticallymodifiedplants.
Materialsandmethods
RearingofH.hampeistockonanartificialdiet
H.hampeiusedinthebioassayswererearedandmaintained in Petri plates containing artificial diet (Villacorta and Barrera, 1993).Theplateswerekeptinadarkclimatechamber(25±2◦C, 60±10%RH) at theLaboratory of Ecological Pest Management of theAgronomic Instituteof Paraná-IAPAR. After30 days,the Petri plates were opened to collect and transfer eggs to new plateswithartificialdietinwhichtheyremaineduntilsufficient numbersoffirstinstarlarvaewereavailableforuseinthe bioas-says.
Productionofthespore/crystalsuspension
StrainsofB.thuringiensisobtainedfromsoilsamples,storedin afilterpaperintheformofsporesandcrystals,wererecoveredon Petriplateswithnutrientagar(NA)andkeptundercontrolled con-ditions(30◦C)for96htocompletesporulation.Then,thecultures ofeachstrainwerescrapedfromtheculturemediawiththehelp ofaDrigalskispatulaandtransferredintomicrocentrifugetubes. Thesemixturesofsporesandcrystals,quantifiedusingaNeubauer as1×109spores/mLforallstrains,wereusedtoperformthe
bioas-says.
SelectivebioassayofstrainsofB.thuringiensisactiveagainstH. hampei
Thisbioassaywasconductedwith34strainsofB.thuringiensis fromtheCollectionofEntomopathogenicMicroorganisms (Labo-ratoryofGeneticsandTaxonomyofMicroorganisms)oftheState UniversityofLondrina(UniversidadeEstadualdeLondrina–UEL) thatwereidentifiedbythepresenceofCryproteinstoxictothe ordersColeopteraandDiptera.B.thuringiensissubsp.israelensis IPS-82strainandB.thuringiensissubsp.kurstakiHD-1wereprovidedas acourtesyofthePasteurInstitute,Paris,andwereusedaspositive and negativecontrols,respectively (Méndez-López etal.,2003). Forthebioassays,artificialdiet(VillacortaandBarrera,1993)was distributed(3mL/well)inasquarepolymethylmethacrylateplates (10cm×10cm)consistingof16wells(1.8cmdiameterby1.5cm depth).Afterdietsolidification,50Lofthemixtureofsporesand crystalswasaddedtoeachwell.Aftercompletedryingofthe sus-pension,fivefirstinstarlarvaewereplacedineachwell.Theinsects weremaintainedin adarkclimatechamber (25±2◦C, 60±10% RH)for5daysandthenmortalitywasquantified.Thebioassaywas performedinduplicateandconsistedof12repetitionswithfive lar-vaeeach.All12repetitionswereperformedinthesamedishwith 16wells,withtheremaining4wellsusedasnegativeand pos-itivecontrols.Thestrainsthatcausedmortalityabove90%were selectedforfurtherstudies.Mortalitydataweresubmittedtothe Scott–Knotttest(p<0.05)usingSASM-Agrisoftware(Canterietal., 2001).
Estimationofthemedianlethalconcentration(LC50)ofB.
thuringiensisstrains
The dose bioassays involved the 11 most virulent strains and IPS-82 as the standard strain. Seven concentrations (100, 30, 10, 3, 1, 0.3 and 0.1%) of each strain were prepared from theinitialsuspensionofspores andcrystals(100%) usedin the selectivebioassays(1×109spores/mL forallstrains).These
sus-pensions were diluted in sterile water with Tween at 0.01%. For each concentration,12 repetitions were performeddivided amongthreesquarepolymethylmethacrylateplates.Ineachplate, four lines with four wells each were used for different con-centrations. Thus, in the three plates, 12 repetitions of four different concentrations were performed.Each well containing five larvae(n=60) wasusedas one repetition,as in the selec-tive bioassay, and received 50L of a suspension. The insects weremaintainedin adarkclimatechamber (25±2◦C, 60±10% RH) for 5 days, followed by quantification of mortality. The bioassays were repeated twice, and the mortality data were subjected to probit analysis (Finney, 1971) using the Polo-PC program(LeOraSoftware,1987)todeterminethelethal concen-tration.TheLC50bioassayresultswereanalyzedbycheckingfor
overlapofthe95%confidenceintervalsaccordingtoprobit analy-sis.
