Kevin O’Donnell, Individual Experimental Project, UM Master Beekeeping Course, August, 2018

Negative behavioral effects from Oxalic Acid Vaporization on honey bee colony vitality, brood, and honey production in various

sized hives containing VSH queens.

First,Let’sTalkAboutTheEnemy•  Varroa Destructor is considered the leading cause of colony morbidity

and the major factor responsible for colony losses worldwide. 1 •  Varroa mites are parasitic and vector a multitude of diseases, viruses

and bacterial infections, causing deformities, immune deficiencies, behavioral changes, weakening and death to entire colonies

•  Varroa are easily spread hive to hive through movement of migratory pollination services, transporting bees or frames within or between apiaries, swarming, foraging, robbing weaker hives during a dearth, drift, feral and untreated colonies, and by transient drones

•  During active brood seasons left unchecked and untreated, mite loads grow exponentially and can quickly overwhelm a hive even though the manifestation of symptoms may not be recognizable for weeks or months – and by then it’s generally too late!

•  Varroa has cunningly adapted its reproductive cycle to mimic the honeybee lifecycle to optimize survivability

•  Varroa have shown to develop resistance to treatments 1 Martin, S.J., Highfield, A.C., Brettell, L., Villa-lobos, E.M., Budge, G.C., Powell, M., Nikaido, S., and Schroeder, D,C. (2012) Global honey bee viral landscape altered by a parasitic mite. Science. 336: 1304-1306.

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TheenemyDCBC11/27/18

MiteInfestaConSymptoms

•  Impairedflightperformance•  LowerrateofreturntothecolonyaKerforaging•  Reducedlifespan•  Significantlyreducedweightofworkerbees•  ReducConinthehoneybeepopulaCon•  IncreaseddriK•  Reducednectar/honeystorage•  Increasedsupersedureofqueenbees•  ScaOeredandabnormalbroodpaOern•  Sunken,darkened,andchewedcappings•  LarvaslumpedintheboOomorsidesofcells•  Crippled,deformedandcrawlinghoneybeesonframes•  Paralysis•  “k”wing,andshriveledwings(DWV)•  DiscoloraConofadultbees•  AccelerateddeclineinpopulaConorabsconding•  Eventualcolonybreakdownanddeath

VisiblyUndetectable

OvertlyVisible

DoomedtoFail

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LifecycleofV.destructor•  Their entire lifecycle is dependent on their host, the honey bee •  They feed and reproduce solely on honey bees •  Two Stages of Life: Phoretic and Reproductive

–  Phoretic Stage – adult females hitch a ride on adult honey bees. Their only source of food is honey bee hemolymph (bee blood).

–  During brood season this stage lasts 11-14 days.1 They may stay with a single host bee or scurry on to multiple hosts

–  During broodless periods (winter), adult female varroa, although not able to reproduce, can survive between the sclerites (exoskeleton) in a phoretic stage feeding off hemolymph for 5-6 months, 2 especially in cold climates

1&2Ayers,G.S.,etal.,TheHiveandtheHoneyBee,Dadant&Sons,2018ed.p.853

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LifecycleofVarroaDestructor

–  Reproductive Stage – an adult female (foundress) enters a single brood cell (prefers drone cells) containing a larva just prior to capping or while being capped.

–  She submerges under the larva in the liquid mix of royal jelly / bee bread and erects snorkel-like tubes (peretrimes) above the surface of the liquid to breathe

–  Once the cell of the developing larva is capped by workers, the foundress climbs onto the bee larva and begins feeding and after about 60 hours lays a single haploid (male) egg

–  Every 25-30 hours thereafter she lays another egg. All successive eggs are diploid (female)

–  All eggs/larva sexually matures in 6-7 days. The male mates with each of its siblings

–  Females mature as they feed off the developing, weakening, pupa –  Mature bee emerges (21-24 days) along with the foundress and (1-3)

mature female daughters. Males and immature varroa nymphs die in cell

–  During a 6 month brood cycle varroa populations can grow 12X

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WhatisOxalicAcid,(OA)?•  Oxalicdehydrate(C2H2O4),isanaturallyoccurringorganicacid.Commonly

foundinspinach,rhubarb,cashews,beetsandpotatoes,etc.,andistheprimaryingredientinwoodbleach

