Bioassayscydia闪退

SynergisticinteractionbetweencarvacrolandBacillusthuringiensiscrystallineproteinsagainstCydiapomonellaandSpodopteraexiguaEdytaKonecka.
AdamKaznowski.
WeronikaGrzesiek.
PatrykNowicki.
El_zbietaCzarniewska.
JakubBaranekReceived:8August2019/Accepted:23March2020/Publishedonline:4April2020TheAuthor(s)2020AbstractTheaimofourstudywastodeterminetheusefulnessofmixturesofcarvacrolandBacillusthuringiensiscrystallineproteinsCryagainstpestsoftwodifferentspecies:CydiapomonellaL.
(Lepi-doptera:Tortricidae)andSpodopteraexiguaHu¨bner(Lepidoptera:Noctuidae).
Thenoveltyofourworkliesinshowingtheinteractionsbetweencarvacrolandbacterialtoxinsagainstinsectpests.
Moreover,wehavedemonstratedthatcarvacrolappliedviaingestionexertstoxicityagainstlepidopterancaterpillars.
WehaveshownthatthebotanicalcompoundandCryproteinsactinsynergyandtheirmixturesaremosteffectiveinreducingthenumberofL1andL3larvaewhenB.
thuringiensistoxinsconstituteupto0.
1%and0.
05%ofthemixtures,respectively.
Carvacrolandcrystallineproteinsactinsynergyinthesecombina-tionsandhavethepotentialtobeeffectiveinprotectingcropsagainstlepidopteranpests.
Thenatureoftheinteractionbetweenthecomponentsdependedontheproportionoftheirconcentrationsinthemixture.
MixturescontainingCrytoxinconcentrationsequalorhigherthan20%causedlowerobservedmortalityofinsectscomparedtotheexpectedone.
Furthermore,weshowedthatcrystallineproteinsofB.
thuringiensisMPUB9,carvacrolandtheirmixturedidnotaffectthemorphologyofinsecthaemocytes,andadditionally,hadnoeffectontheimmunesystem.
KeywordsBacterialtoxinsPlantsubstanceInsecticidalactivityBiopesticideSynergyIntroductionInterestintheuseofcommerciallyavailablebioin-secticidesandfutureprospectsforthedevelopmentofnewbiologicalpreparationsinplantprotectionhassignicantlyincreasedinrecentyears.
Thisisduetothefactthattheyareanenvironmentallysafealterna-tivetochemicalpesticidesandcanbeeasilydegradedwithlimitedpersistenceintheenvironment(Chat-topadhyayetal.
2017).
Additionally,thecurrentstrategyofIntegratedPestManagement(IPM)pro-gramsassumestheapplicationofallavailableplantprotectionmethods,givingprioritytonon-chemicalapproaches.
Biopesticidescontainnaturalproductsderivedfromanimals,plants,fungi,andbacteria.
Almost90%ofcommercialmicrobialinsecticidesareHandlingEditor:EvertonKortKampFernandesE.
Konecka(&)A.
KaznowskiW.
GrzesiekJ.
BaranekDepartmentofMicrobiology,FacultyofBiology,AdamMickiewiczUniversityinPoznan,UniwersytetuPoznanskiego6,61-614Poznan,Polande-mail:edkon@amu.
edu.
plP.
NowickiE.
CzarniewskaDepartmentofAnimalPhysiologyandDevelopment,FacultyofBiology,AdamMickiewiczUniversityinPoznan,UniwersytetuPoznanskiego6,61-614Poznan,Poland123BioControl(2020)65:447–460https://doi.
org/10.
1007/s10526-020-10011-4(0123456789().
,-volV)(0123456789().
,-volV)basedonaspore-crystalsmixtureofBacillusthuringiensis(DamalasandKoutroubas2018).
Plantproductsarealsorecommendedasefcientbioprepa-rationsagainstinsectpests(Koneckaetal.
2018b,2019;Mossa2016).
Moreover,recenttrendsincropprotectionsinvolvetheuseofinsecticidemixtures(Sharifzadehetal.
2018).
Thisapproachcanleadtothepreventionordelayofthedevelopmentofinsectresistance(Sudoetal.
2017),especiallywhenindividualcomponentsofpreparationshavedifferentmodesofaction(Zhuetal.
2016).
Thisstrategylimitscosts(Das2014)andreducesthedosageofprepara-tionswheninsecticidesenhanceeachother'sactivityandactinsynergy(WraightandRamos2005).
B.
thuringiensisstrainssynthesizeproteincrystalsduringsporulation.
Crystalstogetherwithbacterialsporesareeatenanddissolvedintotheinsectmidgut.
Inthemostcommonmodel,Cryproteinsafteractivationbymidgutproteases,bindtoreceptorsonthesurfaceofepithelialcellsanddestroythemidgut(Saiyad2017),allowingbacterialsporestoenterthebodyandcirculatorysystemofinsects,wherethesporesgerminateandbacteriamultiply(Argolo-FilhoandLoguercio2014).
Insecthaemocytesphagocytizebacterialcellsandformaggregatesthatmaybeencapsulatedbyotherhaemocytesorbycellsthatmaybereleasedfromtheaggregates.
Duringbacterialinfection,differentchangesinhaemocytestructureswereobservedinlepidopteraninsects.