MolecularcharacterizationofB.thuringiensisstrainstoxictoH. hampei
Total DNA samples from strains of B. thuringiensis were extracted according to the method described by Ricieto et al. (2013).Thestrainsweregrownat 30◦C for15honplates con-taining Luria–Bertani broth (LB) (Bertani, 1951). A colony of approximately2mmindiameterwastransferredtoamicrotube containing200LofTEbuffer(10mMTris;1mMEDTA;pH8.0) usingasteriletoothpick.Thesuspensionwashomogenizedand incubatedfor10minina boilingwater bath.Then,the suspen-sionwascentrifugedat12000gfor3min.Thesupernatantwas transferred toa newmicrocentrifuge tube and used as a DNA samplefor PCRamplification reactions(polymerasechain reac-tion).Thepresenceofcry1,cry2,cry3,cry4A, cry4B,cry10,cry11 and cyt1 was evaluated using primers and specific amplifica-tionconditions.Allreactionsunderwent an initialdenaturation step at 94◦C for 2min and a final extension step at 72◦C for 5minperformedasdescribedbytheauthors(Bravoetal.,1998; Céronet al.,1995; Ibarraet al.,2003; Vidal-Quist etal., 2009). DNA amplification was performed using a Techne® Endurance TC-412ThermalCycler(TechneLimited,Staffordshire,UK).Each amplificationreactionwasmixedinatotalvolumeof20L con-taining1UTaqDNApolymerase(Invitrogen,Brazil),buffer(20mM Tris–HCl, pH 8.0, 50mM KCl), 1.5mM MgCl2, 0.25mM dNTPs,
0.5Meach primer,2LofextractedDNA andsterilized Milli-Qwater).Thesamereactionwasusedforallprimersdescribed. The amplified products were visualized by electrophoresis on 1.2%agarosegelinTBEbuffer(89mMTris–borate, 2mMEDTA, pH8.0)stainedwithSYBR® Safe(Invitrogen,UK)usinga100bp markerDNALadder(Invitrogen,UK).Afterelectrophoresis,thegel imageswerecapturedusinga SonyCyber-shot8.1digital cam-era.
CharacterizationofCryproteinsthroughSDS–PAGE
TheproteinprofilesofthecrystalsofB.thuringiensisstrainswere characterizedbyproteinelectrophoresison10%polyacrylamide gel (SDS–PAGE). Initially, the crystals were obtained accord-ing to the protocol described by Lecadet et al. (1992). Each strain wascultivated in NutrientBroth medium (NB) (Downes and Ito, 2001) at 30◦C for 72h at 200rpm, until complete sporulation.Thepreparationsof thestrainspores/crystals were analyzedby SDS–PAGE accordingto theprocedureoutlined by
Laemmli(1970). For electrophoresis in Tris–glycinebuffer at a constant voltage of 30mA for 3h, 15L of solubilized spore-crystal preparationswas used.B. thuringiensis subsp. israelensis strainIPS-82,withaknownproteinprofile,wasusedasa refer-ence.
MorphologicalcharacterizationofB.thuringiensisstrains
Themorphologicalcharacterizationofthestrainswasinitially performedusinganopticalmicroscope(CHSModel;Olympus Opti-calCo. Ltd.,Tokyo,Japan)witha100×phase contrastlens.For electronmicroscopy,thelyophilizedmaterialofthestrainsused inthebioassayswasdepositeddirectlyonmetalsupportscoated withgoldfor180sundervacuum(10-1mbar)atacurrentintensity of40mAinaBAL-TECsputterCoaterModelSCD-050andanalyzed onaPhilipsFEIQuanta200scanningelectronmicroscopeQUANTA 200(EIF)underhighvacuumatavoltageof20kVwitha10.2mm workingdistance.
