McKeeetal.:BiocideandPathologicEffectsonA.mellifera JournalofEconomicEntomology ApicultureandSocialInsects BiocideandPathologicEffectsonApismellifera(Hymenoptera:Apidae):Implicationsfor WidespreadPopulationDecline B.C.McKee,D.W.Poll,andE.C.

Schuyler DepartmentofBiology,NorthernMichiganUniversity,1401PresqueIsleAvenue,Marquette, Michigan49855

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Abstract Recentdeclinesinhoneybee(ApismelliferaL.)populationsmaythreatentheproductionof majorcropswithintheUnitedStatesandEurope.Thesedeclinesposetohinderdomesticagriculture bothecologicallyandeconomically,ashoneybeespollinateroughlyonethirdoftheconsumedworld foodsupply.Thoughmyriadstudieshaveattemptedtodetermineaprimarycauseforthisdecline,none yetrevealasinglecausativefactoraffectinghoneybees.Amongtheknownmajorinfluencesarehoney beepathogens,suchasVarroamitesandNosemafungi.Additionally,asbeesforagetheyare potentiallyexposedtoawidevarietyofbiocidesthatareappliedtocrops,aswellasbiocidesapplied bybeekeeperstocontrolinhivepathogens.Thisliteraturereviewelucidatesthemajoreffectsresulting frombiocidesandpathogensonhoneybees,aswellassomenotablesynergisticeffectsbetweenthe agentgroups.Thousandsofbeesweresampledthroughoutthereviewedstudies.Resultsdisplaydiverse relationshipsduetothedifferingmethodologyusedacrossthestudies.Nevertheless,itisclearthatthese agentsmayhavebothpositiveandnegativeeffectsonhoneybeehealth,bothindividuallyand synergistically.Despitetheseconcerns,prospectivealternativeshavebeenproposed. Keywords:Apismellifera,biocide,Varroa,Nosema,synergistic

Introduction Honeybee(ApismelliferaL.)populationsarecurrentlydeclininginNorthAmericaand Europe.Muchresearchhasattemptedtodeterminetheoriginofthesedeclines,thoughnofindingspoint towardanexclusiveanswer.Itiscrucialtounderstandwhatiscausingthedeclinesbecausehoneybees areresponsibleforpollinatingroughlyonethirdoftheworldsconsumedfoodsupply(Gallaietal. 2009).Thisisrepresentedeconomicallybyadomesticcropvalueofmorethan$15billionperyear (ARS2013).ThepotentialhoneybeedemisemayleaveAmericanagriculturecrippledecologicallyand economically.Foralmonds,completecropfailurecouldensueashoneybeesarethesolepollinatorsof thiscrop.Thus,theabsenceofthispollinatormayresultindrasticallyreduceddomesticcropyields. Thisdeclineinhoneybeepopulationsisthoughttostemfromawidevarietyofinfluences,suchas narrowedgenepools,habitatdestruction,climatechange,andnumerousbiocidesandpathogens (vanEngelsdorpandMeixner2010).Manyofthesefactorscausedramaticeffectsonmanagedhoney beepopulationsaswellasbeekeepingprofitability.Inthisreview,weexploretheeffectsofbiocides andpathogensonhoneybeesbothindividuallyandincombinations. Theinfluenceofbiocideexposurehasbeengainingbothinterestandconcern.Forthisreview, thetermbiocideisutilizedtodescribepesticides,miticides,insecticides,herbicides,fungicides, antimicrobials,etc.Biocidesandtheirmetabolitesarefoundintraceamountsthroughouthoneybee coloniesandwithinhoneybeesthemselves.Biocideexposuremaydirectlyoccurasaresultof beekeeperapplicationtocontrolinhivepathogens.Exposuremayalsoresultfromchemicalsappliedto cropsthatutilizehoneybeesforpollination.Ithasalsobeendocumentedthatavarietyofbiocidesmay bepresentonorwithinhoneybeesifnotintended(Mullinet.al2010).Accidentalbiocideexposureis difficulttocontrolasbiocideresiduesareknowntomigratebetweenhoneycombsofdifferentcolonies.