Forexample,granulocytesweresurroundedbyprecipitatedhaemo-plasmandvarioussizesofclearvesicles.
Moreover,themembraneofgranulocyteswasdisruptedandthenucleuswasdistorted.
Bodiesresemblinglysosomeswereobservedinthecytoplasmofinfectedhaemo-cytes(El-AzizandAwad2010).
SomeofB.
thuringiensisstrainsproducehaemolysinwithlyticpropertiesthatinducedeathofinsecthaemocytesandmacrophages(Tranetal.
2013).
However,spore-freepreparationofcrystallineproteins,Cry1,Cry2,andCry9,didnotcausechangesinthemorphologyoflepidopteranhaemocytes(Koneckaetal.
2018a).
Carvacrolorplantextractswithcarvacrolasthemaincomponentareknowntoexertnegativeeffectsagainstcoleopteran(Szczepaniketal.
2018),homo-pteran(Zekrietal.
2016),dipteran(Andrade-Ochoaetal.
2018;deMesquitaetal.
2018;Giatropoulosetal.
2018),lepidopteran(Meloetal.
2018),neuropteran(Castilhosetal.
2018),andhemipteran(Gaireetal.
2019;Rizvietal.
2018)insects.
Thisphytochemicalwasshowntoexhibitcontact(Castilhosetal.
2018;Meloetal.
2018;Rizvietal.
2018),repellent(Giatropoulosetal.
2018),andfumigant(Gaireetal.
2019)toxicity.
Thecarvacrol-containingessentialoilshowedneurotoxiceffectsandwasfoundtoinhibittheacetylcholinesteraseenzymeinthecontactapplicationagainstinsectpests(deMesquitaetal.
2018;Rizvietal.
2018).
CarvacrolhasbeenalsoreportedtohaveinsecticidalactivityuponingestionagainstHemiptera(Parketal.
2017)andLepidoptera(Tangetal.
2011).
OnestudyincorporatedextractsofOriganumvulgare(Lamiales)andArtemisiadracunculus(Asterales)withhighcarvacrolcontentintotheinsectdietandtheplantcompoundcausedahighlossofbodyweightofAlphitobiusdiaperinus(Coleoptera)whenappliedthroughconsumption(Szczepaniketal.
2018).
Dataaboutthetoxicityofcarvacroltolepidopteraninsectsdeterminedbasedoningestionarestillnotcompre-hensive.
Themechanismofcarvacrolactionappliedtotheinsectintestinaltractisunknown.
TheaimofthisresearchwastodeterminetheusefulnessofmixturesofB.
thuringiensiscrystallineproteinsandcarvacrolagainstlepidopteranpests.
Wecombinedbacterialandplantproductsindifferentratiosandestablishedthenatureofinteractionsbetweenthem.
ThestudyappliedcrystalsoftwoB.
thuringiensisstrains:MPUB9andMPUB54.
TheMPUB9strainhadalargenumberandvarietyofgenescodingforinsecticidalcrystallineproteinsCry:cry1Aa,cry1B,cry1C,cry1D,cry1I,cry2Ab,cry9B,andcry9E(Koneckaetal.
2007b).
Moreover,massspectrometryanalysisrevealedthatMPUB9crystalscontainedCry1Aa,1Ba,1Ca,1D,and9Etoxins(Baraneketal.
2017).
Bacterialcrystalsaloneorwithsporesshowedhighinsecticidalactivityagainstinsectpests(Koneckaetal.
2018c,2015,2012,2007b).
B.
thuringiensisstrainMPUB54harboredthefollowinggenes:cry1Aa,cry1Ab,cry1C,cry1D,cry1I,cry2Ab,cry9B,andcry9E.
CrystalsofthestrainMPUB54exhibitedsignicantinsecticidalactivityagainstmoths(Koneckaetal.
2018c).
Theinsecticidalactivityofcrystal-carvacrolmix-tureswasdeterminedagainstinsectsoftwodifferentspecies:Cydiapomonella(Lepidoptera:Tortricidae)andSpodopteraexigua(Lepidoptera:Noctuidae).
C.
123448E.
Koneckaetal.
pomonellaisafruitpestandattacksmainlyapples,butalsopears,apricots,cherries,plums,peaches(Alstonetal.
2010),andevenwalnutsandchestnuts(Hagstrumetal.
2013).
Caterpillarsfeedonthesurfaceofthefruitforarelativelyshorttimeandsubsequentlyenterthefruitanddigtunnelsinsideit(Alstonetal.
2010).
Larvaeremainthereuntilcompletingtheirdevelopment(Pajacˇetal.
2011),andthusarehardlyavailablefortheactionofinsecticides.
Recently,adecreaseinthisinsectsensitivitytochemicalpreparationshasbeennoted(Grigg-McGufnetal.
2015).
S.
exiguaisaphy-tophagousspecies.
Itfeedsmainlyonvegetables,butalsoongrainsandornamentalplants,bothinopeneldsandingreenhouses.
Caterpillarsskeletonizefoliageandconsumefruits(Capinera1999).
Bothpestspeciescauseconsiderableeconomicagriculturallosses(Pajacˇetal.
2011;Huaetal.
2013).
DamagesofplantsduetoeatingplantseedlingsandcottonbollscausedbyS.
exiguaslowthegrowthofcommercialcropssuchascottonandreducecropyield,whichhasnegativeimpactonthetextileindustry(Huaetal.