•  RelaCvelysafetohumansusedasdirected

•  OAincrystalformcanbeappliedtohivesinsoluConorbysublimaCon•  Usedtocontrolvarroamiteloadswithinhives•  Hightoxicitytomites,lowtoxicitytohoneybees•  MosteffecCveduringbroodlessperiods•  LongtermsublethaleffectsoncoloniesareoKenoverlooked

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DesignofExperiment

•  Ques8on:DorepeatedapplicaConsofOxalicAcidtreatmentsforVarroamiteshavespecificsecondaryorsub-lethaleffectsonacCve,growingcoloniesofhoneybees?

•  Ifso,observe,idenCfyandquanCfythesecondaryeffects

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Materials&Methodologystudysubjects,datesandloca8on

•  SixsubjectLangstrothstylehiveswereselectedforthestudyeachconsisCngof2,10-framedeepbroodboxes

•  3testgroup=OAtreated,3controlgroup=notreatment•  SubjectpopulaConsconsistedofApismelliferacarnicawithVarroa

SensiCveHygiene(VSH),overwinteredqueens•  TheexperimentranfromJuly10toAugust07,2018,at44°43’26”N

87°20’35”W,610K.elevaCon,inUSDAPlantHardnessScale5a

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Materials&Methodology,cont.prepara8onsfortreatedhives

•  Treatedatseven-dayintervalsoverathree-weekperiod,foratotalofthreetreatmentsspanningtwenty-onedays(Day0,Day7,andDay14)

•  2g99.6%Oxalicaciddehydratecrystalswereappliedasistherecommendedthresholdfor2storyhives.1

•  OAapplicaConbysublimaConofcrystalsonaVARROX12V,15Wpanvaporizer

•  EntrancesandscreenedboOomboardsclosedofduringapplicaConf•  Honeysupers(ifany)wereremovedpriortotreatmentandreturnedto

theirrespecCvehives24hoursposttreatment

1Rademacher,E.,&Harz,M.,Oxalicacidforthecontrolofvarroosisinhoneybeecolonies-areview,FreeUniversityofBerlin,Dept.ofBiology/Chemistry/Pharmacy,Neurobiology,Königin-Luise-Str.28-30,14195Berlin,Germany.Apidologie37(2006),p.116.

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Materials&Methodology,cont.aconsistent,repeatabletreatmentprocessiskey

•  Panvaporizerwasconnectedtoa12VtruckbaOerywithenginerunningtoensureconsistentcurrent

•  Currentwasconnectedfor2minutesand30secondstoallowthepantoheatsufficientlyandvaporizeOAcrystals

•  Thepanremainedinternaltothehivefor2addiConalminuteswithnocurrent,toensuresublimaConofanyresidualcrystals

•  ThepanwasthenremovedfromthehiveandquenchedinabucketofcoolwaterandpreparedforthenexthiveapplicaCon

•  Entrancesremainedclosedoffoneachtreatedhive(#8,#10,#12)foranaddiConal10minutesbeforereopeningallentrances

•  Thisprocesswasrepeated3X,atseven-dayintervalsthroughoutthe28-daystudy

•  Oddnumberedhives(#7,#9,#11)servedasthecontrolreceivingnoVarroamitetreatment

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Materials&Methodology,cont.miteboardrepara8ons

•  Mitecountswereperformedandrecordedonallsubjecthivesthemorningofthefirsttreatmentviathe‘sugarshake’methodperoperaCnginstrucConsfromtheUniversityofMinnesota,usingtheirGizmodevice1

•  Mite/debrisboardswereindeliblylabeledwiththecorrespondinghivenumber,sprayedwithalightcoaCngofvegetableoil,andinsertedintotheirrespecCvescreenedboOomboardsofeachsubjecthivepriortoOAVtreatments

•  Mite-dropcountswereperformedandrecordedonindividualworksheetsforallsubjecthivemiteboards,whethertreatedornot,24hoursposttreatmentapplicaCon,thencleaned,oiled,andreinsertedandinspectedagainat48hoursposttreatment

