Thepossibleroleofhoney beesinthespreadof pollenfromfieldtrials

March2012

ameco|bees@wur

honeybees&pollen

Colophon

Title:

Thepossibleroleofhoneybeesinthespreadof pollenfromfieldtrials
AmecoEnvironmentalServices Koningslaan60 3583GNUtrecht TheNetherlands Tel.+31(0)302545840 info@amecout.nl|www.amecout.nl bees@wur|PlantResearchInternational|WageningenUR POBox16 6700AAWageningen TheNetherlands Tel.+31(0)317481279 bijen@wur.nl|www.bees.wur.nl Ameco drs.H.A.W.Kleinjans(Rik),S.J.vanKeulenMSc.(Sjoerd) bees@wur dr.T.Blacquire(Tjeerd),dr.ir.C.J.H.Booij(Kees),C.H.HokAHin(Chula),ing. A.C.M.Cornelissen(Bram),dr.J.A.vanDooremalen(Coby) NetherlandsCommissiononGeneticModification(COGEM) POBox578 3720ANBilthoven TheNetherlands Tel.+31(0)302742777 info@cogem.net|www.cogem.net M.Bovers|NetherlandsCommissiononGeneticModification(COGEM) J.N.M.Calis|WageningenUniversity&Researchcentre/Inbuzz D.C.M.Glandorf|NationalInstituteforPublicHealthandtheEnvironment (RIVM)/GMOoffice P.Hogervorst|MinistryofInfrastructureandEnvironment(I&M) T.J.deJong|LeidenUniversity N.M.vanStraalen|VUUniversityAmsterdam(chairman) Thehoneysurveydatawerepartlyfinanciallysupportedintheproject:KB05 001017InzetbijentegenallergiebytheformerDutchMinistryofAgriculture, NatureConservationandFoodQuality(LNV). Inaddition,theresearchersthankdr.J.D.Kerkvlietforcriticallycommentingon Chapter5ofthisreport. ThisreportwascommissionedbyCOGEM.Thecontentofthispublicationisthe soleresponsibilityoftheauthorsanddoesnotnecessarilyrepresenttheviewsof COGEM. Utrecht,March2012

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honeybees&pollen

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honeybees&pollen

Preface

Infieldtrialswithgeneticallymodifiedplants,visitsbyhoneybeescancontributetothedispersalof pollenofsuchplantstoareasoutsidethefieldtrialarea.Ifthispollencouldleadtohybridization betweentheGMplantandwildrelativesinthesurroundingsofthefieldtrial,orifpollencouldend upinhoneyproducedbylocalbeekeepers,pollendispersalbyhoneybeesshouldbeconsideredin theriskassessment.TheGMOOfficeofTheNetherlandsandtheNetherlandsCommissionon GeneticModification(COGEM)havethereforecommissionedastudyontheroleofhoneybeesin thedispersalofpollenwithanemphasisonthedistancesoverwhichpollencanbetransferredin relationtocrosshybridizationandtheconcentrationsofpolleninhoney.Thisreportcontains valuablebackgroundinformationontheforagingbehaviourofhoneybees,thedistancesoverwhich theyfly,andthepracticesofbeekeepersinTheNetherlandsthatinfluencethetransportofpollen.It alsoincludesanewsurveyontheconcentrationsofpolleninhoneysmanufacturedinThe Netherlands.Iamconvincedthatthesedatawillstrengthentheriskassessmentprocedures presentlydoneforfieldtrialsofGMcrops. NicoM.vanStraalen Chairmanadvisorycommittee

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honeybees&pollen

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honeybees&pollen

Contents

Glossaryofterms ..................................................................................................9 Executivesummary .............................................................................................11 Samenvatting ......................................................................................................19 1

1.1 1.2 1.3

Introduction .................................................................................................27
Background........................................................................................................................ 27 Objectiveofthereport ...................................................................................................... 27 Structureofthereport ...................................................................................................... 28

2
2.1

Honeybees...................................................................................................29
2.1.1 2.1.2 2.1.3 2.2 2.3 2.4 Introduction....................................................................................................................... 29 TheEuropeanhoneybee ............................................................................................... 29 Twosexesandtwocastesofhoneybees ...................................................................... 31 Honeybeelifestages..................................................................................................... 32 Theforageofhoneybees .................................................................................................. 34 Pollinationbyhoneybees.................................................................................................. 38 Thefateofpollenasmanagedbyhoneybees .................................................................. 38 Historyinbrief ................................................................................................................... 43 Dutchbeekeepers:hobbyistsandprofessionals ............................................................... 43 Dutchbeekeepingassociations ......................................................................................... 44 Utilizationofhoneybeesforpollinationofcrops ............................................................. 45 Honeyandotherbeeproducts.......................................................................................... 48

3
3.1 3.2 3.3 3.4 3.5

BeekeepinginTheNetherlands ....................................................................43

4
4.1 4.2 4.3 4.4 4.5

Foragingandpollentransport ......................................................................53
Introduction....................................................................................................................... 53 Keystudies......................................................................................................................... 55 Cropspecificinformation .................................................................................................. 57 Otherpollinatinginsects.................................................................................................... 59 Concludingremarks ........................................................................................................... 60

5
5.1 5.2 5.3 5.4

Polleninhoney.............................................................................................62
Introduction....................................................................................................................... 62 Materialsandmethods...................................................................................................... 64 Results................................................................................................................................ 68 Discussion .......................................................................................................................... 73

6 7 8 9

Conclusions ..................................................................................................77 Listofreferences ..........................................................................................81 Listofinterviewedpersons...........................................................................85 Annexes........................................................................................................87

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honeybees&pollen

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honeybees&pollen

Glossaryofterms
Anther Aphid Thepollenbearingpartofthestamenofaflower Small,softbodiedinsectsofthefamilyAphididae(Homoptera)thathave mouthpartsspeciallyadaptedforpiercingandfeedingbysuckingphloemsap fromplants;alsoknownasplantlouse Theegg,thelarvaandthepupastagesinthelifeofhoneybees Thebreedingareaofthecombthatcontainslarvaeandpollen Amassofhexagonalwaxcellsbuiltbyhoneybeestocontainlarvaeandstore honeyandpollen Corbicula Cross pollination Dioecious Flower constancy Genetic modification Aflatorhollowareaborderedwithstiffhairsonthehindlegs(tibia)ofabee, suitedtocollectandtransportpollen;alsoknownaspollenbasket Thetransferofpollenfromthemalereproductiveorgan(anantheroramale cone)ofoneplanttothefemalereproductiveorgan(astigmaorafemale cone)ofanotherplant Plantsthatdevelopmaleandfemaleflowersonseparateindividuals Thetendencyofindividualpollinatorstoexclusivelyvisitcertainflower speciesormorphswithinaspecies,bypassingotheravailableflowerspecies thatcouldpotentiallybemorerewarding(i.e.containmorenectar) TheprocessofusingrecombinantDNA(rDNA)technologyforthepurposeof alteringandcontrollingthegeneticmakeupofanorganism,byintroducing neworreinforcedtraitstoacropthatareagronomicallyorotherwise desired;alsoknownasgeneticengineering Aspecializedorganthatproducesasubstanceandsecretesthisforfurther use Theareaofthecombinwhichhoneyisstored Asugarrichstickyliquid,secretedbyaphidsandsomeothersmallscale insectswhentheyfeedonphloemsapfromplants Periodinwhichhoneybeecoloniescancollectaverylargeamountofhoney Partofacommercialbeehivethatisusedtocollecthoney

Brood Broodcomb Comb

Gland Honeycomb Honeydew Honeyflow Honeysuper

Hypopharyngeal Glandsthatsynthesizeandsecreteforashortperiodoftheadultworkers gland lifethesocalledroyaljelly;locatedintheheadofthehoneybeebelowthe pharynx Imaginaldisc Mandible Micropyle Oneofthepartsofaninsectlarvathatwillbecomeaportionoftheadult insectduringpupaltransformation Apairofappendagesneartheinsectsmouth Asmallopeningatthebroadendoftheeggofahoneybee,whichallowsfor passageofsperm