Evenwhenappliedinsublethaldoses,concentrationsbelowtherespectiveLD50,biocideshavebeen notedtocausemodifiedbeeperformanceinaspectsofgrowth,longevity,behavior,andsusceptibilityto pathogens(Wuetal.2011). Honeybeesareexposedtoawidevarietyofpathogenswhichinfesthivesandcauseinfectious diseases.Thoughothersareadditionallyprevalent,thetwomostnotedhoneybeepathogensare endoparasiticVarroahivemitesandNosemamicrosporidiafungi.Varroamiteinfestationsstrongly affecthoneybeehivesduringthewinter(Dainatetal.2012).Thesemitesshareanectoparasitic relationshipwithbroodlarva.Theylayeggswithincombcellsthateventuallyyieldnewlyemergedmites whichfeedonlarvalhemolymph.Nosemafungalinfestationsareoneofthemostprevalenthoneybee pathogens,oftenassociatedwithhighlydiseasedcolonies(Paxton2010).VarroamitesandNosema fungiareoftentreatedwithbiocidesinattempttocontrolinfestations. Synergisticeffectsofmultiplebiocidesandpathogensmayalsohaveunknownnegativeeffects onhoneybees.Theseincludebiocidebiocide,biocidepathogen,andpathogenpathogeninteractions exposuretomultipleagentssimultaneouslymayaffectbeesdifferentlywhencomparedtoindividual factors.TheobjectiveofthisstudywastoreviewvariouspublicationsandsurveysonexposureofApis melliferatobiocidesandpathogens.Anadditionalobjectivewastoexaminehowthesefactorsaffect honeybeesbothindividuallyandsynergistically.WeprimarilyfocusedonbiocidesrelatedtoVarroa andNosemapathogens,astheyhavethemostdocumentedinformation. MaterialsandMethods Searchstrategyandstudyselection.Themainmethodforobtainingresourceswas searchingwithGoogleScholar.Whenstartingthisreview,wereadavarietyofliteratureonallaspects

ofdeclininghoneybeepopulationstodecidewhichtopicweshouldfocuson.Aftersettlingonbiocides andpathogens,werefinedoursearches,usingkeyphrasessuchasApismelliferabiocideorhoney beebiocide.AdditionalsearchesweredonebyaddingVarroaorNosematothesearches.Dueto theambiguityofthetermbiocide,searcheswerealsoalteredbyreplacingbiocidewithpesticide, miticide,fungicide,etc. Whenarticleswerefoundpertainingtobiocideandsynergisticeffectsonhoneybees,theywere savedtodesignatedfoldersoutliningtheirspecificfocusinZoterothisisanopensourceresearchtool whichassistsincollecting,organizing,analyzing,citingandsharingscientificresources.Allauthorshad accesstooursharedlibraryandcouldthusreadandreviewthecompiledliterature.Inadditionto Zotero,datafromreviewedarticleswereextractedandpostedinsharedviaGoogleDrive.Google DocumentsandSpreadsheetswereutilizedforfurtherreferenceanddataextraction. WealsocompileddataonmiticideuseandmeanpercentcolonylossesfromBeeInformed NationalManagementSurveys.TheBeeInformedPartnershipisaprojectaimedatdetermining preferredhivestrategiesthroughsurveysofbeekeeperhivelosses,managementpractices,andbiocide use.Thisallowedexaminationofsuggestivedataontheeffectsindividualmiticideshaveonhoneybee colonies. EligibilityCriteria.Allliteraturechosenforthisreviewwaspeerreviewedandpublishedina crediblejournal,orcitedbytheUnitedStatesDepartmentofAgricultureAgriculturalResearch Service(USDAARS)orApiaryInspectorsofAmerica(AIA).Dueinparttotimeconstraints,our resultsreview11journalarticlesonthetopicofbiocidesand/orpathogensandtheireffectsonhoney beehealth.Thesearticlesweregroupedandevaluatedinconcordancewiththeguidelinesbelow:

 Biocideanalysesofhoneybeesandtheirproducts Varroamites oVarroabiocidesynergisticeffects Nosemafungi oNosemabiocidesynergisticeffects Results BiocideExposure.Inthereviewedstudies,samplesofbeewax(broodcomb/foundation comb/honeycomb),beepollen(beebread/corbicularpollen),andadultbeeswereanalyzedforbiocide detections.Acomprehensivebiocideanalysisof749samples(259wax,350pollen,and140adultbee samples)foundalarmingbiocidedetectionswithinbeeproductsacrossNorthAmerica(Mullinetal. 2010).Samplescontainedatotalof118differentbiocidesandbiocidemetabolites(Fig.1),withmean sampledetectionsof8.0forwax,7.1forpollen,and2.5forbees(Fig.2).Usingthecombination formula ( n r ) withthenumberofbiocidesdetectedforeachsampletypeandtheaveragesample detection,thenumberofpossiblebiocidecombinationsforaveragesampleswere58,433,559,570for wax,1,258,931,596,832forpollen,and45,540forbees.Asimilaranalysisofbroodcombwax detectedatotalof39differentbiocides,withameansampledetectionof10biocides(Wuetal.2011). Inanothercase,all19collectedcorbicularpollensampleswereshowntocontainpesticides,witha meansampledetectionof9.1biocides.Thesepollensamplescontainedsignificantlyhigherfungicide loadsthananyotherbiocidesubcategory(Pettisetal.2013). VarroaMites.Miticidesareoftenimplementedbybeekeeperstoexterminatepathogenichive mites.Johnsonetal.(2013)studiedtherelativetoxicityoffivemiticidescommonlyusedbybeekeepers

(Fig.3).Thisassessmentfurtherexaminedinteractionsbetweensublethalmiticideandfungicide pretreatmentswithmiticideposttreatmentsonbeemortality(Fig.3).Themostlethalmiticide (Amatraz)hadthefewestinteractionswithothermiticideandfungicidepretreatments,whilethethird mostlethal(Taufluvalinate)interactedwiththemostpretreatments,includingall5miticidesand4of5 fungicides(Johnsonetal.2013).Miticidepairshavebeendetectedinhighamountsinbeeproducts, suchasfluvalinateandcoumaphoswhichwerefoundin77.7%ofanalysedsamples(Mullinetal.2010). Althoughmiticidesmaybetoxictobeesbothindividuallyandsynergistically,treatingVarroa infestedhivescanlowermortalityrates.Onestudylookedatoverwinteringcolonysurvivalratesfortwo honeybeepopulationsinfestedwithVarroamites,oneleftuntreatedandonetreatedwithorganicacids (Dainatetal.2012).All11oftheuntreatedhivesdidnotsurvivethewinter,whileonly2ofthe16 treatedhivesperished.AlthoughtreatingVarroainfestedhivesmayleadtoloweroverwinteringhive losses,Varroamitescanalsoactasvectorsforotherpathogens,suchasthestrongpositiverelationship withDeformedwingvirus(DWV)(Lockeetal.2012).Evenafterapplyingtaufluvalinate,amiticide,to removeVarroafromhives,highpupaeDWVinfectionspersistedasaresultofthispathogenpathogen interaction(Lockeetal.2012). Miticideshavealsobeenshowntohavenegativebehavioralandphysicaleffectsonhoneybees. Inparticular,coumaphosconcentrationhasbeenshowntohaveapositiverelationshipwithlarvalqueen rejectionrates.Whencoumaphosconcentrationsof100ppmwereappliedtobroodwax,aqueen rejectionrateof50%wasobserved.Atconcentrationsof1000ppmorgreater,queenswererejected in100%ofcases.Coumaphosexposurealsoresultedinanaveragequeenweightof132mgcompared tocontrolqueensat270mg,a49%reductioninthosetreated.Treatmentqueenfecunditywasalso lowerthanqueensrearedinthenegativecontrol,orcoumaphosfree,environment(Pettisetal.2004).