Table1
MortalitycausedbyB.thuringiensisagainstfirstinstarlarvaeofH.hampeionthefive daysoftheselectivebioassay(darkchamber,25±2◦C,60±10%RH).
Strain Mortality(%)a Strain Mortality(%)a
IPS-82b 100.00a BR127 10.00c BR80 100.00a BR145 10.00c BR81 100.00a BR78 8.75c BR140 98.80a BR164 8.75c BR58 98.75a BR07 7.50c BR67 98.75a BR83 6.25c BR135 98.75a BR141 6.25c BR137 98.75a BR143 6.25c BR139 98.75a S1269 5.00c BR147 98.75a BR03 5.00c BR146 97.50a BR05 3.75c BR138 96.25a BR87 3.75c BR37 21.25b BR43 1.25c BR79 21.25b BR148 1.25c BR18 20.00b BR09 0.00c BR131 17.50b BR187 0.00c BR149 16.25b HD1c 0.00c BR105 15.00b BR38 15.00b Controlsample 1.25c
aMeansfollowedbythesameletterinthecolumnsdonotdifferfromeachother
accordingtotheScott–Knotttest(p<0.05).
bB.thuringiensissubsp.israelensisstandardIPS-82strain-positivecontrol. c B.thuringiensissubsp.kurstakistandardHD-1strain-negativecontrol.
Results
SelectivebioassayofB.thuringiensisstrainsagainstH.hampei Thirty-fourstrainsofB.thuringiensiswereincludedinthe selec-tivebioassay.Amongthesestrains,11(32.3%)causedmortalityto H.hampeilarvaethatexceeded96%,whichdidnotdiffer signif-icantlyamongthe11strainsorfromthestandardIPS-82strain. These11strainsunderwentquantitativebioassaystodetermine themedianlethalconcentration(LC50).Theremaining23strains
(67.6%)causedmortalityratesbelow21.2%.Amongthesestrains, BR09and BR187and thestandard strainB. thuringiensissubsp. kurstakiHD-1werenon-pathogenictoH.hampei(Table1).
Estimationofmedianlethalconcentration(LC50)ofB.
thuringiensisstrains
TheLC50fortheH.hampeilarvaerangedfrom0.037×109to
0.956×109spores/mL. ThelowestLC
50wasfortheBR58strain,
whichdidnotdiffersignificantlyfromthestandardIPS-82.No sig-nificantdifferencesweredetectedamongtheLC50valuesofBR58,
BR137,IPS82,BR80andBR67strains,whichshowedthebestresults andwerethemosteffectiveagainstH.hampei(Table2).
In a second group classifiedby the LC50 values, the strains
(BR147,BR135andBR146)differedfromthestandardIPS-82strain andfromthestrainsthathadthelowestLC50 values(BR58and
BR137).TheexceptionwastheBR81strain,whichdidnotdiffer significantlyfromtheIPS-82strainbutwasalsoinsertedinthis group.
ThethirdgroupcomprisedtheBR138andBR139strains,which showedanLC50valuehigherthanthatofthefivemosttoxicstrains.
Finally,theBR140strain,withthelowesttoxicity,differedfromall otherstrainsandhadthehighestLC50value.Astheexception,the
BR81strainLC50valuewasapproximatelythreefoldhigherthan
thatoftheBR58andBR137strainsbutwasnotdifferentfromthose oftheBR80andBR67strainsandfromthatofthestandardIPS-82 strain.
TheBR58strainwasapproximately25-foldmoretoxicthanthe BR140strain,whichshowedthehighestLC50valueanddiffered
Table2
Medianlethalconcentration(LC50)ofB.thuringiensisstrainsagainstfirstinstar
lar-vaeofH.hampei(n=60)onthe5daysofthedose–responsebioassay(darkchamber, 25±2◦C,60±10%RH).