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 Monoecious Moulting Nectar

Plantsthatdevelopmaleandfemaleflowersonthesameindividual,orcarry bisexualflowers Themannerinwhichinsectsandotherarthropodsroutinelycastofftheir cuticleduringlarvalstages Asugarrichliquidsecretedbynectariesinoroutsideflowersofvarious plants,consumedbypollinatorsandgatheredbyhoneybeesasenergy source;honeybeesprocessthenectarintohoneyandstorethisasenergy reserveforthewintertime Thesugarsecretingglandonplants,insideoroutsideaflower,thatattracts andfeedspollinators(anddefenders) Invascularplants,phloemisthelivingtissuethatcarriesorganicnutrients, particularlycarbohydrates(sugars),synthesizedinthegreentissues,toall partsoftheplantwhereneeded Themalegermcellsproducedbyallfloweringplantsforfertilizationand plantembryoformation Thestrawliketongueofthehoneybeeconsistingofmodifiedmouthparts, amongothers,usedforsuckingliquids Apolyphenolrichresinoussubstancewithantibacterialandantifungal properties,collectedbyhoneybeesfrombotanicalsources,tobeusedasa sealanttocoverunwantedopenspaces,cellsofthecombandtheinnerwalls ofthenest Akindofvalvebetweenthehoneystomachandtherealstomach (ventriculus)ofahoneybee,whichfilterssolidparticlesfromthecontentsof thehoneystomach Asetofscoresthatisnotequalonbothsides:thisresultsfromafewscores inadatasetfallingfarthertooneendortheother Thereceptivetipofthecarpel(i.e.theovuleandseedproducing reproductiveorganinfloweringplants),whichreceivespollenatpollination andonwhichthepollengrainsgerminate

Nectary Phloem

Pollen Proboscis Propolis

Proventriculus

Skewed distribution Stigma

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Executivesummary
Honeybeesareimportantpollinatorsinagriculturalcrops,homegardens,orchardsandwildlife habitats.Astheyflyfromflowertoflowerinsearchofnectarandpollen,theytransferpollenfrom planttoplant,thusfertilizingtheplantsandenablingthemtobearfruit.Inlightofthis,honeybees couldbeafactorinspreadingpollengrainsderivedfromgeneticallymodified(GM)plantsinfield trials.Theextenttowhichpollendispersaloccursandthedistancesachieveddependsonmany factors.Knowledgeofthesefactorsmaybeimportantfor(future)riskassessmentsofGMplants. Anoverviewofrelevantinformationconcerningtherelationshipbetweenhoneybeesandpollenis presented,basedonaliteraturesurvey,adatabaseofpollencompositionofDutchhoneysanda conciselaboratoryexperiment.Moreinparticular,thisreportencompasses: Informationaboutthespreadofpollenbyhoneybees,inconnectionwithdeterminingthe possibilityofpollentransportandpotentialcrosspollinationwithrelated(wild)plantsinthe surroundingenvironment Informationaboutthepresenceofpolleninhoney Anexperimentalestimationofthepollencontentofhoneys(ingpollen/ghoney) InformationaboutbeekeepersandbeekeepinginTheNetherlands

Honeybees
Amongthemanyspeciesofbees,honeybeesrepresentbeesfromthegenusApis,primarily distinguishedbytheproductionandstorageofhoneyandtheconstructionofperennial,colonial nestsoutofwax.InTheNetherlands,theEuropean,Western,orCommonhoneybee(Apis mellifera)isanativespecies.ThesubspeciesApismelliferamellifera,whichexistsinThe Netherlands,isalsoknownastheEuropeandarkbee. Twosexes(male(i.e.drones)andfemale)andtwocastesoffemalebees(queensandworkers)make upthepopulationofabeehive,eachhavingitsowncharacteristics,roles,andtaskswithinthehive. Thevastmajorityofhoneybeesareworkerbees.Theworkerbeessequentiallytakeonaseriesof specifictasksduringtheirlifetime,suchascleaningandforaging.Foragerstakecareofbringingfrom theenvironmenteverythingthatthecolonyneedstothehive:pollen,nectar,waterandpropolis: Pollen Pollenistheplantproteinsourceforthelarvaeandyoungbees,providingnitrogen,phosphorus, aminoacids,andvitaminsessentialfordevelopmentofthebees.Polleniscollectedinthefurof thebeeandstoredfortransportinpollenbaskets(corbicula)onthetibiaeoftheworkers'rear legs.

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 Nectar Nectar,obtainedfromfloralnectaries,sometimesdeepinsideflowers,orfromextrafloral nectaries,providesanalmostpurecarbohydratesourceforhoneybees. Water Honeybeescollectonlylittleamountsofwater,becausetheirmusclesproducealotofwater whenburningsugars,andthenectarcollectedcontainsalotofmoisture.Sometimesextra waterisneededforcoolingthehive. Propolis

Propolis,anothersubstancebroughtintothehive,isalsocalledbeeglue.Itisaplantresinused tocoverthehivewallsandcombstoclosecracksandholes.Ithasantibacterialandantifungal properties. Foragingofhoneybeesishighlyorganized:throughtrophallaxis(i.e.theexchangeoffood)andthe communicationbythebeedance(waggledanceandrounddance),thereexistsawarenessofthe needsandthespatialdispositionofresourcesintheenvironmentamongtheworkersinacolony. Pollinationbyhoneybees Pollinationisthetransferofpollengrains,themalesexcellsofaplant,fromtheantherwherethey areproducedtothereceptivesurface,orstigma,ofthefemaleorganofaflower.Honeybeesare amongtheinsectsthattransferpollenbetweenflowersandbetweenplantsandareoftenregarded asbeingthemostimportantpollinators,hencethewordpollinationishabituallyusedtodescribe theserviceofprovidingbeestopollinatecropplants. Honeybeesareefficientpollinatorsforseveralreasons.Theirhairybodieseasilytrappollenand carryitbetweenflowers.Thebodysizeofhoneybeesenablesthemtopollinateflowersofmany differentshapesandsizes;however,sometimesnopollinationoccursdespitenectarorpollen removal,forinstancebecausestigmasarenottouched.Thepollinationpotentialofhoneybeesis alsohighbecausetheycanbemanagedtodeveloplargepopulations. Polleninthepelletsinthebeespollenbasketmayremainviableforseveralhours,butarelostfor pollination.Somepollenonthebeebodyisalsoabletogerminateafterseveralhours,andsocanbe transferredinviablestateoverlargedistances,butmostofitisdepositednearbyflowersofthe sameplantoronotherplantscloseby.Typicalnumbersofpollengrainsfoundonthebeesbody amountto1,000to10,000grainsperbee.Pollenlongevityvariesconsiderablybetweenplant species.

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 Powderbox Theairinsidethebeehive,aswellasitsinhabitants,appearstocontainpollenfromallsources visitedbythebees.Duetothispowderboxeffect,individualbeesmaycarrysignificantamountsof pollenfromplantsnotactuallyvisitedbythem.Althoughthenumberofpollengrainsexchanged insidethehiveverylowcomparedtothenumberdirectlydepositedonforagingbees,itmaybe ofsomesignificanceforcrosspollinationbetweenplantsindifferentspotsthatarenotindirect flightconnectionofindividualbees.Thispathwaywithconnectingflights,throughwhichpollenis carriedintothehivebyoneindividualmaybecarriedtoadifferentflowerpatchbyanother individualmightimplythatthemaximumpollenflowdistanceactuallycanbetwicethemaximum flightdistanceoftheindividualhoneybees. Pollenendingupinhoney Naturalhoneyalwayscontainspollengrains,andoftensporesoffungiaswell.Pollengrainscanend upinhoneythroughthreeroutes: 1. 2. 3. Pollenfromtheflowerthatisvisitedbythebeefallsintothenectarandisharvestedbythe bee; Pollenonbeesandintheairinsidethehivecontaminatethenectarduringprocessingby thebees(packing,drying,conserving);and Pollenpackedincellsinthesamecombasthehoneymaybemixedwiththehoneybythe beekeeperduringcentrifugationorpressingofthehoneyfromthecombs.Especially pressingofhoneycombsmayaddalotofpollen;however,thismethodisseldomappliedin commercialhoneyproduction. Honeyflows Insomeperiodsveryrichhoneyflowsareavailable,fromwhichbeecoloniescaneasilyharvesta lotofhoneyandpollen,andwhichboostthedevelopmentofcolonies.Thesehoneyflowsallowthe beekeepertoharvesthoney.Inbetweenhoneyflows,thebeespartlyrelyontheirreservesand forageonseveralplantspeciesthatjustdeliverenoughresourcestocoverthemaintenanceofthe colony. Mosthoneyflowsoccurduringspring,fewerduringsummerandthebeginningofautumn.The

opportunisticnatureofthehoneybeesresultsindifferentlimitsforasourceinordertobeattractive forthebees.