OverwinteringHiveLosses.ThecompiledBeeInformedNationalManagementSurveydata onbeekeepermiticideusesuggestsanoveralldecreaseinmeanpercentcolonylossesforbeekeepers applyingmiticidesbetween2010and2013(BIP2012,2013,2014)(Fig.4a).Splittingthemiticide dataintoindividualmiticidesrevealsinmoreclaritythedifferingeffectsofeachpesticideused(Fig.4b). BeekeeperswhousedApiGuard,ApiLife,orformicacidtocontrolVarroamiteshadlowermean percentoverwinteringcolonylossescomparedtobeekeepersusingaproductotherthanthesefor Varroamitecontrol.ApiGuardandApilifearethymolcompounds.Beekeepersthatusedeither fluvalinateorsucrocidesawslightlyhighermeanpercentoverwinteringcolonylosseswhencomparedto beekeepersusingotherproducts.Thepercentcolonylossesofthesetwomiticidesarecomparableto beekeeperswhodidnotuseanyVarroacontrolproducts. NosemaFungi.Theantimicrobialfumagillinhasbeenfoundtobe100%effectiveincontrolling Nosemainfestations(Higesetal.2008).Inthisstudy,alltreatedhiveswinteredingoodhealth,while hivewidebreakoutoccurredinalluntreatedhives.Alltreatmenthiveeggsbecamehealthycolonieswith 10combsfullofbees,untilthenextspringwhentheyweresubsequentlytreatedagainwithNosema. Althoughfumagillintreatmentsignificantlyreducedhoneybeedepopulationrisksoveraoneyearperiod, itdidnotpreventNosemareinfectionthefollowingyear. AnotherstudyobservedvariousbiocideeffectsonNosemainfectionratesafterfeedingonbee collectedcorbicularpollensamples(Pettisetal.2013).Thesesamplescontainedupto35different biocides,22ofwhichwerefoundtohaverelativeriskvaluessignificantlydifferentfromcontroldiets. IncreasedNosemaprevalencewasobservedforeight,withdecreasedprevalencefortheremaining14. TwoofthebiocidesassociatedwithincreasedNosemaprevalencewerethemiticidesDMPFand fluvalinate.BeesfedpollencontainingeitherofthesemiticidesresultedinovertwicetheNosema

prevalencewhencomparedtobeesfedthenegativecontroldiet.Thefungicideloadofpollenwasalso foundtosignificantlyaffectNosemaprevalence,withsimilarresultsseenusingonlychlorothalonil,the mostabundantfungicideoftheirpollensamples(Pettisetal.2013).Additionally,miticide/fungicidepairs havebeendetectedinhighpercentagesofbeeproductsamples,including41.2%ofsamplescontaining boththemiticidefluvalinatewiththefungicidechlorothaloniland39.1%ofsamplescontainingthe miticidecoumaphoswiththefungicidechlorothalonil(Mullinetal.2010). Thecommerciallyavailableinsecticides,fipronilandthiacloprid,havebeenshowntosignificantly increasemortalitywhenappliedatsublethalquantitiestobeespreviouslyexposedtoNosemaceranae fungi.WhilethepercentageofbeemortalityratesforbeesonlyexposedtoN.ceranaereacheda maximumof47%,maximumpercentagebeemortalityratesforbeesexposedtoN.ceranaeandeither thiaclopridorfipronilincreasedto71%and82%respectively(Figure5).Fipronilapplicationdecreased sporeproductionby33%,whereasthiaclopridincreasedsporeproductionby40%.Despitefipronil decreasingsporeproduction,thischemicalhadthehighestoverallpercentmortalityrateswhen combinedwithN.ceranae(Vidauetal.2011). Theneonicotinoidimidacloprid,oneofthemostwidelyusedpesticides(Yamamoto1999),can alsohaveinteractiveeffectsonhoneybeesexposedtoNosema.Comparedtobeesonlyinfectedwith Nosemaoronlyexposedtoimidacloprid,significantlyincreasedbeemortalityrateshavebeenobserved followingsynergisticNosemaimidaclopridexposure(Alauxetal.2010).Followingexposureto sublethalimidaclopridconcentrationsof120ppb,foragingbeesweremorelikelytolosetheirwayback tothehivewithanearinstantonsetofsymptoms.Allhoneybeesexposedtoconcentrationsof300+ ppb(lethaldose 280ppb)didnotreturntothehive,orperishedwithinthehourtheywereobserved (Yangetal.2008).