2013).
C.
pomonellainfestationcanreach60–80%forpearsandapples,respectively,inorchardswithoutpestcontroltreatment(Vreysenetal.
2010).
Ithasbecomeaworldwidepestduetothevarietyofhostsandtheincreaseininternationalfruittrade(Thaleretal.
2008).
Theinsectimmunesystemcanbeatargetfordevelopmentofnovelstrategiesofsuppressingpestviability(Czarniewskaetal.
2019a),butthereisnoinformationhowcarvacrolandamixtureofcarvacrolandcrystallineproteinsofB.
thuringiensisaffectinsect'simmunity.
Inthiswork,weexaminedtheinvivoeffectofcarvacrolandthemixtureofcarvacrolandbacterialCryproteinsonmorphology,viability,andcellularimmuneresponse(phagocytosisandnoduleformation)ofthethirdinstarofS.
exiguacaterpillarstodeterminewhethercarvacrolandamixtureofcarvacrolwiththebacterialtoxinsarecytotoxictoimmunocompetentcellsandaffectinsectimmunity.
Forthispurpose,weusedaverysensitivehaemocytebiotestthatwepreviouslydeveloped,throughwhichitispossibletodetectchangesinthemorphology,adhesion,viability,andimmunologicalfunctionofhaemocytesthatcanbeinducedbyvariousbioticandabioticfactors(Czarniewskaetal.
2012,2018,2019a,2019b;Kuczeretal.
2016;Koneckaetal.
2018a;Kowalik-Jankowskaetal.
2019).
OurresultsshouldexplainthemechanismofinteractionbetweencarvacrolandB.
thuringiensisCryproteinsinthemixtureagainstinsects.
MaterialsandmethodsBacteriaTwoBacillusthuringiensisstrainswereusedinthestudy:MPUB9andMPUB54.
ThestrainsweredepositedintheBacteriaCollectionoftheDepartmentofMicrobiology,AdamMickiewiczUniversityinPoznan,Poland.
B.
thuringiensisMPUB9wasisolatedfromtheintestinaltractofdeadcaterpillarofcodlingmothCydiapomonelladuringepizooticscausedbynaturallyoccurredbacterialinfectioninthelabora-tory-culturedlineoftheinsect(Koneckaetal.
2007a).
B.
thuringiensisMPUB54wasculturedfromsoilsample(Koneckaetal.
2018c).
PlantsubstanceAplantsubstance–carvacrol–wasusedinthestudy.
Thepreparationofcarvacrol(purityC98%)waspurchasedfromSAFC,St.
Louis(USA).
InsectsTheactivityofbacterialcrystalsandcarvacrolweredeterminedagainsttwolepidopteranspecies:thecodlingmothC.
pomonellaL.
andthebeetarmywormSpodopteraexigua(Hu¨bner),representingTortricidaeandNoctuidae,respectively.
CaterpillarsofcodlingmothandbeetarmywormcamefromstandardizedlaboratoryculturelinesrearedattheFacultyofBiologyatAdamMickiewiczUniversity,Poznan,Poland.
Insectswererearedonasyntheticmedium(McGuireetal.
1997)at26°CwithaL:D16:8photoperiodand40–60%RH.
DeterminationofinteractionbetweenbacterialcrystallineproteinsandcarvacrolagainstinsectsIsolationofB.
thuringiensiscrystallineproteinsTheisolationprocedurewasdescribedpreviously(Koneckaetal.
2012,2015,2019).
Thestrainswereculturedonamediumforbacteriasporulation123SynergisticinteractionbetweencarvacrolandBacillusthuringiensiscrystallineproteinsagainst…449(LecadetandDedonder1971)at308Cforsevendays.
Thecollectedcrystalsandsporesweresuspendedin1MNaCl,10mMKCl,pH7.
5.
Subsequently,thecrystalswereseparatedfromthespore-crystalmix-turesbysucrosedensitygradientcentrifugation(Guzetal.
2005).
Crystalslayerwascollected,washedinsteriledistilledwaterandweighed.
ThepurityofcrystalswascheckedbystandardlightmicroscopywhichwasprecededbyastainingwithamidoblackandZiehl'scarbolfuchsin(Smirnoff1962).
Estimationofinsecticidalactivityofcarvacrol,B.
thuringiensiscrystallineproteinsandtheirmixturesCarvacrolwashomogenizedwithTween80(1:0.
5ratio).
Then,thedilutionsofthebotanicalpreparation,B.
thuringiensisproteins,andtheirmixtureswerepreparedinsteriledistilledwater(Table1)andappliedtoC.
pomonellaandS.
exiguacaterpillarsoftherstinstar(L1)andadditionallytoS.
exigualarvaeofthethirdinstar(L3).
CaterpillarsofC.
pomonellaandS.
exiguaoftherstinstarwerefedwith:(1)carvacrol,(2)B.
thuringiensisMPUB9Cryproteins,(3)B.
thuringiensisMPUB54Cryproteins,(4)mixturesofcarvacrolorMPUB9proteins,and(5)mixturesofcarvacrolandMPUB54proteins.