Strain LC50(Spores/mL) 95%CI Slope±SE
Lower Upper BR58 0.037×109a* 0.017×109 0.070×109 1.410±0.124 BR137 0.039×109a 0.023×109 0.067×109 1.227±0.108 IPS-82 0.040×109ab 0.017×109 0.080×109 1.519±0.137 BR80 0.049×109abc 0.024×109 0.089×109 1.402±0.122 BR67 0.113×109abc 0.050×109 0.104×109 1.543±0.163 BR81 0.114×109bcd 0.074×109 0.169×109 0.998±0.097 BR147 0.148×109cde 0.089×109 0.243×109 1.292±0.107 BR135 0.164×109de 0.122×109 0.217×109 1.430±0.128 BR146 0.196×109de 0.134×109 0.273×109 1.232±0.137 BR138 0.251×109e 0.180×109 0.344×109 1.348±0.159 BR139 0.297×109e 0.203×109 0.424×109 1.159±0.140 BR140 0.956×109f 0.624×109 1.60×109 1.049±0.183 *Meansfollowedbythesameletterinthecolumndonotdifferfromeachother
byoverlappingof95%confidenceintervals,accordingtoprobitanalysis.
Table3
GeneticandproteinprofilesofB.thuringiensisstrainsselectedinabioassayagainst H.hampei.
Strain Geneticprofile Proteinprofile(kDa)
BR58 cry4A,cry4B,cry10,cry11,cyt1 130/70 BR137 cry4A,cry4B,cry10,cry11,cyt1 130/65 IPS-82 cry4A,cry4B,cry10,cry11,cyt1 130/70/28 BR80 cry4A,cry4B,cry10,cry11,cyt1 130/70
BR67 cry3 65/70
BR81 cry4A,cry4B,cry10,cry11,cyt1 130/70 BR147 cry4A,cry4B,cry10,cry11,cyt1 130/65 BR135 cry4A,cry4B,cry10,cry11,cyt1 130 BR146 cry4A,cry4B,cry10,cry11,cyt1 130 BR138 cry4A,cry4B,cry10,cry11,cyt1 130/65 BR139 cry4A,cry4B,cry10,cry11,cyt1 130 BR140 cry4A,cry4B,cry10,cry11,cyt1 130/65
lethalconcentration(LC50)valuesweredeterminedwereselected
forfurthermolecular,morphologicalandproteincharacterization. MolecularcharacterizationofB.thuringiensisstrainsagainstH. hampei
In the molecular characterization, differentcry gene groups weredetectedintheB.thuringiensisstrains.Thecombinationcry4A, cry4B, cry10,cry11and cyt1 was observed in 10 strains, which exhibitedanamplificationproductsimilartothatexpectedforB. thuringiensissubsp. israelensis(cry4Aa,cry4Ba,cry10Aa,cry11Aa, cyt1Aa,cyt1Caandcyt2Ba,locatedonasingleplasmidof72MDa) (Berryetal.,2002;Guerchicoffetal.,1997;Ibarraetal.,2003).BR67 wastheonlystrainthatdidnotshowanyamplificationproduct fortheabovegenesandamplifiedonlyforthecry3gene,which hasinsecticidalactivityagainstcoleopterans(Schnepfetal.,1998) (Table3).
Morphologicalandproteincharacterization
Theanalysisoftheproteinprofilesofsporeandcrystal mix-turesbyelectrophoresison10%polyacrylamidegel(SDS–PAGE)of thestrainsmoretoxictoH.hampeirevealedbandsof28,65,70and 130kDa(Table3).Theproteinprofileofthestrainusedasthe stan-dard,B.thuringiensissubsp.israelensis(IPS-82),wassimilartothat previouslyreported,withbandsat27,65,128and135kDa(Becker andMargalit,1993).
Allprofilesanalyzed,exceptthatforBR67,hadamolecularmass of130kDa,whichisoftenrelatedtothecrystalproteinsofCry4A and Cry4B classes witha molecularweight range 128–135kDa
(Lereclusetal.,1989).BR58strainpresentedpeptidesof130kDa, whichmightbecorrelatedwithCry4proteins.TheBR58strainalso hadpeptidesof70kDa,whichcouldbeassociatedwithboththe Cry10(∼78kDa)(Thorneetal.,1986)andCry11proteins(∼72kDa) (Delécluse et al., 1995). Almost all strains revealed molecular massesfrom65to70kDa,confirmingthepresenceoftheexpected genes(cry10andcry11).