 Discussion Resultsshowthatbeeswaxandpollencontainahighabundanceanddiversityofbiocides

(Mullinetal.2010,Wuetal.2011,Pettisetal.2013).Thissuggeststhatbeecollectedpollenmay containthebiocidesappliedbyfarmerstoeradicateplantpathogens(Mullinetal.2010,Pettisetal. 2013).Thus,uponbeesreturningtothehivewithfieldcollectedpollen,biocidescontainedinthepollen mayaccumulateinbeehivewax.Beekeepersarelikelynotapplyingthehighdiversityofbiocides detectedinwaxsamples,suggestingthattheyoriginatefromadifferentsource.Thus,beekeepersmay needtoconsidernotonlywhatchemicalstheysprayontheirhives,butalsothechemicalssprayedon thecropfieldsthatthehoneybeespollinate.ThisthoughtisfurtheredwiththefindingsofMullinetal. (2010),inwhichpollensamplescontainedthehighestbiocidedetections.Thesebiocidesandtheir metabolitesareadditionallyseenwithinthebeesthemselves(Mullinetal.2010).Thisgoestoshowthat beesarenotonlyexposedtothesebiocides,butareingestingthemaswell. Miticidesusedbybeekeeperstoeradicateinhivemites,suchasVarroa,havebeenfoundto havestronginteractionsonbeemortalitywhenappliedsynergisticallywithothermiticidesorfungicides. Thehighpercentageofbeeproductsamplescontainingboththemiticidesfluvalinateandcoumaphos causegreatconcernforhowtheimmeasurablecombinationsofbiocidesareaffectinghoneybees (Mullinetal.2010).However,lowerVarroainfestationshavebeennotedasaresultoftreatinghives withmiticides(Dainatetal.2012).Althoughtheseresultsaremerelysuggestive(i.e.unknownor uncontrollableenvironmentalvariabilitybetweenlocations),BeeInformedsurveydatasuggestsan overalldecreaseinhivelosseswhentreatedforVarroamites,whileindividualbiocideshavevariable effectsonhivelosses(BeeInformed2011,2012,2013).Togiveanideaofhowmuchthedata

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covered,therewere10,142surveyresponsescovering968,576beecoloniesandanestimated 312,061numberofcolonieslost.Nevertheless,evenifhiveshavebeeneradicatedofVarroa,thereisa chancethatVarroamitesmayhavealreadybroughtadditionalpathogenstohives,asseenwithDWV (Lockeetal.2012).ThehighlarvalDWVinfectionratefollowingVarroaremovalsuggeststhatevenif Varroainfestationsareeradicatedfromhives,theycanbringadditionalpathogenswhichareableto persistandnegativelyaffectthehealthofthehive(Highfieldet.al.2009).Treatinghiveswithmiticides mayalsocausenegativeeffectsonbeesalone,asseenwiththehighqueenrejectionrateandoverall lowerweightwhenqueenbeesweretreatedwithcoumaphos(Pettisetal.2004).Thishighrejection ratemaysuggestqueenlarvaehealthisaffectedbycoumaphosandthatnursebeescansense abnormalitiesandhygienicallyremovetheseindividualsaccordingly.Thisrejectionbehaviorshouldbe furtherstudiedinordertounderstandwhatnursebeesarerecognizing,whetheritbepathogenic, biocidal,orboth. Althoughthebiocidefumagillinhasbeen100%effectiveincontrollingNosemainfestations (Higesetal.2008),thisisnotthecaseforallbiocides.Further,thetrueinteractionswithotherfactors remainunknown.SomechemicalshavebeenfoundtoincreaseNosemaprevalence,suchasmiticides, fungicides,andinsecticides(Pettisetal.2013,Vidauetal.2011).Additionally,notallbiocidesfoundin corbicularpollensampleswerefoundtoincreaseNosemaprevalence(Pettisetal.2013),although decreasedNosemaprevalencedoesnotnecessarilymeantherewillbeadecreaseinbeemortality.This wasseeninbeesexposedtobothNosemaandtheinsecticidefipronil,inwhichthiscombinationsaw thegreatestpercentbeemortality(Vidauetal.2011).Inthiscase,fipronildecreasedNosema prevalence,suggestingthatNosemaandbiocideexposuremayhavesynergisticnegativeeffectsonbee health,regardlessofwhetherornotNosemaabundancechanges.Nosemawasalsoshowntohave