Theactivityofeachconcentrationofthepreparationswasdeterminedfor30C.
pomonellacaterpillarsofrstinstar(threesimultaneousreplicateswithtenlarvaeeach)or30S.
exiguacaterpillarsofrstinstar(threesimultaneousreplicateswithtenlarvaeeach).
S.
exigualarvaeofthethirdinstarwerefedwith(1)carvacrol,(2)B.
thuringiensisMPUB9Cryproteins,or(3)mixturesofcarvacrolandMPUB9proteins.
Theactivityofeachconcentrationofthepreparationswasdeterminedfor30S.
exiguacaterpillarsofthirdinstar(threesimultaneousreplicateswithtenlarvaeeach).
BioassayswithC.
pomonellaandS.
exiguawereconductedininsectcultureboxes,asdescribedpreviously(Koneckaetal.
2012,2015,2018a,c,2019).
Eachboxwasdividedinto12separatewells–onewellperonelarva.
Syntheticmediumforcodlingmothandbeetarmywormrearingwasplacedintothewells.
ItscompositionwasthesameasdescribedbyMcGuireetal.
(1997),butwithoutformaldehydetoavoiditseffectontheactionofCryproteins.
Consideringinsectbiology,themediumwaspouredintowellsorformedintocylindricalpiecesof5mmindiameterandaheightof3mmforC.
pomonellaorS.
exiguabioassays,respectively.
Eachdilutionofbotanical,bacterial,andmixedpreparationswasappliedonthedietsurfaceinavolumeof10llforS.
exiguaor50llforC.
pomonellabioassays.
Differentvolumeswereusedtorefertovariousspeciesduetotheuseofvarioussurfaceareasofthemediumontowhichthepreparationwaspoured.
Bioassayswithallmixedpreparations,aswellastheirindividualcomponents,atconcentrationsatwhichtheywereincorporatedintothemixturewereper-formedsimultaneously.
TheTween80inconcentra-tionof10mgml-1andsteriledistilledwater,insteadoftheinsecticidalsubstance,wasgivento30cater-pillarsasanegativecontrol.
Insectswererearedat26°CwithaL:D16:8photoperiodand40–60%RH.
Sevendaysafterpreparationapplications,deadandliveinsectswerecounted.
Calculationofthesynergism,antagonismandadditionbetweencarvacrolandcrystallineproteinsagainstpestsTheexpectedmortalityofinsects(MEXP)causedbythemixtureoftheplantcompoundandB.
thuringien-siscrystallineproteinswascalculatedaccordingtotheequation:MEXP1BC100%,whereBistheproportionofinsectsthatsurvivedtheexposuretocrystallineproteinsinrelationtothetotalnumberofinsectsused,andCistheproportionofinsectsthatsurvivedtheexposuretocarvacrolinrelationtothetotalnumberofinsectsused(Tabashniketal.
2013).
Thecharacteroftheinteractionbetweentheplantandbacterialsubstancesagainstinsectswasdeter-minedaccordingtoTabashniketal.
(2013).
Syner-gismbetweencomponentswasrecordedwhenthevalueoftheobservedmortalitywasstatisticallysignicantlyhigherthanthevalueoftheexpectedmortalityofinsects.
Antagonismwasobservedwhentheobservedmortalitywasstatisticallysignicantlylowerthantheexpectedone.
Ifthedifferencebetweenthevaluesoftheexpectedandobservedmortalitywasnotstatisticallysignicant,theinteractionwasrecog-nizedasadditional.
Fisher'sexacttest(https://www.
graphpad.
com/quickcalcs/contingency1/)wasemployedtodeterminestatisticallysignicant123450E.
Koneckaetal.
differences(P\0.
05)betweentheresults(Tabashniketal.
2013).
ImpactofCryproteins,carvacrolandtheirmixtureonhaemocytesandinsectcellularimmuneresponseWeexaminedthehaemocytes'morphology,viability,adhesion,andphagocyticactivityaswellasnoduleformationintheS.
exiguaL3larvaefedwith(1)250ngofbacterialtoxinsperlarva,(2)500,000ngofcarvacrolperlarva,or(3)theirmixture.
Tween80inconcentrationof10mgml-1insteadoftheinsectici-dalsubstancewasusedasanegativecontrol.
Thehaemocytes'morphology,viability,andadhesionstudywasperformedaccordingtothemethoddescribedbyCzarniewskaetal.
(2012)andKoneckaetal.
(2018a).
Thephagocyticabilityofhaemocytesofcontrolandexperimentalcaterpillarswasanalyzedinvivobyusinguorescentlatexbeads(Sigma-AldrichL1030)accordingtothemethoddescribedbyCzarniewskaetal.
(2019a).
Caterpillarsanaesthetizedanddisinfectedwithethanolwereinjectedwith2lloflatexbeadssuspendedinphysiologicalsolution(274mMNaCl,19mMKCl,9mMCaCl2)(2:1000v/v)throughtheabdominalforelegbyusingaHamiltonsyringe(HamiltonCo.
,Reno,Nevada,USA).
Thecaterpillarswereanaesthetizedagain,disinfectedandwashedindistilledwatertwohoursaftertheinjectionofthelatexbeadsandthesamplesofhaemolymph(2ll)werecollectedanddilutedin20llofsaline.
Haemolymphsampleswereincubatedonthepoly-L-lysine-coatedglasses(SigmaP4707)for30minatroomtemperatureinthedark.