•  Miteboardswerethencleanedandstored,andpreppedasabovethedayofthenexttreatment

1Reuter,G.S.,Lee,K.,Spivak,M.,GizmoOpera8ngInstruc8ons,Methodtoaccuratelymeasure300beesanddeterminetheinfesta8onofvarroamites,UniversityofMinnesotaInstrucConalPoster#171,DepartmentofEntomology,UniversityofMinnesota,www.extension.umn.edu/honeybees

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Materials&Methodology,cont.broodcell,honey&nectar,andmiteloaddocumenta8on

•  FabricateabroodcellcounCngtemplatemadeofclearLexan®inscribedwith1inchsquaregridlines(12inch=30cells)

•  OnthemorningofeachOAVapplicaCon,eachframeineachhivewasinspected(SideA,southsidefacingandSideB,northsidefacing)forthenumberofcells/framecontainingopenorcappedbrood

•  ResultswerefieldrecordedonindividualhiveworksheetsandthedatalatertransferredtoaMicrosoKExcelfile

•  SideAandSideBofeachframecontainingnectarandcappedhoneyfromeachsubjecthivewasalsocounted,fieldrecorded,andsummedtothenearest0.5frame

•  MiteloadesCmatesoneachhivewereconductedaKer28days(sugarshakemethod)

•  Excelfilesweretabulatedandanalyzedtodeterminechangesinmiteload,broodproducConandnectar/honeystoresoncontrolhivesvs.OAVtreatedhives

1Reuter,G.S.,Lee,K.,Spivak,M.,GizmoOpera8ngInstruc8ons,Methodtoaccuratelymeasure300beesanddeterminetheinfesta8onofvarroamites,UniversityofMinnesotaInstrucConalPoster#171,DepartmentofEntomology,UniversityofMinnesota,www.extension.umn.edu/honeybees

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HighLevelResults•  BroodReduc8on-HivestreatedwithOAVresulted,ingeneral,ina

dramaCcdecreaseintheamountofbroodpresenttheweekfollowingeachtreatmentintwoofthethreegroups

•  NectarCollec8onInhibi8on/HoneyProduc8onReduc8on-ThestrongesthivesshowedthegreatestdisparityinnectarcollecConandhoneyproducConinuntreatedvs.treatedhiveswithuntreatedhive(#7)farexceedingallothersubjecthivesinthetestintotalvolume

•  UnexpectedlyLowMiteLoads&MiteDrops-Miteloadspre-andpost-testforallsubjecthiveswerefarlessthanexpected.Mitecountspost-treatmentforalltreatedhiveswerefarbelowwhatwasexpected.

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Brood•  Thecontrolhive(#7),receivingnoOAVtreatment,gainedasmallbut

steadyincreaseinbroodweek-to-weekwitha16%increaseinbroodoverallbythe21stday

•  Thetreatedhive(#8),showedaprecipitous81%dropinbroodproducConoverthecourseofthe21-dayexperiment

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Brood•  Thecontrolhive(#9),swarmedatanundeterminedCmepriorthestartof

thetest.BroodproducConmodulatedwithanoveralldecreaseof5%overthecourseofthe21-daytest

•  Thetreatedhive(#12),madefromaSpringsplitwhosequeenonlybeganlayingtheweekbeforethestartoftheexperiment.OAVtreatmentsappearedtohaveminimaleffectonbroodproducCon.However,thelayingpaOernbecameincreasinglyununiformedweek-to-week.

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Brood•  Thecontrolhive(#11),acapturedswarmfrommyapiary9dayspriorto

thestartofthetest.BroodproducConeventuallyacceleratedduringthesecondandthirdweekstoanoverallincreaseof278%

•  Thetreatedhive(#10),showedevidenceofemergedqueencellsbutfailedtoproducealayingqueen.UndeterminedifOAVtreatmentwasacontribuCngcause.EvenaKertheintroducConoftwoframesofbroodfromanucleuscolonyaKerDay7,thehivefailedtomaterializeaviablequeen.

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NectarCollecCon/HoneyProducCon•  AdecreaseinnectarcollecConandsubsequenthoneyproducConinthe

OAVtreatedhivescomparedtohivesthatwentuntreated.

Swarmedpriortotesttaking½foragerswithit!