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increasednegativeeffectswhencombinedwiththeinsecticide,imidacloprid,resultinginanoverall increasedsusceptibilitytodiseaseandrateofmortality(Alauxetal.2010).Imidaclopridalonecanhave lethaleffectsathighdosesandneurologicaleffectsatsublethaldoses.Naturalforagingbehavior exposesbeestothiscommoninsecticidewhichisappliedtothecropstheypollinate.Workerbeesare exposedtolowdosesinrealworldscenariossoitispossiblethatsublethalexposuremaystillbe affectingbeesneurologically. Inconclusion,therearediverserelationshipsbetweenbiocides,biocidebiocidecombinations, andbiocidepathogencombinationsonhoneybeehealth.Thehighdiversityofsynergisticexposures makeitdifficulttopinpointhoweachbiocideaffectshoneybees.Biocidescanhavevariedeffects, someshowingnotablylessharmfuleffects,whileothersshowmoredrasticlethaleffects.Synergistic biocidebiocideeffectsareofparticularinterestbecausehoneybeesarenotgenerallyexposedtoany singlechemicalinrealworldsituations.Additionalresearchisalsoneededontheeffectsof biocidepathogencombinationsonbeehealth.Biocidesoftentargethoneybeedetoxificationsystemsin whichenzymesthatotherwisebreakdownforeignchemicalsarealtered.Whiletheapplicationofa biocidemayeliminateahivepathogen,theeffectsthatthisbiocidehasonhoneybeesmaymakethem moresusceptibletootherpathogens,ifnotharmingthebeesoutright.Ithasalsobeenfoundthatnative croppollinators,suchasbumblebeesandbeetles,canbemoreeffectivepollinatorscomparedtothe nonnativehoneybee(Garibaldietal.2013),providinganalternativeroutetocroppollinationifthe honeybeeepidemicisnotresolvedbeforetheirpossibledemise.

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ReferencesCited Alaux,C.,J.L.Brunet,C.Dussaubat,F.Mondet,S.Tchamitchan,M.Cousin,J.Brillard,A. Baldy,L.P.Belzunces,andY.LeConte.2010.InteractionsbetweenNosemamicrospores andaneonicotinoidweakenhoneybees(Apismellifera).EnvironmentalMicrobiology12: 774782. (BIP)BeeInformedPartnership.2012.BeeInformedNationalManagementSurvey20102011. <http://beeinformed.org/2012/03/beeinformednationalmanagementsurvey20102011/> (BIP)BeeInformedPartnership.2013.BeeInformedNationalManagementSurvey20112012. <http://beeinformed.org/2013/07/beeinformednationalmanagementsurvey20112012/> (BIP)BeeInformedPartnership.2014.BeeInformedNationalManagementSurvey20122013. <http://beeinformed.org/2014/03/beeinformednationalmanagementsurvey20122013/> Dainat,B.,J.D.Evans,Y.P.Chen,L.Gauthier,andP.Neumann.2012.Deadoralive: deformedwingvirusandVarroadestructorreducethelifespanofwinterhoneybees.Applied andEnvironmentalMicrobiology78:981987. Gallai,N.,Salles,JM.,SetteleJ.,andVaissire,B.E.2009.Economicvaluationofthe vulnerabilityofworldagricultureconfrontedwithpollinatordecline.EcologicalEconomics68: 810821. Garibaldi,L.A.,I.SteffanDewenter,R.Winfree,M.A.Aizen,R.Bommarco,S.A. Cunningham,C.Kremen,L.G.Carvalheiro,L.D.Harder,O.Afik,I.Bartomeus,F. Benjamin,V.Boreux,D.Cariveau,N.P.Chacoff,J.H.Dudenhffer,B.M.Freitas,J. Ghazoul,S.Greenleaf,J.Hiplito,A.Holzschuh,B.Howlett,R.Isaacs,S.K.Javorek, C.M.Kennedy,K.M.Krewenka,S.Krishnan,Y.Mandelik,M.M.Mayfield,I.