Thehaemo-cyteswerewashedwithsalinetoremoveunphagocy-tizedbeadsandstainedwithDAPIsolutionfor5mininthedarktovisualizecellnuclei.
PhagocyticactivityofhaemocyteswasexaminedwithaNikonEclipseTE2000-Uuorescencemicroscope.
ThephotosofhaemocyteswerecapturedwithaNikonDS-1QMdigitalcameraandanalyzedwiththeImageJsoftware(version2).
Theresultswereexpressedasapercentageofphagocytichaemocytesinthetotalnumberofhaemocytesintheimages.
NoduleformationinthehaemoceolofcontrolandexperimentalcaterpillarswasstudiedaccordingtothemethoddescribedbyCzarniewskaetal.
(2018).
Briey,caterpillarsanaesthetizedanddisinfectedwithethanolwereinjectedwithStaphylococcusaureus(SigmaS2014SaintLouis,Missouri,USA)suspen-sioninphysiologicalsaline(2ll,2:1000v/v)throughtheabdominalproleg.
Caterpillarsweredissectedtwodaysaftertheinjectionofbacteriatoexposethenodulesonthedorsalsideofthehaemocoel.
Forthispurpose,theinsect'sbodywaspinnedontoaSYLGARDRlledPetridish,andthenthefatbody,Malpighiantubules,andthedigestivetractwereremoved.
ThenumberofnoduleswascountedbyusinganOlympusSZX12stereoscopicmicroscopy(OlympusCo.
,Tokyo,Japan),andthreeimagesofeachcaterpillarwerecapturedwithanOlympusU-LH100HGdigitalcamera(OlympusCo.
,Tokyo,Japan).
Thenumberofnodulesonthedorsalsideofthehaemocoelwascountedinallinsects.
TheimageswereanalyzedwiththeImageJ(version2)software.
ResultsDifferenttypesofinteractionswereobservedbetweenB.
thuringiensiscrystallineproteinsandcarvacroldependingontheconcentrationproportionofthecomponentsinthemixtures.
ThecombinationofB.
thuringiensistoxinsandplantcompoundinaratioof1:25,000and1:50,000resultedinasynergybetweentheseproductsagainstCydiapomonellaL1andcausedfrom1.
5-to1.
9-foldhigherinsectmortalitythanexpected,dependingonbacterialstrainCryproteinsused.
ThesametypeofinteractionwasobservedagainstS.
exiguarstandthirdinstarswhentheconcentrationsofcarvacrolinthemixtureswererelativelyhigher.
Theconstituentscombinedin1:1000,1:2000,and1:10,000ratiosactedinsynergyagainstS.
exiguarstinstarandcausedfrom1.
5to1.
8-foldhigherinsectmortalitythanexpected.
Syner-gisticinteractionswerealsonotedbetweencrystallinetoxinsandthephytochemicalin1:2000and1:5000ratiosagainstS.
exiguaL3,astheobservedinsectmortalitywasfrom2to2.
4-foldincomparisontotheexpectedrate.
Bacterialandplantproductsinaratioof1:2000exhibitedsynergismagainstS.
exiguaL1andthethirdinstarlarvaeofS.
exigua(Table1).
123SynergisticinteractionbetweencarvacrolandBacillusthuringiensiscrystallineproteinsagainst…451Table1ActivityofB.
thuringiensiscrystallineproteins,carvacrol,andtheirmixturesagainstlepidopteraninsectsInsectPreparationConcentration(ngperinsect)*ProportionCry:carvacrolMortality(%)P**EffectObserved(±SE)ExpectedC.
pomonellaL1CryMPUB92010––20(±5.
77)10(±0)––––––CryMPUB542010–––56.
7(±3.
33)46.
7(±8.
82)––––––Carvacrol500,0005000––23.
4(±3.
33)10(±5.
77)––––––CryMPUB9carvacrol20500,00020500010500,0001050001:25,0001:2501:50,0001:50070(±0)30(±5.
77)60(±5.
77)23.
4(±3.
33)38.
42831190.
037(s)1.
00(ns)0.
037(s)1.
00(ns)SynergisticAdditiveSynergisticAdditiveCryMPUB54carvacrol20500,000205000105000001050001:25,0001:2501:50,0001:50096.
7(±3.
33)83.
4(±3.
33)93.
4(±3.
33)56.
7(±8.
82)66.
160.
458.
451.
40.
006(s)0.
084(ns)0.
048(s)0.
796(ns)SynergisticAdditiveSynergisticAdditiveControl***––0–––S.
exiguaL1CryMPUB950010050–––13.
4(±3.
33)13.
4(±6.
66)3.
4(±3.
33)–––––––––CryMPUB54200100––30(±11.
55)26.
7(±8.
82)––––––Carvacrol100,0001,000500–––36.
7(±8.
82)20(±10)13.
4(±3.
33)–––––––––CryMPUB9carvacrol500100,0005001000500500100100,00010050050100,000505001:20001:21:11:10,0001:51:20001:1073.
4(±3.
33)13.
4(±6.
66)20(±5.
77)66.
7(±3.
33)10(±0)66.
7(±3.
33)20(±5.
77)44.
330.
424.
344.
324.
337.
915.
60.
035(s)0.
209(ns)1.