Foragingandpollentransport
Findingrewardingspots Honeybeesareverywellequippedtolocateplacesinalldirectionsaroundthebeehivewhereplants arefloweringandlotsofpollenandnectaristobefound.Assoonasscoutshavefoundarewarding

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 foragingplace,thisiscommunicatedinthebeehive,whichenablesnectarforagersandpollen foragerstovisitthesespotsaswell. Nearbyorfaraway?

Whenenoughpollenandnectarisavailablenearthebeehiveorfoodisconcentratedinasmallarea, beestendtoflyaslittleaspossibleandbringtheirpollenornectartothecolonyrightaway,Asthis isoftenthecase,mostofthetimethereisnoneedtoflyfurtherthanafewhundredmeters. However,whennearbyfoodislimitedandgoodpatchesareavailableatlargerdistances,honey beesareknowntoflymorethan5,uptoeven10kilometres. Thedistanceoverwhichpollenaretransporteddependsnotonlyofthepotentialflyingcapacityof honeybees,butisalsodependentonthecomplexforagingbehaviourofbees.Theforaging behaviourdependsona)thetimeoftheyear,b)thestateofthecolony,c)thedistributionoffood patchesinthelandscapesurroundingthebeehive,andd)onthevariationinactivitybetween individualbees.Thequantificationofpollentransportintermsofamountsinspaceandtimeis thereforemuchmorecomplexthanonlymeasuringflyingdistances. Spatialactivitypatterns Therangeanddistancecoveredbybeesisextremelyvariable.Thewayinwhichbeesdistributetheir activityintheenvironmentaroundthebeehivelargelydependsonhowthemostrewardingfood sourcesaresituatedinthelandscape.Onlyafewstudiesonbeesandhoneybeeshaveanalysed theseactivitypatternsindetail.Morerecently,modelshavebeendevelopedtodescribethese patternstopredictthetransportofpollenfromflowertoflowerandfromfieldtofield. Transportofpollenandcrosspollination Whenvisitingflowerstheforagingbeescontinuouslytakeup,butalsolosepollen.Whenflyingto anotherpatchortothehive,thousandsofpollengrainsaretransported.Inpractice,mostpollenis transportedoversmalldistancesanddroppedagain.Oftenonlyasmallfraction(lessthan1%)is carriedlongenoughtomediatepollentransferbetweenplantsmorethan1kmapart.Itisdifficultto quantifytheseeventsandtheirimpactongeneflow.Theprobabilityofpollentransportand potentialcrosspollinationisexpectedtodecreaseexponentiallywithdistance,notonlybecause onlyfewbeestendtoflylongdistances,butalsobecausepollenislostandreplacedbynewlocal pollenandbecauseoflossofpollenviability.Experimentsandmodellingapproachesgiveinteresting andencouragingresults,butsimulatingpollinationpatternsinheterogeneouslandscaperemainsan elusivegoal.

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Polleninhoney
Realhoneyalwayscontainspollengrains,originatingfromcontaminationofthenectarinsidethe flower,contaminationofnectarandhoneybythebeesduringprocessingandfromcontamination bythebeekeeperduringhoneyharvestfromthehoneycombs.Contaminationbythebeesduring processinginsidethehiveisthepredominantroute. Thebotanicaloriginofthehoneyistosomeextentreflectedinthepollenprofileofahoney.Tobe qualifiedasaunifloralhoney,aminimumshareofspecificpollenspeciesneedtobemet.Almost200 honeysamplesfroma2008surveyalloverTheNetherlandswereanalysedfortheirbotanicalpollen profiles.Morethan50plantfamilieswererepresented.Inthisreportanoverviewispresented, focussingonthepresenceofpollenfromSalicaceae,Fabaceae,Rosaceae,Brassicaceae,Solanaceae, Chenopodiaceaeandmaize.Pollenfrommaizewasfoundintwosamples(1.54.2%ofthegrains). BothRosaceaeandBrassicaceaewerewellrepresented,butitisnotknownwhichpartofthese representappleandoilseedrape.ThefamilySolanaceae,towhichpotato(Solanumtuberosum) belongs,wasrepresentedinseveralhoneys.However,itcannotbedeterminedwhetherpotato pollenwasinvolved.Chenopodiaceaepollenwasrelativelyrareinthehoneysamples,andthe chanceofitbeingpollenofbeet(Betavulgaris)isverysmallsincebeetisnotattractivetobeesand becauseithardlyeverflowers. Fivehoneysamplesoftheabovementionedsurveywereusedtoquantitativelydeterminethe abundanceofpollengrains,usingacytometriccountingchamber.Thenumbersrangedfromafew hundredto28,000pergramofhoney.Intheliteraturedata<2000to10.000arementioned.Further researchisnecessaryandthemethodforsamplepreparationandcountingstillneedssome improvement. Toexpresspollenabundanceonamassbasis,acalibrationexperimentwasconductedandthe specificweightofpollengrainswasestimatedforfiveplantspecies.Itwasfoundthatindividual pollengrainsofthechosenspeciesweighedfrom0.0043gto0.067g.Togetherwiththecounted numberofpollengrainsinhoney,thisimpliesthatahoneymaycontain43to670gpollenper gramofhoney.Forunifloraloilseedrapehoneyandfruitblossomhoney,thecontentofthespecific pollen(oilseedrape,appleandpear)wouldbeabout60g/g.

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Conclusions
Themainconclusionsofthereport,clusteredpertopic,arethefollowing: BeekeepinginTheNetherlands BothhobbyistandprofessionalbeekeeperspracticebeekeepinginTheNetherlands.Ofthe approximately8,000Dutchbeekeepers,about6,900areorganisedinthreebeekeeping associations,ofwhichtheDutchBeekeepersAssociation(NederlandseBijenhoudersvereniging NBV)isbyfarthelargestwith6,000members. Hobbyistbeekeepershaveanaverageoffivecolonies,professionalbeekeeperswelloverone hundred. Aroundtenbeekeepingcompaniesareprofessionallyinvolvedinthepollinationofcropswith honeybeesandmakealivingoutofit.Overall,itisestimatedthatsomethinglike32,000honey beecoloniesareutilizedforpollinationinTheNetherlandsbyapproximately1,700beekeepers. Hobbyistbeekeepersseldommovetheirhives(exceptwhenhiredforpollinationinfruit orchards),whilstprofessionalsregularlymovetheirhives(every4weeksonaverage). InTheNetherlands,honeybeesarebeingutilizedinfruitorchards,inthehorticultureindustry andforseedproduction.Professionalbeekeepersorbeekeepingcompaniescovernearlyall pollinationinclosedgreenhousesandpolytunnels.However,regularcollaborationtakesplace withhobbyistbeekeepers,inordertoguaranteethesupplyofbeecolonies.Pollinationinthe fruitsectorismostlycoveredbynoncommercial,hobbyistbeekeepers. Theannualvalueofpollinationfeesisroughlyestimatedat4millionEuroforoutdoor cultivation,and7millionEuroforcultivationingreenhousesandpolytunnels.However,the indirecteconomicvalueorbenefitofpollinationbyhoneybeesandotherinsectpollinatorsis consideredtobemuchhigher. Honeybeesarenotonlyutilizedforpollination.Severalproductsthatareproducedbybeescan beharvestedbythebeekeeper.Obviously,themostrecognizableproductmadebyhoneybees ishoney.Otherproductsincludebeeswax,propolis,royaljelly,beepollenandbeevenom.Allof thesebeeproducts(could)contain(traceamountsof)pollen. Foragingandpollentransport Honeybeesvisitflowersforpollen(theirprotein,fatandmineralsource)andnectar(energy source).Individualbeesspecializeanddonotforagefornectarandpollensimultaneouslyand devotetheirforagingtoonlyoneplantspeciesatatime. Foragingisnotonlyoptimizedattheindividualforagerlevel,butonthewholecolonylevel.This isachievedthroughcommunicationandtaskdivision. MosthoneyontheDutchmarket(92%)isimportedfromamongstothersChinaandArgentina; thetwolargesthoneyproducingcountriesintheworld.