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Motzke,T.Munyuli,B.A.Nault,M.Otieno,J.Petersen,G.Pisanty,S.G.Potts,R. Rader,T.H.Ricketts,M.Rundlf,C.L.Seymour,C.Schepp,H.Szentgyrgyi,H. Taki,T.Tscharntke,C.H.Vergara,B.F.Viana,T.C.Wanger,C.Westphal,N. Williams,andA.M.Klein.2013.WildPollinatorsEnhanceFruitSetofCropsRegardlessof HoneyBeeAbundance.Science339:16081611. Higes,M.,R.MartnHernndez,C.Botas,E.G.Bailn,A.V.GonzlezPorto,L.Barrios, M.J.delNozal,J.L.Bernal,J.J.Jimnez,P.GarcaPalencia,andA.Meana.2008. HownaturalinfectionbyNosemaceranaecauseshoneybeecolonycollapse.Environmental Microbiology10:26592669. Johnson,R.M.,L.Dahlgren,B.D.Siegfried,andM.D.Ellis.2013.Acaricide,fungicideand druginteractionsinhoneybees(Apismellifera).PLoSONE8:e54092. Locke,B.,E.Forsgren,I.Fries,andJ.R.deMiranda.2012.Acaricidetreatmentaffectsviral dynamicsinVarroadestructorinfestedhoneybeecoloniesviabothhostphysiologyandmite control.AppliedandEnvironmentalMicrobiology78:227235. Mullin,C.A.,M.Frazier,J.L.Frazier,S.Ashcraft,R.Simonds,D.vanEngelsdorp,andJ.S. Pettis.2010.HighlevelsofmiticidesandagrochemicalsinNorthAmericanapiaries: implicationsforhoneybeehealth.PLoSONE5:e9754. Paxton,R.J.2010.DoesinfectionbyNosemaceranaecauseColonyCollapseDisorderinhoney bees(Apismellifera)?JournalofApiculturalResearch49:8084. Pettis,J.S.,A.M.Collins,R.Wilbanks,andM.F.Feldlaufer.2004.Effectsofcoumaphoson queenrearinginthehoneybee,apismellifera.Apidologie35:605610. Pettis,J.S.,E.M.Lichtenberg,M.Andree,J.Stitzinger,R.Rose,andD.vanEngelsdorp.

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2013.Croppollinationexposeshoneybeestopesticideswhichalterstheirsusceptibilitytothe gutpathogenNosemaceranae.PLoSONE8:e70182. (USDAARS)U.S.DepartmentofAgricultureAgriculturalResearchService.2013.Honey beesandcolonycollapsedisorder.USDAARS,Beltsville,Maryland. <http://www.ars.usda.gov/news/docs.htm?docid=15572> vanEngelsdorp,D.,andM.D.Meixner.2010.Ahistoricalreviewofmanagedhoneybee populationsinEuropeandtheUnitedStatesandthefactorsthatmayaffectthem.Journalof InvertebratePathology103:S80S95. Vidau,C.,M.Diogon,J.Aufauvre,R.Fontbonne,B.Vigus,JL.Brunet,C.Texier,D.G. Biron,N.Blot,H.E.Alaoui,L.P.Belzunces,andF.Delbac.2011.Exposuretosublethal dosesoffipronilandthiaclopridhighlyincreasesmortalityofhoneybeespreviouslyinfectedby Nosemaceranae.PLoSONE6:e21550. Wu,J.Y.,C.M.Anelli,andW.S.Sheppard.2011.Sublethaleffectsofpesticideresiduesin broodcombonworkerhoneybee(Apismellifera)developmentandlongevity.PLoSONE6: e14720. Yamamoto,I.1999.Nicotinetonicotinoids:1962to1997,pp.327.InI.YamamotoandJ.E. Casida(eds.),Nicotinoidinsecticidesandthenicotinicacetylcholinereceptor.SpringerJapan KK,Osaka,Japan. Yang,E.C.,Y.C.Chuang,Y.L.Chen,andL.H.Chang.2008.Abnormalforagingbehavior inducedbysublethaldosageofimidaclopridinthehoneybee(Hymenoptera:Apidae).Journal ofEconomicEntomology6:17431748.