Themorphological analysisbyopticaland scanningelectron microscopyconfirmedthetypicalcharacteristicsofstrainsofthe speciesB.thuringiensis,i.e.,spores,crystalsandrod-shaped vegeta-tivecells.Theimagesrevealedthatallstrainshadsphericalcrystals, verysimilartothosefoundinB.thuringiensissubsp.israelensis. Discussion
Among the34strainstested against H.hampeiin the selec-tivebioassay,11werethemosttoxicandpresentedmorphological (sphericalcrystals)andmolecular(cry4A,cry4B,cry10,cry11and cyt1)characteristicsandproteinprofiles(130/70–65kDa)similar tooneanotherandtotheB.thuringiensissubsp.israelensisstandard IPS-82(Table3).Thismolecularprofileofthestrainsisresponsible forthehightoxicitytoDiptera.Nevertheless,thiscombinationof morphological,molecularandproteinprofileshaspreviouslybeen citedasacontrolagentagainstColeoptera,suchasH.hampeiand A.grandis(Méndez-Lópezetal.,2003;Monneratetal.,2012).The strainscontainingcry1,cry2andcry3,suchasstrainBR145(Ricieto etal.,2013),whichhasactivegenesagainstColeoptera,showed lowtoxicitytoH.hampeicausingonly10%mortality(Table1).
Méndez-Lópezetal.(2003)achievedsimilarresults.After eval-uating170B.thuringiensisstrainsforthecontrolofH.hampei,these authorsfoundthatonly32causedmortalitybetween90and100%. Theyalsoobserved thattheDiptera-specificstrainshad similar molecular,morphologicalandproteincharacterizationandwere activeagainstAedesaegyptiLinnaeus,1762(Diptera:Culicidae).The authorsalsotestedninestrainswithmosquitocidalactivityagainst H.hampeiprovidedbythePasteurInstitute.TheyobservedthatB. thuringiensissubspeciesisraelensis andthompsoniobtained100% mortalityand themorrisoni (PG14) and malaysiensis subspecies causedamortalityrateabove80%anddidnotdiffersignificantly fromthemosttoxicstrains.
TheBR58andBR137strains,whichhadthelowestLC50values,
amplifiedthecry4A,cry4B,cry10,cry11andcyt1genes.Thesestrains alsoexhibitedbandsof130and65–70kDa,whichmightberelated tothecry4A andcry4Bgenesandtothecry10and cry11genes, respectively(Monneratetal.,2012).TheBR135,BR146andBR139 strainsshowedthesamegenepoolasthemostvirulentstrainsbut presentedonlyonebandof130kDa.Thesethreestrainsresultedin higherLC50values,suggestingthatthelowtoxicitywaseitherdue
tolowlevelsofortothelackofexpressionofthecry10,cry11and cyt1genes.
ThePCRtechniqueprovidesarapidandsuperficialmolecular characterizationbasedontheprimersused.However,the tech-niquecannotidentifyallgenesinastrainordetectwhetheragene isexpressed.Forexample,thecry10,cry11andcyt1amplifiedgenes ofBR135,BR146andBR139strains,respectively,mightbepresent atverylowlevelsorbedisrupted,mutatedorunderthecontrol ofadefectivepromoter.Therefore,thecontributiontothelethal effectagainstthepestwaslow,ortheexpressionofthesegenes wasinsufficienttoformdetectableproteinlevelsintheprotein characterization.
Thegenescry4andcyt1werefoundinalmostallstrainsinthis study,whicharealsothegenesresponsibleforthehighmortalityof A.grandis(Monneratetal.,2012).Thesegeneswerealsoidentified inthestrainsB.thuringiensissubsp.israelensisandsubsp.morrisoni (PG14)(Choietal.,2004;Waalwijketal.,1985),whichare con-sideredtoxictoH.hampei(Méndez-Lópezetal.,2003).Therefore,
thesestudiesprovideevidenceoftheimportantroleofthesetoxins inactivityagainsttheorderColeoptera.