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 Onlyasmallpartofthepollenthatiscollectedonflowersisavailableforpollination:beesuse morethan99%asfood. Thedistanceuptowhichbeesforageforwater,pollenandnectarmayrangeupto2,5,1014 kmrespectively.However,ifarichresourceisnearby,mostoftheforagingtripswillbemuch shorter(0.51km).Iffoodnearbythecolonyisscarceandveryrewardingpatchesarefurther away,largeflightdistancesarelikelytobecomefrequent. Insidethehiveofhoneybees,pollenisexchangedunintendedbetweenindividuals:thismay leadtopollentransportbetweendistantflowerpatchesthatarenotvisitedbythesame individualforager. Foroilseedrape,ithasbeenshownthata)beesmayflylongerdistancestoforageonsuch fields,andb)beestendtostayclosetotheirhiveifthehiveislocatednexttoanoilseedrape field.

Appleisaveryattractivecropforhoneybeestoforageon.However,itisnotdocumentedwhich distanceshoneybeesflytoforageonapple,becauseitiscommonpracticetoplacecoloniesin (orverynear)appleorchardsinordertooptimizepollination.

Honeybeesarenotattractedtothecroppotato,unlessaphidsfeedonthepotatoplants. However,farmersrarelytolerateaphidsthatareattractivetohoneybees,andevenifaphidsare present,aphiddensitiesoftenpeekbeforeflowering.

Sugarbeetcropsarenotmeanttoflowerandarethereforenotattractivetohoneybees. Maize,althoughitisawindpollinatedplant,isalsovisitedbyhoneybees.However,nodatais availableaboutthedistancesbeescoverformaize.Inaddition,hardlyanyofthemaizepollen collectedmayresultinpollination,sincehoneybeesarenotatallinterestedinfemalemaize flowers.

Thoughpotentialflightdistancesofhoneybeeshavebeenmeasuredandestimatedinanumber ofdetailedstudies,itisalmostimpossibletosetupageneralmodelthatpredictsactivityand pollinationpatternsforspecificcases,asthenumberofconditionsthatcaninfluencesuch patternsisalmostunlimited.

Itshouldbekeptinmindthatbesideshoneybees,alsobumblebeesandmanyotherinsectsare involvedinpollentransport.Therefore,muchinformationonwhatthismeansatalandscape scaleisneededtoestimateitsimpactontheoutcrossingphenomena.

Polleninhoney Thepresenceofpolleninhoney Honeycontainssmallamountsofpollen.Thispollenhasenteredthehoneythroughthree pathways:directcontaminationofnectarattheflower,contaminationbybeesinsidethehive, andcontaminationbythebeekeeperduringhoneyharvestingandprocessing.Contaminationby thebeesduringprocessinginsidethehiveisthemostimportantrouteofpollenintohoney.

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 Althoughthebotanicaloriginofthepollengrainsinhoneycanbeanindicationofthenectar sourceofthehoney,thisisnotstraightforwardduetothecontaminationpathsmentioned above.Blendingofcommercialhoneysmayalsobeanadditionalobstacletotraceability. About200honeysamples,fromasurveythroughoutthecountryinTheNetherlands(2008), carriedpollengrainsofatleast50plantfamilies,windpollinatedaswellasinsectpollinated species. Rosaceaepollen(includingapple),Brassicaceaepollen(includingoilseedrape)werevery abundant.Chenopodiaceae(towhichBetabelongs)andSolanaceae(towhichpotatoSolanum tuberosumbelongs)andPoaceae(grassfamily,includingmaize)werealsorepresentedinthe honeys.Maizepollenwasonlyencounteredtwice(outof200)andinverysmallquantities. Experimentalestimationonthepollencontentofhoneys Thenumbersofpollengrains,countedinfiveoftheDutchhoneysfromthesurvey,

correspondedwellwiththosereportedinliterature,generallytoppingat10,000grainspergram honey. Thereliabilityofpollencountingdependsonthedilutionofthesuspensionusedincounting chambersandneedstobefurtherimproved. Astrongandsignificantrelationshipwasfoundbetweentheamountofpollen(grams)addedto anartificialhoney,andthecountednumberofgrainsinthesehoneys.Thehighlysignificant calibrationfactorswereusedtoestimaterealpollenconcentrations(g/g)ofrealhoney. Preliminaryresultsindicatethatonlytraceamountsofpollenarepresentinhoney,rangingfrom 43gto670gpollen/ghoney. Basedonthecalibrationcurves,oilseedrapehoneyandfruitblossomhoneywerecalculatedto containatleast60and58gofthespecificpollen(oilseedrape,appleandpear)pergramof honeyrespectively.

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Samenvatting
Honingbijenbestuivenlandbouwgewassen,plantenintuinen,boomgaardenenplantenindewilde natuur.Alszezichverplaatsenvanbloemnaarbloem,opzoeknaarnectarenstuifmeel(ookwel aangeduidmethetoorspronkelijkEngelsewoordpollen,inhetNederlandshetpollen),wordter stuifmeelvandeenenaardeandereplantovergebrachtwaardoorbevruchtingkanplaatsvinden.In hetlichthiervanzoudenbijeneenrolkunnenspelenindeverspreidingvanstuifmeelafkomstigvan veldproevenmetgenetischgemodificeerde(GG)planten.Dematewaarindezeverspreidingkan optredenendedoorbijentebehalenfoerageerafstandenzijnvanvelefactorenafhankelijk.Kennis vandezefactorenkanvanbelangzijnvoor(toekomstige)risicobeoordelingenvanGGplanten. Inditrapportwordteenoverzichtgegevenvanrelevanteinformatiemetbetrekkingtotderelatie tussenhoningbijenenpollenverspreiding,gebaseerdopeenliteratuurstudie,eendatabasemet stuifmeelgegevensvanNederlandsehoningeneenbeknoptlaboratoriumexperiment.Eris ondermeergekekennaardevolgendeonderwerpen: Informatieoverdeverspreidingvanpollendoorhoningbijen,inverbandmethetbepalenvande mogelijkheidvanpollentransporteneventuelekruisbestuivingmetverwante(wilde)plantenin deomgeving. Informatieoverdeaanwezigheidvanpolleninhoning. Eenexperimenteleschattingvandestuifmeelinhoudvanhoning(ingpollen/ghoning). InformatieoverdeimkersendebijenteeltinNederland.

Honingbijen
BinnendevelebijengeslachtenvertegenwoordigenhoningbijenhetgeslachtApis.Ditgeslacht onderscheidtzichvananderedoordeproductieenopslagvanhoningenhetbouwenvannestenvan was.InNederlandisdeEuropeseofwestersehoningbij(Apismellifera)eeninheemsesoort.De ondersoortApismelliferamelliferadieinNederlandvoorkomtwordtookwelEuropesezwartebij genoemd. Tweegeslachten(mannelijk(darren)envrouwelijk)entweekastenvanvrouwelijkebijen (koninginnenenwerksters)vormensameneenbijenvolk.Deovergrotemeerderheidvan honingbijenzijnwerkstersendezezijngedurendehunlevenverantwoordelijkvooreenaantal specifieketaken,zoalsschoonmakenenfoerageren.Foerageerdersverzamelenalleswathetvolk nodigheeftvanuitdeomgevingvandekast:pollen,nectar,waterenpropolis.