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MiteCounts•  Miteloadsinallsubjecthives,asevidencedbytheiniCalsugarshakemite

loadcount,wereminimalattheonsetwith0-1mite/300bees.Thefinalmiteloadtakenonday28,7daysfromthefinalOAVtreatment,werealsowellwithinmanageablerange0-4mites/300bees.

•  Mitecountstakenat24hoursand48hoursaKereachtreatmentwerewidelyvariableandatCmesindiscerniblefromhivesthatdidnotreceiveOAVtreatment,suggesCngliOleornoresidualeffect

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Discussion

•  Whilethestronghivesreflectconsistencywithpreviouspublishedstudies,overallreportedresultsareonlymoderatelyconsistentwithexpectaConsassomesubjecthiveswereunstabl.

•  Alargersamplesize,equalizedhivestrength,aretestoraparalleltestsitewouldbenecessarytoconfirmtrendsandmakemoredefiniCveconclusion

•  BeginninghivestrengthvariabilitywasnotaconsideraConoftheoriginalprotocolbutwasnecessitatedduetosubjectavailabilityandCme.ThepropensityofA.miliferacarnicatoswarmmorefrequentlythanA.miliferaligus8cawouldseemtoindicatethatthelaOerbeabeOerchoicesubspeciesforthistest

•  Miteloadspre-andpost-testwerefarlessthanexpected.Mitecountspost-treatmentwerefarbelowwhatwasexpected.ItisundeterminedwhetherVSHqueengeneCcsplayedanysupporCngrole

•  ConsideraConmustbegiventotechnique,accuracyandreliabilityofthesugarshakemethodtodeterminemiteloads

•  ConsideraCon,too,mustbegiventothequalityandpotencyoftheOxalicacidcrystals,andtotheCmingoftreatment.Itisunknownwhetheraconsistentseven-dayintervalisopCmal.Highermitecountdropswereexpected.Inhindsite,asugarshaketestshouldhavebeenperformedpriortoeachOAVapplicaContogainfinerdetailanddetermineweeklyefficacy.ThiswasanunintenConaloversight

•  TheclearestresultsofthetestwereintheformofnectarcollecConinhibiConandreducedhoneyproducConindicaCngthatqueenegg-layingdecreasedandworkeracCvityandnursingwasnegaCvelyimpactedtoanindefinableextent

•  EgglayingbecamelesspaOernedandmorearbitrary,raisingthequesConastowhetherOAVinterferedwiththequeen’sabilitytomaximizebrooddensitypaOerns,orifshewasfailingasaresultofotherreasons.AddiConally,severaldeadanddyingbeeswereobservedontheinnercoversandscreenedboOomboardsofthetreatedhives24hoursaKertreatmentmoresothanonthecontrolhivesalthoughtheirnumberswerenotsignificantlydifferent

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Conclusions•  OxalicacidvaporizaConisaneffecCvetoolinanoverallstrategyforthe

controlofVarroamites•  OAVworkedmoreeffecCvelyonhiveswithliOleornocappedbroodthanon

hiveswithsignificantcappedbrood,consistentwithpreviouspublishedresults

•  OAVappearedtoaffectworkerbeebehaviortotheextentthatitresultedindecreasedbroodproducCon,foragingandsubsequenthoneystoresintwoofthreegroupstested,andalteredlayingpaOerninoneofthethreetreatedhives

•  Accuracyandreliabilityofthesugarshakemethodologyfordeterminingmiteloadsissuspect,atleastasfarasthisexperimentshows,astherewastoomuchvariabilityinmitedrops,andmitecounts

•  ThereisnoclearindicaConthatVSHqueensaOributedtoanymeaningfulreducConinmiteloadsasthiswasbeyondthescopeofthetest

•  Furtherstudywithmorefocusedprotocolsandlessvariabilityshouldbeexamined

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Q&A

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ThankYou!KevinO’Donnell,fadoauthor@gmail.com

847-363-8847

CerCfiedMasterBeekeeperUniversityofMontanaConCnuingEducaConProgram

Endorsedby:MontanaStateBeekeepersAssociaCon,

AmericanHoneyProducersAssociaCon,andProjectApism.

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