Thus,amongotherhypotheses,thetoxiceffectofthestrains againstH.hampeimight beduetotheaction ofCry4and Cyt1 proteins. Although only toxic to Diptera in vitro (Höfte and Whiteley,1989),theCyt1proteinshavealsoshowntheirpotential againstH.hampei,A.grandisandChrysomelascriptaFabricius,1801 (Coleoptera: Chrysomelidae), when individually tested against thesebeetles(FedericiandBauer,1998).
Inadditiontotheirinsecticidalactivity,theCyt1proteinsalso suppresshighselectionlevelsof C.scriptapopulationsresistant totheCry3Aprotein(FedericiandBauer,1998).Thisactionmay beduetothestructureanddifferentiatedmodeofactionofCyt proteins,whichmakethempowerfulalliesinthemanagementof cross-resistanceofinsectpestsbothinthecompositionofbiological insecticidesandtheconstructionofgeneticallymodifiedplants.
Delécluse et al. (1991) showed that the Cyt1A protein was notessentialfortheentomopathogenicactivityofB. thuringien-sissubsp.israelensisagainstA.aegyptiandCulexpipiensLinnaeus, 1758(Diptera:Culicidae),becausethesuppressionof thecyt1A generesulted ina strain similartothewildentomopathogenic strains.However,thefrequentoccurrenceofCyt1Aproteincrystals suggests that it may be a significant component of the ento-mopathogenicfeatureofB.thuringiensissubsp.israelensisstrains; forexample,insynergisticactioninconjunctionwithother pro-teinsorothervirulencefactorscapableofincreasingthetoxicityof thestrain(ChilcottandEllar,1988).
ThesynergismbetweenthetoxinsproducedbyB.thuringiensis subsp.israelensiswasalsoobserved betweentheCry4and Cyt1 proteinsbyTabashnik(1992)whoestimatedtheLC50valuesofCry4
andCyt1proteinsagainstA.aegyptilarvae.Thevalueswere10-fold lowerwhentheseproteinsweretestedincombinationthanwhen eachwasusedseparately.
GiventhefunctionsandroleoftheCyt1protein,theprotein islikelyanimportantcomponentoftheproteinassemblyofthe toxicstrainsagainstH.hampei.TheCyt1proteinshouldbetested separatelyandcombinedwithotherproteinsinfutureresearch. Similarly,inadditiontoassessingstraintoxicitytoapest,the syn-ergisticrelationshipsoftheCyt1proteinwithotherproteinsand theeffectonsuppressionordelayincasesofresistanceofinsects tootherproteinsofB.thuringiensisinsertedintotransgenicplants requirefurtherinvestigation.
Thisobservationmay assistresearchers indecidingwhether theinsertionofoneormorecryandcytgenesintocoffeeplants willyieldplantsmoreresistanttoH.hampei,becauseinaddition tothesynergismbetweentheCryandCytproteins,someauthors showthatexpressionofmultipleB.thuringiensistoxinscanreduce instancesofselectionofinsectpopulationsresistanttoCry pro-teins(Lietal.,2014;Moaretal.,1990).Facedwiththenecessityto studythegenepoolfoundinmorestrains,theknowledgeofeach crygenefunctionpublishedinpreviouspapersisofutmost impor-tancefortheselectionofgenesforthecontrolofH.hampei,which shallinitiallybesubmittedtobioassays,togetherandindividually. ThestrainsmosttoxictoH.hampeiselectedinthisstudy,except forstrainBR67,amplifiedthecry4Aandcry4Bgenes.Asshownin previousworks,thesegenesshowtoxicitynotonlytoDipterabut alsotoColeoptera,whichissignificant,becauseinadditiontothe importantindividualtoxicityofCry4AandCry4Bproteins,reports indicatesynergisticactivityagainstmosquitolarvae(Bravoetal., 2007).