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 Pollen Stuifmeelofpollenisdeplantaardigeeiwitbronvoordelarvenenjongebijen.Hetlevert essentilebouwstoffenvoordeontwikkelingvanbijen,zoalseiwitten,vetten,mineralenen vitamines.Pollenwordtverzameldindevachtvandebijenopgeslagenvoortransportin stuifmeelkorfjes(corbicula)opdeachterpotenvandewerksters. Nectar Nectarwordtverkregenuitdehoningklierenvanbloemenenbiedteenbijnazuiverebronvan koolhydratenvoorhoningbijen. Water Honingbijenverzamelendoorgaansslechtskleinehoeveelhedenwater,aangezienhunspieren veelwaterproducerenbijdeverbrandingvansuikers.Daarnaastbevatdeverzameldenectar veelvocht.Echter,somsiserextrawaternodigvoorhetkoelenvandekast. Propolis Propolisiseenlijmachtigesubstantie,verzamelddoorhoningbijenvandeschubbenvan knoppenvanbomen,meestalpopulieren.Propoliswordtdoorbijengebruiktomongewenste kierenenopeningeninhetnesttedichten.Propolisremtdegroeivanbacterinenschimmels. Hetfoeragerenvanhoningbijenistotopzekerehoogtegeorganiseerd:viatrophallaxis(het uitwisselenvanvoedsel)endoorcommunicatie,onderanderedebijendans(kwispeldansen rondedans),verkrijgendewerkstersinformatiemetbetrekkingtotdebehoeftevanhetbijenvolken deomliggendevoedselbronnenwaaropgefoerageerdkanworden. Bestuivingdoorhoningbijen Bestuivingisdeoverdrachtvanstuifmeelkorrels(waarindemannelijkegeslachtscellenofde spermakernenwordengevormd)uitdehelmhokjesvandemeeldraadnaardestempelvande

stamper.Honingbijenbehorentotdeinsectendiestuifmeeloverbrengentussenbloemenenplanten enwordenvaakbeschouwdalsdebelangrijkstebestuivers.Vandaardathetwoord'bestuiving' doorgaanswordtgebruiktvoorhetinzettenvanbijenvolkenbijhetbestuivenvangewassen. Honingbijenzijngeschiktvoorbestuivingomverschillenderedenen.Hunbehaardelichaam vergemakkelijkthetopnemenvanpollenenhettransporterenervantussenbloemen.Doorhun lichaamsgroottezijnbijeninstaatbloementebestuivenvanverschillendevormengrootte. Echter,somsvindtergeenbestuivingplaatsondankshetfeitdatnectarofpollenzijnmeegenomen, bijvoorbeelddoordatdestigma'snietzijnaangeraakt.Hetbestuivingspotentieelistevenshoog, doordathoningbijeningrotekoloniesleven. Pollenindestuifmeelkorfjesvanbijenkanenigeurenlevensvatbaarblijven,maarisverlorenvoor bestuiving.Stuifmeelophetlichaamvandebijisookinstaatomnaeenpaaruurnogtekiemen;het

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 eenbloemvaneenandereplantopeengrotereafstand.Deaantallenpollenkorrelsdiezijn aangetroffenophetbijenlichaamliggengemiddeldtussende1.000en10.000korrelsperbij.De levensduurvanstuifmeelkanaanzienlijkverschillentussenplantensoorten. Poederdoos Deluchtinbijenkastenenhethaarkleedvandebijen,blijktstuifmeeltebevattenvanongeveeralle doordekoloniebezochtevoedselbronnenuitdeomgeving.Doordit'poederdooseffect'kunnen individuelebijenaanzienlijkehoeveelhedenstuifmeelmetzichmeedragenvanplantendienietdoor henzelfbezochtzijn.Hoewelhetaantalstuifmeelkorrelsdatwordtuitgewisseldindekastzeerlaag isinvergelijkingmethetaantaldatdirectterechtkomtopfoeragerendebijen,kanhetvanenige betekeniszijnvoorkruisbestuivingtussenplantenopverschillendelocaties,dienietopdedirecte vliegroutesvanindividuelebijenliggen.Hetkandusvoorkomendathetpollenvandeenebijinde kastwordtopgepiktdooreenanderebijenvervolgenswordtgetransporteerdnaareenveldinde tegenovergestelderichting.Ditzoubetekenendatdeafstandwaaroverpollenverplaatstkan worden,infeitetweemaaldemaximalevluchtafstandvanindividuelebijenis. Hoepollenterechtkomtinhoning Natuurlijkehoningbevataltijdpollenkorrelsenvaakooksporenvanschimmels.Pollenkorrels kunnenopdriemaniereninhoningterechtkomen: 1. 2. 3. Pollenvandebloemwaaropwordtgefoerageerdbelandtindenectarendezenectarwordt vervolgensgeoogstdoordebij; Pollenopdebijenenindeluchtvandekastverontreinigtdenectaralsdezewordt verwerktdoordebijen(verpakken,drogen,opslaan); Pollendatisopgeslagenindecellenvandezelfderaatalswaarindehoningisopgeslagen kanindehoningterechtkomenbijhetslingerenofpersenvanderaatdoordeimker. Vooralhetpersenvanderaatzorgtervoordatveelpollenindehoningbelandt;deze methodewordtechternauwelijksmeertoegepastbijcommercilehoningproductie. Dracht Inperiodenvangoededrachtzijndrachtplantenaanwezigdieopdatmomentgrotehoeveelheden nectar,stuifmeelen/ofhoningdauwbieden.Eengoededrachtisoverhetalgemeenbevorderlijk voordegezondheidvanhetbijenvolkensteltdeimkerinstaatdehoningteoogsten.Tussen drachtenmakenbijendeelsgebruikvanhunreservevoorradenenfoeragerenzeopbepaalde plantensoortendienetgenoegvoedselleverenomdekolonietekunnenonderhouden. Demeestegoedehoningdrachtenvindenplaatsinhetvoorjaareninminderematetijdensdezomer enhetbeginvandeherfst.Hetopportunistischekaraktervandehoningbijenresulteertin

isechterwaarschijnlijkerdathetwordtafgezetopeennabijgelegenbloemvandezelfdeplantdanop

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 eenhoge,indezomereenlageredrempel.

verschillendedrempelwaardenvoorgewassenomaantrekkelijktezijnvoordebijen:inhetvoorjaar

Foerageergedragenpollentranport
Hoewordendebestevoedselplekkengevonden? Honingbijenzijnheelgoedinstaatomplekkenrondomdekastentevindenmetveelbloeiende plantenwaarstuifmeelennectartehalenis.Zodradeverkennerseenvoedselrijkeplekhebben gevondenvliegenzeterugnaardekastwaarzeaandeanderebijenviadebijendanslatenweten waardezeplekkenzichbevindenzodatanderepollenennectarverzamelendebijendezegaan bezoeken. Dichtbijofverweg? Omenergietesparenenefficintvoedselteverzamelenvliegenbijenhetliefstzominmogelijk zodatalletijdbenutkanwordenvoorhetfoerageren.Ditkanechteralleenalservoldoendevoedsel aanwezigisnabijdekastofalsvoedselvoldoendegeconcentreerdisopnplek.Omdatdekasten veelalopgunstigeplekkenwordengeplaatstzaldevliegafstanddoorgaansnietverderzijndaneen paarhonderdmeter.Alsdehoeveelheidvoedselindenabijheidvandekastechteronvoldoendeis energoedeplekkenopafstandaanwezigzijnblijkenhoningbijenprobleemloosafstandenvan5tot 10kmtekunnenoverbruggen. Deafstandwaaroverstuifmeelwordtgetransporteerdhangtnietalleenafvandevliegcapaciteitvan debijenmaarwordtmedebepaalddooranderefactorenrondhetfourageergedrag.Hoeveren waarheenbijenvliegenhangtonderandereafvana)detijdvanhetjaar,b)degezondheidstoestand /fitheidvandekolonieenc)deverdelingvanvoedselrijkeplekkenindeomgeving.Erisooksprake vanverschillentussenindividuen.Hetbepalenvandematevanstuifmeeltransportinruimteentijd isveelmoeilijkerdanhetmetenvanalleenvliegafstanden. Ruimtelijkeactiviteitspatronen Uithetvoorgaandemogeblijkendatdeafstandendiebijenoverbruggenomvoedselteverzamelen indepraktijkergvariabelzijn.Demanierwaaropbijenvanuitdekasthunomgevingexploiteren hangtvooralafvandeliggingvandebestevoedselplekkeninhetomringendelandschap.Erzijn slechtseenpaarpublicatiesbekendwaarindefoerageerpatronengedetailleerdzijnonderzochten beschreven.Recentzijnmodelstudiesgedaanomdevliegpatronentevoorspellenopzowelkleine schaal(vanbloemtotbloem)alsovergrotereafstanden(tussenpercelen). Stuifmeeltransportenkruisbestuiving Terwijlbijenvanbloemtotbloemvliegenwordtsteedsmeerstuifmeelopgenomenmaareendeel gaatdaarbijookweerverloren.Tijdenslangerevluchtenwordenduizendenstuifmeelkorrels getransporteerd.Indepraktijkwordtvooralveelstuifmeeloverkorteafstandenverplaatst.Slechts

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 inuitzonderlijkegevallenzaleenkleinefractie(minderdan1%)overgrotereafstanden(>1km) wordenverspreid.Dekansdatstuifmeelwordtverspreidentotkruisbestuivingkanleidenneemt exponentieelafmetdeafstand,nietalleenomdatslechtsweinigbijengroteafstandenoverbruggen maarookomdatveelstuifmeelonderwegverlorengaat.Experimentenenmodelstudiesbieden steedsmeerinzicht,maarechtgoedevoorspellingenoverdematewaarinstuifmeeltransportin complexelandschapssituatiestotkruisbestuivingkanleidenzijnopbasisvandebeschikbare gegevensnietmogelijk.