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 Fig.1.NumberofdifferentbiocidesdetectedinhoneybeeproductsacrossNorthAmerica(Mullinet al.2010).Thenumberofbiocidedetectionswere87in259waxsamples,98in350pollensamples, and46in140adultbeesamples.Atotalof118differentbiocidesweredetectedacrossall749bee productsamples.

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 Fig.2.Averageandmaxnumberofdifferentbiocidesdetectedinhoneybeeproductsamplesacross NorthAmerica(Mullinetal.2010).Averagebiocidedetectionswere8.0across259waxsamples,7.1 across350pollensamples,and2.5across140adultbeesamples.Themaximumbiocidedetectionofa singlesamplewas39forwax,31forpollen,and25foradultbee.

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 Fig.3.Relativebeetoxicitytofivetopicallyappliedmiticidesandtheirinteractionswithmiticideand fungicidepretreatments(Johnsonetal.2013).Resultsoftherelativetoxicitystudyonthefivemiticides alonearenumberedontheyaxisinorderofmosttoxictoleasttoxic.Beeswerethenpretreatedwith oneoffivemiticidesorfourfungicidesandsubsequentlytreatedwithoneofthefivemiticidesfromthe listontheyaxis.Thepercentofsignificantpretreatmentinteractionsaredisplayedonthexaxis,with fungicidesingreyandmiticidesinblack.Interactionswerequantifiedbyobservingsignificantincreases inbeemortalityafterapplyingpretreatmentandposttreatmentbiocides,comparedtoacontrol acetonepretreatmentandmiticideposttreatment.

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 Fig.4.CompiledBeeInformedNationalManagementSurveysfor20102011,20112012,and 20122013onpercentoverwinteringcolonylossusingproductssoldtocontrolVarroamites.a) Comparisonofmeanpercentagecolonylossforbeekeepersthattreatedtheirhiveswithaknown VarroamitecontrolproductversusbeekeepersnotusinghivetreatmentproductstocontrolVarroa miteinfestations.b)Meanpercentagecolonylossforbeekeepersthattreatedtheirhiveswithdifferent Varroamitecontrolproducts.Thiscomparesmeanpercentagecolonylossusingaspecific VarroacontrolproductversususingadifferentVarroacontrolproduct.

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 Fig.5.EffectofN.ceranaeonhoneybeesensitivitytothaiclopridandfipronil(Vidauetal.2011). Percentageofhoneybeemortalityof(i)uninfectedcontrol(lightgreensquare),(ii)N.ceranaeinfected (darkgreensquare),(iii)uninfectedandchronicallyexposedtofipronil(lightredcircle),(iv)uninfected andchronicallyexposedtothiacloprid(lightbluetriangle),(v)N.ceranaeinfectedthenchronically exposedtofipronil(lightredcircle),(iv)uninfectedandchronicallyexposedtothiacloprid(darkblue triangle),(v)N.ceranaeinfectedthenchronicallyexposedtofipronil(darkredcircle),and(vi)N. ceranaeinfectedthenchronicallyexposedtothiacloprid(darkbluetriangle).Thearrowindicatedthe timeofexposuretoinsecticides(10dayspostinfection).Datarepresentthepercentageofcumulative mortalitycalculatedfrom3cages,eachcontaining50honeybees.doi:10.1371/journal.pone.0021550

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