Thecry11genethatwasamplifiedinalmostallevaluatedstrain, encodestheCry11proteinwhichisoneofthemostactive pro-teinsagainstA.aegypti (Crickmoreetal.,1995).However,when individuallytestedagainstthecoleopteranA.grandis,theprotein showedlow toxicity(Monneratetal.,2012).Additionally, stud-iesconductedbyMéndez-Lópezetal.(2003)revealedthatstrains
containingCry11proteins,i.e.,B.thuringiensissubsp.fukuokaensis (strainT03C-001),jegathesan(strainT28A-001)andmedellin(strain T30-001)(Crickmoreetal.,1998),werenottoxictoH.hampei.Thus, weinferredthatthecry11genealonemightnotberesponsiblefor thetoxicitytoH.hampeiinthepresentstudy.
TheCry3protein,whichhasknownbiologicalactivityagainst Coleoptera(VanFrankenhuyzen,2009),isalsotoxictoH.hampei (López-Pazosetal.,2009).Furthermore,thisproteinwasidentified inaB.thuringiensisstrainincoffeeplantationsinCostaRica(Arrieta etal.,2004).StrainBR67wastheonlyonetoobtainan amplifica-tionproductforthecry3genealone.However,theLC50valueof
thisstraindidnotdiffersignificantlyfromthoseofthestandard IPS-82strainandtheotherstrainsinthegroupofgreatertoxicity. StrainBR67alsoshowedasinglebandbetween65and70kDain theproteinprofile,whichcorrespondstothecry3gene.
IncontrasttotheresultsreportedbyMéndez-Lópezetal.(2003)
andthoseinthepresentstudy,López-Pazosetal.(2009)foundno mortalityofH.hampeicausedbyB.thuringiensissubsp. israelelen-sisIPS-82.Thelatterauthorsexplainedthattheabsenceofdeath wasmost likely due todifferences in theIPS-82 strain caused bychanges in thecoloniesof B. thuringiensis.Such divergences canresultfromdifferentconditions ofcropcultivation,suchas nutritionandincubationtemperature,whichleadtoalossof mul-ticellularattributes.Thesedissimilaritiesmayalsobeattributable toanymutationoccurringinthegenepromotersessentialforthe toxicityofthebacteria.
Othermethodologicaldivergencesmightalsohaveinfluenced theresults;forexample,thebioassaysconductedbyLópez-Pazos etal.(2009)wereperformedonlywithCryrecombinantproteins. Nevertheless, theworkof Méndez-López et al. (2003) and this study,withtheinitialobjectivetoanalyzetheentirecontentsofthe strains,usedsporeandcrystalmixtures.Thus,toxicitytotheinsect couldberelatednotonlytoCryproteins,butalsotothevirulence factors,suchasproteases,chitinases,proteinsin thevegetative phaseandCytproteins.Thesamplepurificationprotocolcanalso resultindifferencesbecausethetoxincontentcanvarybetween 20and90%inunpurifiedpreparations.Additionally,the purifica-tionmethodcaninfluencetheoutcomesincasesofcomparisonof toxicitywhenusingthesamestrain.
Microscopicanalysisofthemorphologyofcrystalsofastrain canprovideinformationonitsinsecticidalactivity(Lereclusetal., 1993;Saadounetal.,2001;Tailoretal.,1992).AllstrainstoxictoH. hampeiselectedinthisstudyhadthesamemorphologyofspherical crystals,whichusuallyidentifystrainstoxictoDiptera,ascitedby
Rohetal.(2007).
Amongthemosttoxicstrainsassessedinthiswork,theLC50
valuesof fourdidnot differsignificantlyfromthat ofthe stan-dardIPS-82.Amongthesestrains,BR58andBR137exhibitedhigh potentialtocontrolH.hampei,becausealthoughthemolecular pro-filedidnotdiffer,theyhadlowerLC50 valuesthanthestandard
strain.Ibarraetal.(2003)observedsimilarresultsinidentifying newstrainsofB.thuringiensiswithtoxicactivitytowardsA.aegypti, withfourstrainsthatweremoretoxicthanthestandardlevelsof B.thuringiensissubsp.israelensisbutpresenteda similargenetic profile.