Stuifmeelinhoning
Echtehoningbevataltijdstuifmeelkorrels,dieomdiverseredenenindehoningterechtkunnenzijn gekomen:alindebloemindenectar,onbedoeldtijdensdehoningbereidingdoordebijenzelf,of tijdensdehoningoogstdoordeimker.Hetmeestestuifmeelindehoningkomtvandebijenzelfin dekasttijdensdehoningbereiding. Debotanischesamenstellingvandestuifmeelkorrelsindehoningweerspiegelttotopzekerehoogte dediversiteitvanplantensoortenwaarvandenectarverzameldis.Omtewordengekwalificeerdals plantspecifiekehoningdientereenbepaaldminimumaandeelvanhetstuifmeelvandieplantinde honingvoortekomen.In2008isdoorbijen@wurin200honingmonstersdeaanwezigheidvan verschillendesoortenstuifmeelgeanalyseerdwaarbijvooralgekekenisnaarstuifmeelvan Salicaceae,Fabaceae,Rosaceae,Brassicaceae,Solanaceae,Chenopodiaceaeenmas. Maspollenwerdinslechtstweehoningmonstersaangetroffen(max.1.54.2%vandekorrels). PollenvanRosaceaeenBrassicaceaewerdveelgevondenmaarhetwasnietvasttestelleninwelke mateditpollenvanappel,respectievelijkkoolzaad,was.PollenvandeSolanaceae,waartoeook aardappelbehoort,werdookinverschillendemonstersgevondenmaarookhierisnietzekerofer aardappelstuifmeelbijzat.StuifmeelvanChenopodiaceaewerdvrijwelnietaangetroffenendekans dathetsuikerbietstuifmeelbetrofisergkleinomdatdezeplantnauwelijksbloeitenookniet aantrekkelijkisvoorbijen. Invijfvandebovengenoemdemonsterswerdendeaantallenstuifmeelkorrelsdoormiddelvande cytometrischetelkamerzonauwkeurigmogelijkgeschat.Hetaantalkorrelspergramhoningbleekte varirenvaneenpaarhonderdtot28.000.Ditstemtredelijkovereenmetdeliteratuurwaarineen rangevan<2.000tot10.000wordtbeschreven.Erismeeronderzoeknodigomdemonsterbereiding entelmethodenteverbeteren. Omdestuifmeelhoeveelhedeninhoningnietalleeninaantalkorrelstekunnenuitdrukkenmaarook ineengewichtaanstuifmeel,werdhetgewichtperstuifmeelkorrelvastgesteldvoor5 plantensoorten.Hetgewichtperkorrelloopttussendezevijfsoortensterkuiteenvan0.0043gto 0.067g.

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 Wanneerdezegetallenwordengecombineerdmetdeaantallenkorrelspergramhoning,blijktdat honing43tot670gstuifmeelpergramkanbevatten.Voorplantspecifiekekoolzaadhoningof fruithoningwordtgeschatdatpergramongeveer60gkoolzaadrespectievelijkappelstuifmeel aanwezigzalzijn.

Conclusies
Hierondervolgendebelangrijksteconclusiesvanhetrapport,geclusterdperonderwerp. BijenteeltinNederland InNederlandzijnzowelhobbymatigealsprofessioneleimkersactief.Vandeongeveer8.000 imkerszijnercirca6.900aangeslotenbijdriebijenhoudersverenigingen.DeNederlandse Bijenhoudersvereniging(NBV)isverrewegdegrootstemet6.000leden. Eenhobbyimkerheeftgemiddeldvijfbijenvolken,professioneleimkersmeerdanhonderd. Erzijnongeveertienimkerbedrijvenactiefopdebestuivingmarktenverdienenhierhunbrood mee.Erwordtgeschatdatcirca32.000bijenvolkenwordeningezetvoorbestuivinginNederland doorongeveer1.700imkers. Hobbyimkersverplaatsenzeldenofnooithunkasten,tenzijzewordeningezetvoorde bestuivingindefruitteelt(boomgaarden).Professioneleimkersverplaatsenhunkastenechter zeerregelmatig:gemiddeldelkevierweken. InNederlandwordenbijeningezetvoordebestuivingvanboomgaarden,indetuinbouwsector enindezaadteelt.Professioneleimkersnemennagenoegallebestuivingonderglas(kassen)en intunnelsvoorhunrekening.Nietteminwordterregelmatigsamengewerktmethobbyimkers omdeaanleveringvanbijenvolkentegaranderen.Bestuivingindefruitteeltwordttraditioneel vooralverzorgddoordehobbyimkers. Dejaarlijkseomzetvandebestuivingsgeldenwordtgrofweggeraamdop4miljoeneurovoor buitenteelten7miljoeneurovoorteeltinkassenentunnels.Deindirecteeconomischewaarde ofdebijdragevanbestuivingdoorbijen(enanderebestuivers)wordtvelemalenhogergeschat. Bijenwordennietenkelingezetvoorbestuiving.Verschillendeproductendiedoorbijenworden geproduceerdkunnendoordeimkerwordengeoogst.Hetmeestherkenbareproductishoning. Andereproductenzijnbijenwas,propolis,Koninginnegelei,pollenkorrelsenbijengif.Aldeze bijenproductenkunnensporenvanpollenbevatten. Foerageergedragenpollentransport Honingbijenbezoekenbloemenvoorstuifmeel(datdientalsbronvooreiwit,vetenmineralen) ennectar(alsenergiebron).Individuelebijenzoekenofwelnectarofwelstuifmeelenrichten zichdaarbijopnplantensoortgedurendededag. DemeestehoningopdeNederlandsemarkt(92%)wordtgemporteerduitonderandereChina enArgentini,detweegrootstehoningproducerendelandenindewereld.

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 Nietalleenfoeragerenbijenalsindividuzoefficintmogelijkmaarookhetopkolonieniveauis hetfoerageergedraggeoptimaliseerd.Ditwordtbereiktdoorcommunicatietussendebijenen dooreenoptimaletaakverdeling. Slechtseenkleindeelvanhetverzameldestuifmeelwordtgebruiktvoordebestuivingvan bloemen.Meerdan99%wordtdoordebijenalsvoedselgebruikt. Deafstanddiebijenkunnenoverbruggenomwatertehalenis2km,voorstuifmeel5kmen voornectar1014km.Alserechtergoedevoedselbronnennabijzijnblijvendemeeste foerageervluchtenvandebijenbinneneenstraalvan0,51kmrondomdebijenkast.Alser echterweinigvoedselindebuurtisenergoedeplekkenopgrotereafstandenaanwezigzijn, danwordensteedsvakerlangeafstandsvluchtengemaakt. Binnendebijenkastwordtonbedoeldstuifmeeltussenbijenuitgewisseld.Ditkanleidentot pollentransporttussenverderafgelegenplaatsendiedoorverschillendebijenwordenbezocht.

Voorkoolzaadisaangetoonddatbijenlangereafstandenoverbruggendoordeaantrekkelijkheid vanzulkevelden.Wanneerkastennabijbloeiendekoolzaadveldenstaanzullendebijendaar vlakindebuurtfoerageren.

Appelistijdensdebloeieenergaantrekkelijkgewas.Hetisechternietbekendhoeverzevoor deboomgaardenzullenvliegenomdatbijenkastenmeestalinofnabijdeboomgaardworden geplaatstomdebestuivingteoptimaliseren.