00(ns)0.
038(s)0.
299(ns)0.
037(s)1.
00(ns)SynergisticAdditiveAdditiveSynergisticAdditiveSynergisticAdditiveCryMPUB54carvacrol200100,0002001000200500100100,0001005001:5001:51:2.
51:1,0001:560(±10)33.
4(±3.
33)30(±10)83.
4(±3.
33)30(±10)57.
146.
441.
753.
635.
61.
00(ns)0.
0430(ns)0.
422(ns)0.
047(s)0.
785(ns)AdditiveAdditiveAdditiveSynergisticAdditiveControl***––0–––S.
exiguaL3CryMPUB9500250–10(±10)3.
4(±3.
33)––––––Carvacrol500,000250,000––20(±5.
77)13.
4(±3.
33)––––––123452E.
Koneckaetal.
Reducedconcentrationsofcarvacrolinmixturestoratioslowerthan1:1000resultedintheadditiveeffectbetweenB.
thuringiensisproteinsandtheplantsubstanceagainstpests.
Similarvaluesoftheobservedandexpectedinsectmortalitywererecordedinthemajorityofsamplesshowinganadditiveeffectbetweenthecomponents,inwhichCrytoxinsandthephytochemicalweremixedin1:10,1:250,and1:500ratios.
However,asignicantdecreaseinthevalueoftheobservedmortalityincomparisontotheexpectedonewasnotedinabioassaywithmixturescontaining71%orlowercarvacrolconcentration.
Thevaluesoftheobservedmortalitywerefrom1.
05-toeven2.
3-foldlowerthantheexpectedmortalitywhenbacterialandbotanicalproductswereappliedin1:1,1:2,1:2.
5,and1:5ratiostotheinsects(Table1).
B.
thuringiensistoxins,carvacrol,andtheirmixturehadnoeffectonthemorphology,viability,adhesion,andimmunologicalfunctionofinsecthaemocytes.
InsecticidalagentsdidnotexertapoptoticandcaspaseactivityinS.
exiguahaemocytes.
Cellshrinkageandfragmentationofnucleiwerenotvisibleinthehaemocytes.
Thecytoskeletonofthecellswaswelldeveloped,andthushaemocytesadheredtothecoverslipandformednumerouslopodia,similarlyascontrolhaemocytes(Fig.
1).
Additionally,nodiffer-encesincellularimmuneresponsewereobservedincaterpillarstreatedwithbacterialproteins,plantcompound,andtheirmixtureincomparisontothecontrolinsects.
Thepercentageofphagocytichaemo-cytes(Fig.
2)andthenumberofnodulesformedininsecthaemocoelduringimmuneresponsedidnotdiffersignicantlywhencomparedtothecontrolinsects(Fig.
3).
DiscussionThedemonstrationofinteractionsbetweenB.
thuringiensiscrystallineproteinsandcarvacrolagainstinsectpestsisthenoveltyofourresearch.
Addition-ally,wehaveshownthatcarvacrolappliedviaingestionistoxictolepidopterancaterpillars.
AlthoughtherearestudiesonthetoxicityofmixturesofB.
thuringiensisandplantproductsagainstpests(Abedietal.
2014;Amizadehetal.
2015;Nouri-Ganbalanietal.
2016),noneofthemconcerntheuseofcarvacrolasacomponentofthesecombinations.
OurstudyisinlinewiththesearchforeffectivebiologicalplantprotectionproductsandharmonizeswithIPMprograms.
Theuseofamixtureoftwoinsecticidalsubstancesthatactsynergisticallyismoreprof-itablethanemployingtwodifferentpreparationsinseparateapplications.
Ourapproachwillnotonlyreducethecostsofplantprotection,butalsoenableapplyinglowerdosesofpesticidesandreducingtheoccurrenceofinsectresistance,whichisofgreatimportanceduetotheemergenceofinsectresistancetoCryproteins(PeraltaandPalma2017).
OurresearchshowedthatCrytoxinsandcarvacrolactedinsynergyandtheirmixtureswerethemosteffectiveinreducingthenumberofL1andL3pestsTable1continuedInsectPreparationConcentration(ngperinsect)*ProportionCry:carvacrolMortality(%)P**EffectObserved(±SE)ExpectedCryMPUB9carvacrol500500,000500250,000250500,000250250,0001:50001:5001:20001:100056.
7(±3.
33)26.
7(±8.
82)53.
4(±3.
33)20(±11.
55)2821.
722.
415.
60.
035(s)1.
00(ns)0.
033(s)1.
00(ns)SynergisticAdditiveSynergisticAdditiveControl***––0–––ssignicantatP\0.
05,nsnon-signicant*ngofCryin50llor10llofpreparationappliedviaingestionagainstC.
pomonellaorS.
exigua,respectively(seeMethodsfordetails)**ProbabilitythatthedifferencebetweenobservedandexpectedmortalityoccurredbychancebasedonFisher'sexacttest(Tabaschniketal.
2013)***TheTween80inconcentrationof10mgml1andsteriledistilledwater,insteadoftheinsecticidalsubstance,wasgiventoinsectsasanegativecontrol123SynergisticinteractionbetweencarvacrolandBacillusthuringiensiscrystallineproteinsagainst…453whenbacterialtoxinsconstitutedupto0.
1%and0.