Theabove resultsdemonstratethat bioassays of strainsand crygenesmustbeconductedagainstinsectsbothseparatelyand together,becauseevenwiththesamegenecontent,theycancause differentlevelsoftoxicity.Alternatively,strainsmaycontaingenes thatarethedeterminingfactorfortoxicityagainstthepestbutare notidentifiedbyPCR.
Inadditiontocryandcytgenes,othervirulencefactorsinB. thuringiensisstrainscancontributetobacteriatoxicity(Vilas-Bôas etal.,2012).Moreover,synergisticinteractionscanoccuramong theCryproteinsorevenamongtheseproteinsandspores.However, becauseof thevariationin toxicityofeachprotein,establishing
anindividualcontributiontothetoxiceffectofa strainis diffi-cult(GlareandO’Callaghan,2000;Visseretal.,1990).Thesetypes of interactioncouldalso explain thedifferentlevels of toxicity obtainedbythestrainstested,whichalthoughinasimilargene pool,showeddifferentLC50valuesforH.hampei.
Severalgroups have focused theirresearchonsearching for highlytoxicstrainsofB.thuringiensiscontainingmultipleCry pro-teinswiththeaimtoobtainspecificityinthecontrolofdifferent insectpestsandmoreoptionsforthemanagementofinsects resis-tanttocertaingenes(Bobrowskietal.,2003).Thus,strainsofB. thuringiensistoxictoH.hampeiexpressingadiversifiedgene con-tent,containingmorethanonegenethatmightbetoxictothepest, areofextremeimportanceforthemanagementofinsectresistance togeneticallymodifiedplants.
Asobserved inthis study,for mostof thetoxic strains, this genediversitymaypermittheuseofcombinedgenesbyadding theexpressionproductofmorethanonegenetobindtodifferent membranereceptorsinsertedinthesameplant(VanRie,1999). Consequently,theprobabilityofaninsectbecomingmore resis-tanttoatoxinwillbereduced,whichfavorslonger-termviabilityof geneticallymodifiedcoffeeplants(DegrandeandFernandes,2006). Furtherstudiesshouldconsiderthepossibilityofinsertingone ormoregenesintothecoffeeplanttocomplementthiswork.With theresultsobtainedtodate,i.e.,theselectionof11strains viru-lentagainstH.hampeiandidentificationoftheirgenepools,new insightswilllikelybeyieldedinfuturegeneticinvestigationsthat examinetheeffectofeachgene.Moreover,thesesurveyswillhelp researchersunderstandtherelationshipsamongthesestrainsand leadtofurtherstepstowardsincreasingtheirtoxicityoreven pre-venttheselectionofresistantinsects.Additionally,studiesaiming attheselectionofcrygenescanbeextendedtoincludeother impor-tantcoffeepests,suchasLeucopteracoffeella(GuérinMènevilleand Perrottet,1842)(Lepidoptera:Lyonetiidae).
Conclusions
Amongthe34strainsstudied,11causedmortalitytoH.hampei larvaethatexceeded96%.Fromtheselectivebioassay,strainsBR58, BR137,BR80andBR67hadthelowestLC50valuesanddidnotdiffer
fromthestandardIPS82.Theproteinprofilesproducedbandswith molecularmassesof28,65,70and130kDa.Themolecular char-acterizationshowedthepresenceofcry1,cry2,cry3,cry4A,cry4B, cry10,cry11andcyt1genes.Themorphologicalanalysisrevealed thatallstrainshadsphericalcrystals,verysimilartothosefound inB.thuringiensissubsp.israelensis.Asaresult,theseisolateshave potentialforbiotechnologicalcontrolofH.hampeiandshouldbe importantcandidatesformorestudiesandforuseasgenesources fortheconstructionoftransgeniccoffeeplants.
Conflictsofinterest
Theauthordeclaresnoconflictsofinterest.
Acknowledgments
TheauthorswishtothanktheConselhoNacionalde Desenvolvi-mentoCientíficoeTecnológico(CNPq)forfinancialsupport.
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