Aardappelisgeenaantrekkelijkgewasvoorhoningbijen,tenzijerveelluizenopvoorkomen. Boerenlatenechterzeldeneendusdanigeluizenbelastingvanhungewastoedathetvoor honingbijenaantrekkelijkwordt.Bovendienpiekendemeesteluizensoortenvoordebloeivan aardappel.

Suikerbietenkomennormaalgesprokenniettotbloeienzijndaaromnietattractiefvoorbijen. Hoewelmaiseendoorviadewindbestovengewasis,wordendebloeiendeplantenweldoor bijenbezochtvanwegederijkdomaanstuifmeel.Erwerdenindeliteratuurechtergeenharde datagevondenoverdeafstandenwaaropmaisveldenbijenaantrekken.Ookbij maispollentransportisdekansoplangereafstandbestuivingkleinomdatdevrouwelijke bloeiwijzentotaalnietaantrekkelijkzijnvoorbijen.

Inverschillendediepgaandestudieszijnvliegafstandenvanbijengemetenenviaverschillende methodengeschat.Hetisechtervrijwelonmogelijkommodellentemakenomdeactiviteitsen bestuivingspatronenpreciestevoorspelleninconcretesituatiesdaardecomplexiteitvanhet landschapenhetaantalbenvloedendefactorengrootis.

Hetisbelangrijkingedachtentehoudendaternaasthoningbijenenhommelsernogveel andereinsecteninstaatzijnstuifmeeltetransporterenenbijtedragenaanbestuiving.Mede daaromisermeerinformatienodigoplandschapsschaalomhetbelangvanstuifmeeltransport ophetprocesvanuitkruisinggoedtekunneninschatten.

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 Stuifmeelinhoning Deaanwezigheidvanstuifmeelinhoning Honingbevatkleinehoeveelhedenstuifmeel.Ditstuifmeelkanviadriewegenindehoning terechtzijngekomen:directeverontreinigingvandenectaralindebloem,verontreiniging veroorzaaktdoordebijenindekast,enverontreinigingdoordeimkerbijhetoogstenen

verwerkenvandehoning.Hetmeestestuifmeelkomtindehoningterechtdoordeactiviteitvan debijenindekastmethetbewerkenenopslaanvandehoning. Debotanischeherkomstvanhetstuifmeelinhoningkaneenaanwijzinggevenoverde nectarbronwaarvandehoningafkomstigis.Tochisdezeaanwijzingnietnopn,gedeeltelijk doordeanderebovengenoemderouteswaarmeestuifmeelinhoningterechtkomt.Doordat honingpakkersenhandelarenverschillendesoortenmengenwordthetsomsnogmoeilijkerom ietsvandeoorsprongtezeggen. Ongeveer200honingen,afkomstigvaneenmonitoringin2008overheelNederland,bevatten stuifmeelkorrelsvanminstens50plantenfamilies.Datwarenzowelwindbestuivendeals insectenbestuivendeplantensoorten. StuifmeelvanRosaceae(inclusiefappel)enBrassicaceae(inclusiefkoolzaad)kwamenheel frequentenveelvoor.Chenopodiaceae(waartoe(suiker)bietbehoort)enSolanaceae(waartoe aardappelSolanumtuberosumbehoort)andPoaceae(grassenfamilie,inclusiefmais)werden ookinhoningaangetroffen.Stuifmeelvanmaiswerdslechtsin2vande200monsters aangetroffeneninheellagepercentages. Experimentelevaststellingvanhetstuifmeelgehaltevanhoning DeaantallenstuifmeelkorrelsindevijfNederlandsehoningenvandemonitoringin2008 kwamengoedovereenmetaantallenindeliteratuur.Meestalmaximaal10.000korrelsper gramhoning. Debetrouwbaarheidvanhettellenvanstuifmeelkorrelshangtafvandeverdunningvande suspensiediewordtgebruiktindetelkamers.Demethodemoetdaaromnogverbeterdworden. Erwerdeensterkensignificantverbandaangetoondtussendehoeveelheid(grammen) stuifmeeldiewerdtoegevoegdaaneenkunstmatigehoning,enhetdaarnaviatellen teruggevondenaantalkorrelsindiehoning.Desterksignificanteijkfactorenwerdengebruikt omdehoeveelheidstuifmeelinechtehoning(g/g)vasttestellen.Deeersteresultatengeven aandaterslechtheelkleinesporenstuifmeelinhoningvoorkomen,varirendvan43gtot670 gstuifmeel/ghoning. Opgrondvandeijklijnenwerduitgerekenddateenkoolzaadhoningeneenfruitbloesemhoning minstens60en58gvanhetsoortspecifiekestuifmeel(koolzaadenappel)bevattepergram honing.

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1
1.1 Background

Introduction

Honeybeesareflyinginsectsthatareknownfortheirroleingatheringnectartoproducehoney.In additiontothis,honeybeespollinateagriculturalcrops,homegardens,orchardsandwildlifehabitat. Ashoneybeestravelfromflowertoflowerinsearchofnectarandpollen,theytransferpollenfrom planttoplant,thusfertilizingtheplantsandenablingthemtobearfruit.Manycropsrelyonhoney beesandotherbeesforpollination. Inlightofthis,honeybeescouldbeafactorinspreadingpollengrainsstemmingfromgenetically modified(GM)plantsinfieldtrials.Thiscouldpossiblyleadtocrosspollinationofwildrelatives,and pollengrainscouldendupinthehoneyofthesebees.Furthermore,ifanewlyproducedproteinina GMplanthasatoxicorallergeniceffect,thisactivitycouldbeexpressedinthepollenaswell.The extenttowhichthedisseminationcanoccurandthedistancesachieveddependsonmanyfactors, includingthetypeofcrop,thedistanceoverwhichhoneybeesflyandthetimethatpollengrainsare viable.Knowledgeofthesefactorsmaybeimportantfor(future)riskassessmentsofGMplants.

1.2

Objectiveofthereport

Thisreportpresentsanoverviewofrelevantinformationconcerningtherelationshipbetweenhoney beesandpollen,basedonaliteraturesurvey,adatabaseofpollencompositionsofDutchhoneys andaconciselaboratoryexperiment.Moreinparticular,thisencompasses: Informationaboutthespreadofpollenbyhoneybees,i.e.foragingdistancesandthespecific cropsthatarevisited,inconnectionwithdeterminingthepossibilityofpollentransportand potentialcrosspollinationwithrelated(wild)plantsinthesurroundingenvironment. Informationaboutthepresenceofpolleninhoney,i.e.fromwhichcropscanpollenbefoundin honeyandinwhatamountsandpercentages,andwhichfactorsinfluencethesenumbers.Inthis regard,thefocusisonhoneyproducedinTheNetherlandsandonthefollowing(GM)crops, relevantforTheNetherlands:corn,potato,oilseedrape,sugarbeetandapple. Anexperimentalestimationonthepollencontentofhoneys(ingpollen/ghoney). InformationaboutbeekeepersandbeekeepinginTheNetherlands,bothfromahobbyistanda professional(orcommercial)pointofview.

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 Theabovementionedinformationwillbeusedinthecontextofbothfieldtrialsandcommercial cultivation.Accordingly,COGEMwilldrawconclusionsandmakerecommendationstoThe NetherlandsMinistryofInfrastructureandEnvironment. PleasenotethatthepurposeoftheresearchisnottomapoutpossiblerisksofGMcropsforhoney bees.Hence,theauthorswillnotmakerecommendationsregardingthissubject.

1.3

Structureofthereport

AnintroductiontohoneybeebiologyisgiveninChapter2ofthereport,illustratingamongstother itslifestages.InChapter3,beekeepinginTheNetherlandsiselaboratedbydescribingitshistory,the twokindsofbeekeepers(i.e.hobbyistsandprofessionals),andthemostrelevantbeekeeping associations.Inaddition,thewayinwhichhoneybeesareutilizedforthepollinationofcropsis explainedandadescriptionisgivenabouthoneyandotherbeeproductsinTheNetherlands. Chapter4presentsaspectsthatplayaroleinforagingofhoneybeesandpollentransport.Chapter5 portraystheoutcomesofthelaboratoryexperimentonpolleninhoney.Finally,Chapter6presents someoverallconclusionsthatcanbedrawnfromtheresearch.