05%ofthemixtures,respectively.
Theuseofthesemixturescausedanincreaseintheobservedinsectmortality,uptoapproximately1.
9-foldincomparisonwiththeexpectedone.
However,thetypeoftheinteractionbetweenthecomponentsdependedontheproportionoftheirconcentrationsinthemixture.
IncreasingtheconcentrationofCryproteinsdidnotresultinasynergisticeffectbetweenbacterialandplantproducts.
MixturescontainingconcentrationsofB.
thuringiensistoxinsequalorhigherthan20%causedlowerobservedinsectmortalitythanexpected,asifcarvacrolpreventedtheactionofbacterialtoxinsorreducedtheireffects.
Differenttypesoftheinteractionsbetweensubstancesdependingontheconcentrationofcomponentswerealsoobservedinotherstudies(Koneckaetal.
2018a;RajguruandSharma2012).
WeproposetheuseofacombinationofB.
thuringiensistoxinsandcarvacrolagainstlepi-dopteranpests,becausetheyareconsideredresponsi-bleforthelossofsignicantamountsofplantcrops(Culliney2014).
BacterialproteinsfromtheCry1,Cry2,andCry9groups,whichareproducedbyB.
thuringiensisMPUB9andMPUB54,aswellascarvacrol,werefoundtobeinsecticidalsubstanceswithnonegativeeffectonotheranimalsthanplantpests(Gaoetal.
2018).
Inaddition,thesesubstancesdonotpersistintheenvironmentforalongtime(Mossa2016).
CommercialbiopesticidesbasedonFig.
1FluorescencemicroscopyimagesofhaemocytesofSpodopteraexiguaL3(negativecontrola),fedwith250ngofBacillusthuringiensisCryproteins(b),500,000ngofcarvacrol(c),andtheirmixture(d).
AllhaemocyteswerestainedwiththeSR-VAD-FMKreagentforcaspaseactivitydetection(noredcolor–noactivecaspase),withOregonGreen488phalloidinforF-actincytoskeleton(greencolor)stainingandwithDAPIforDNAstaining(bluecolor)123454E.
Koneckaetal.
B.
thuringiensiscontainbacterialspores.
Wesuggesttheusageofaninsecticidalpreparationwithnobacterialsporesduetothepossibilityofsynthesisofvertebratevirulencefactorsbyvegetativebacterialcellsthatgerminatefromsporesintheinsectbody(Kimetal.
2015).
Thus,inthisstudy,weisolatedB.
Fig.
2Fluorescencemicroscopyimagesshowingthephago-cytosisofuorescentlatexbeads(greencolor)byhaemocytesofSpodopteraexiguaL3(negativecontrola),fedwith250ngofBacillusthuringiensisCryproteins(b),500,000ngofcarvacrol(c),andtheirmixture(d).
AllhaemocyteswerestainedwithDAPItovisualizeDNA.
Thegraphshowsthepercentageofphagocytichaemocytesinhaemolymphofthestudiedcaterpil-lars(e)(means±SE,n=5).
Barswiththesameletterdonotdiffersignicantly(PC0.
05)123SynergisticinteractionbetweencarvacrolandBacillusthuringiensiscrystallineproteinsagainst…455thuringiensiscrystalsfromspore-crystalmixturesbysucrosedensitygradientcentrifugation.
Mixturescanbeadvantageouscomparedtoindi-vidualconstituents,becausetheymayhavedifferentmodesofactionandmaydelaythedevelopmentofresistance.
ThegeneralmodeofactionofB.
thuringiensisCryproteinsisknown(Jouzanietal.
2017;Saiyad2017).
However,manyimportantques-tionsconcerningthemechanismbywhichinsectcellsarekilledbysomeofthecrystallinetoxinsremainpoorlyunderstood(Vachonetal.
2012).
MostCryproteinsdamageepithelialcellpermeabilityandcellularintegrityoftheinsectmidgut(Meloetal.
2016).
Themodeofactionofcarvacrolappliedtoinsectsviatheintestinaltrackisnotexplained.
Itremainsunresolvedwhetherthephytochemicalisinvolvedinthedestructionofinsecttissuesoftheintestinaltrackorwhetherithasanimpactonothercellsthatwereexposedtocarvacrolafterthedestruc-tionofmidgutcellsbyCryproteins.
WeattemptedtoexplainthelatterpossibilityanddeterminetheimpactofcarvacrolanditsmixtureswithMPUB9Cryproteinsoninsecthaemolymphcells.
TheeffectofB.
thuringiensisMPUB9toxinsonthemorphologyandviabilityofinsecthaemocyteshasbeendeterminedpreviously(Koneckaetal.
2018a).
However,weperformedsimilarexperimentsinthisstudyasacomparativeassay.
Tothebestofourknowledge,thereisnodataontheinuenceofcarvacrolanditsmixturewithbacterialtoxinsoninsecthaemocytes.
Furthermore,wealsoconsideredtheconsequencesfortheinsect'simmunesystem,resultingfromtheactionoftheinsecticidalagents.
WeshowedthatcrystallinetoxinsofB.
thuringiensisMPUB9,carvacrol,andtheirmixturedidnotaffectthemorphology,viabilityofinsecthaemocytes,and,additionally,theyhadnoFig.
3ImagesshowingthenoduleformationinhaemocoelofSpodopteraexiguaL3(negativecontrola),fedwith250ngofBacillusthuringiensisCryproteins(b),500,000ngofcarvacrol(c),andtheirmixture(d)inducedbyStaphylococcusaureussuspensioninjection.
Arrowsindicatenodules.
Thegraphshowstheactivityofcellularresponseinhaemocoelofthestudiedcaterpillars(e).
Thenumberofnoduleswasthedeterminantofcellularresponse(means±SE,n=5).
Barswiththesameletterdonotdiffersignicantly(PC0.
05)123456E.
Koneckaetal.
effectsontheimmunologicalsystem.
Thismayindicatethatcarvacrolactedearlier,beforetheintegrityofthemidgutcellswasdisturbedoraffectedinotherinsecttissuesthanthehaemolymph.
IntopicalandfumigantbioassaysperformedbyGaireetal.
(2019),carvacroldemonstratedneuro-inhibitoryeffectsinhemipteraninsect.
Rizvietal.
(2018)showedthatcarvacrolinhibitedacetylcholinesteraseactivityininsectsofthesameorderbycontactapplication.
SimilaractivityofcarvacrolwasfoundagainstDiptera(deMesquitaetal.
2018;Parketal.
2016).
Inourresearch,therouteofcarvacrolappli-cationwasdifferentandtheinsectsusedrepresentedadifferentorderincomparisonwiththeotherstudiesmentionedabove.
ThemodeofactionofingestedcarvacrolaswellastheexplanationofthesynergybetweenCryproteinsandcarvacrolagainstLepi-dopteraneedselucidationbyadditionalexperimentalstudies.
Inconclusion,thepresentstudyhasrevealedforthersttimethatcrystallineprotein-carvacrolmixtures,containingCrytoxinsinconcentrationsupto0.
05%indietaryapplicationsexhibitstrongerinsecticidalactiv-itiesascomparedtothecomponentsassessedsepa-rately.
Thebacterialandplantproductsactinsynergyinthesecombinationsandhavethepotentialtobeeffectiveinprotectingcropsagainstlepidopteranpests.
CompliancewithethicalstandardsConictofinterestTheauthorshavedeclaredthattheyhavenoconictofinterest.
Researchinvolvinghumanand/oranimalrightsThisarti-cledoesnotcontainanystudieswithhumanparticipantsoranimals(vertebrates)performedbyanyoftheauthors.
OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.
0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproductioninanymediumorformat,aslongasyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicence,andindicateifchangesweremade.
Theimagesorotherthirdpartymaterialinthisarticleareincludedinthearticle'sCreativeCommonslicence,unlessindicatedotherwiseinacreditlinetothematerial.
Ifmaterialisnotincludedinthearticle'sCreativeCommonslicenceandyourintendeduseisnotpermittedbystatutoryregulationorexceedsthepermitteduse,youwillneedtoobtainpermissiondirectlyfromthecopyrightholder.
Toviewacopyofthislicence,visithttp://creativecommons.
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Insects7(1):2EdytaKoneckaisanassociateprofessor,full-timeworkingatDepartmentofMicrobiology,FacultyofBiology,AdamMickiewiczUniversityinPoznan,Poland,oninsecticidalactivityofBacillusthuringiensiscrystallinetoxins,theuseful-nessofbacteriainplantprotection,andmaternallyinheritedmicrobialendosymbiontsinArthropoda.
AdamKaznowskiisaprofessorinMicrobiology,theHeadofDepartmentofMicrobiologyandtheHeadofInstituteofExperimentalBiologyatAdamMickiewiczUniversityinPoznan,Poland.
Hisworkfocusesonthemechanismsofbacterialpathogenicity,antibioticsresistance,andtheuseofbiologicalcontrolagentsasinsecticides.
WeronikaGrzesiekisagraduatestudentofbiologyattheFacultyofBiology,AdamMickiewiczUniversityinPoznan,Poland.
Hermaster'sthesisweredevotedtotheanalysisoftheCry-carvacrolmixturesactivityagainstSpodopteraexigua.
PatrykNowickiisaPhDstudentattheDepartmentofAnimalPhysiologyandDevelopment,AdamMickiewiczUniversityinPoznan.
Heisworkingonthedetectionofnewactivitiesofinsectpeptidesandtheiranaloguesininsects.
El_zbietaCzarniewskaisanassociateprofessorattheDepartmentofAnimalPhysiologyandDevelopment,Adam123SynergisticinteractionbetweencarvacrolandBacillusthuringiensiscrystallineproteinsagainst…459MickiewiczUniversityinPoznan.
Herscienticactivityisfocusedonidentifyingmolecules,e.
g.
insectpeptides,withastrongeffectonreducingtheviabilityandreproductionofharmfulinsectsinordertodesignbioinsecticides.
JakubBaranekreceivedhisPhDdegreeinbiologicalsciences,biotechnologydiscipline.
HewasaresearchfellowatCharlesSturtUniversity,Australia.
CurrentlyheisanassistantprofessorattheDepartmentofMicrobiology,AdamMickiewiczUniversityinPoznan,Poland.
Hisresearchworkisfocusedonbiologicalcontrolofinsectpestsandchitinolyticactivityofmicroorganisms.
Publisher'sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalafliations.
123460E.
Koneckaetal.