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2
2.1 Introduction

Honeybees

HoneybeesareasubsetofbeesinthegenusApis, primarilydistinguishedbytheproductionand storageofhoneyandtheconstructionofperennial, colonialnestsoutofwax.Honeybeesaretheonly extantmembersofthetribeApini,whichcontains onlyonegenus:Apis(seePicture1).Currently,there aresevenrecognisedspeciesofhoneybeewitha totalof44subspecies,thoughhistorically,anywhere fromsixtoelevenspecieshavebeenrecognised.

Picture1 Apismellifera,oneofsevenrecognised speciesofhoneybee(PhotobyPhilBendle)

Honeybeesrepresentjustasmallfractionoftheapproximately20,00030,000knownspeciesof bees.Severalotherbeesproduceandstoresomekindofhoney,butonlymembersofthegenusApis aretruehoneybees. Twoattributesofthehoneybeethathavebeenessentialtoitsevolutionandbiologyareits aggregationbehaviourand,particularlyinthecaseofthecavitynestingspecies,itsabilitytocoolthe nestbyevaporationofwatercollectedoutside.Thesefeaturesenablehoneybeecoloniestoachieve amarkeddegreeoftemperatureconstancywithinthenestirrespectiveoftheexternaltemperature. Forthisreason,thegenusApiswasabletocoloniseawidevarietyofenvironments,rangingfrom tropicaltocooltemperate(Milner1996). Anotherbehaviouralcharacterofhoneybeesisthecommunicationofinformationaboutfood sourcesviathedancelanguage.Theaccuratedisseminationofinformationconcerningdirection anddistanceofforageareasleadstoefficientexploitationoffoodsources(Milner1996).

2.1.1

TheEuropeanhoneybee

InTheNetherlands,theEuropean,Western,orCommonhoneybee(Apismellifera 1 /A.m.)isa nativespecies.ThesubspeciesApismelliferamellifera,whichexistsinTheNetherlands,isalso knownastheEuropeandarkbee.About8,000yearsago,afterthelasticeage,thisspeciesspread overthewholeofEuropefromtheMediterranean(seeFigure1).

1

ThegenusApisisLatinforbeeandmelliferacomesfromLatinmellihoneyandferretobearhencethescientificname meanshoneybearingbee.

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 Itcanbeassumedthat,inThe Netherlandsandlargepartsof Europe,thenativedarkbeedoes currentlynotexistasapure subspeciesinthewildanymore (Blacquireetal.2009).This becausebeekeepershavebrought ingeneticmaterial(i.e.queens) fromsubspeciesA.m.ligusticaand A.m.carnica.Nevertheless,most ofthegenesofthebeesinThe Netherlandsstillarethoseofan A.m.melliferasubspecies.
Figure1 Distributionofthevarious subspeciesoftheEuropeanhoney bee(Apismellifera),suchasApismelliferaiberica(SpainandPortugal) andApismelliferamellifera(westernandnorthernEurope)

Inaddition,notmanybeecoloniesactuallyliveinthewildanymore,butitisdifficulttosaywhenthe wildpopulationofhoneybeesvanished.Presumably,thenumbersgraduallydeclinedduringthelast century,throughthereductionofwildlifehabitats.Thisparallelstheexpansionandintensificationof theagriculturalsector,throughwhichtheavailabilityoffloweringplantsinseminaturaland functionalsystems(e.g.meadowsandfields)diminished,aswellasthepresenceofnestcavities (hollowtrees). TheaccidentalintroductionoftheVarroadestructormiteinthe early1980sgavethefinalblowtowildcoloniesofthehoneybeein Europe.Varroamitesareexternalparasitesandarethemost importantpestofhoneybeesaroundtheworld.Themites,which areaboutthesizeofapinhead(approx.1,5x1,1mm,seePicture 2),usespecialisedmouthpartstoattackdevelopingbeelarvaeor adultbees,resultingindeformedbees,reducedlifespanand ultimatelythedestructionofthecolony(DAFF2011). From1850untilnow,thenumberofbeecoloniesinTheNetherlandshassteadilydecreasedfrom 200,000toapproximately80,000insummerand40,000inwinter.Thesecolonies,keptby beekeepers,essentiallyrepresentthecontinuationoftheoriginalpopulationofwildhoneybees (Blacquireetal.2009).
Picture2AVarroamiteona beepupa(PhotobyBramCornelissen)

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2.1.2

Twosexesandtwocastesofhoneybees

Twosexes(male(i.e.drones)andfemale)andtwocastesof femalebees(queensandworkers)makeupthepopulationofa beehive,eachhavingitsowncharacteristics,roles,and responsibilitieswithinthehive.Uponcloserexamination,the threetypesofhoneybeeshaveadifferentappearance(see Picture3). 1. Queen

Picture3Threetypesofhoneybees: worker(l),drone(m)andmature queen(r)(PhotobyZachHuang)

Withinahive,thereisonlyonequeen.Itisafemalebeewithafullydevelopedreproductivesystem. Thequeenmatesonlyoncewithseveraldrones,andthenremainsfertileforlife.Aqueencanlive for35yearsandcanlayupto2,000eggsperday.Fertilizedeggsbecomefemale(workers)and unfertilizedeggsbecomemale(drones).Whenthequeendiesorbecomesunproductive,theother beeswillinitiatethedevelopmentofanewqueen.Forqueenbees,ittakes16daysfromeggto emergence(BYBA2011). 2. Worker Aworkerisafemalebeeofwhichthereproductiveorgansareundeveloped,duetoaspecificdiet duringitsdevelopmentstageandthroughtheactivityofqueenpheromoneinthecolony.Thevast majorityofhoneybeesareworkerbees.Workerbeesmaylivefor49monthsduringthewinter season,butonly6weeksduringthedemandingsummermonths.Forworkerbees,ittakes21days fromeggtoemergence(BYBA2011). Theworkerbeessequentiallytakeonaseriesofspecifictasksduringtheirlifetime,asdepictedin Figure2.Theactivitiesofyoungbeesstartinthecentreofthebroodnestwiththecleaningofcells andtendingthebrood.Subsequently,theworkersgototheouteredgesofthenestinordertopack pollenandstorenectar.Untilafteraboutthreeweeks,workersbecomeforagersforanother1020 days.Foragerstakecareofbringingfromtheenvironmenteverythingthatthecolonyneedsinthe hive:pollen,nectar,waterandpropolis(seealsoSection2.2).Someactivitiescanbeexecuted lifelong(e.g.patrolling,resting,andventilatingthenest). 3. Drone Dronesarefertilemalebeesthatarekeptonstandbyduringthesummerformatingwithavirgin queen.Becauseadronehasabarbedsexorgan,whichcannotbepulledoutofthefemalegenital opening,matingisfollowedbydeathofthedrone.Fordrones,ittakes24daysfromeggto emergence(BYBA2011).Becausedronesareofnouseinthewinter,theyareexpelledfromthehive intheautumn.

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Figure2 Thechangingtasksduring thelifeofa workerhoneybee(fromSeeley1995)

2.1.3

Honeybeelifestages

Aswithmostadvancedinsects,honeybeesexhibitacompletedevelopmentormetamorphosis duringtheirlife:theyoungandtheadultslookverydifferent.Thelifestagesofahoneybeeareegg, larva,pupaandadult(seeFigure3).Notethatthecellsaredepictedvertically,butinreality,theyare orientedhorizontally.Thefirstthreestagesarealsoreferredtoasbrood.Developmentfromeggto adultingeneraltakestwotothreeweeks(Stone2005). Egg Theeggsaredescribedashavinganappearancesimilartosausageshapedpoppyseeds.Eachegg hasasmallopeningatthebroadendoftheegg,themicropyle,whichallowsforpassageofsperm. Hatchingtakesplacethreedaysafteregglaying.

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Larva
Figure3 Honeybeelifestages:fromeggtolarva, thentopupaandfinallytoanadultbee (Picturefromgetbuzzingaboutbees.com)

Fromhatchingoftheegg,thelarvalstagelastssixdays.Upon hatching,thelarvaisalmostmicroscopic,resemblingasmall, white,curved,segmentedwormlackinglegsandeyes.Itlies coiledonthebottomofthecell.Larvaearefedroyaljellyand laterbeebread,i.e.nutritionalgranulesofpollenwithadded honeyornectarpreparedbytheworkers(seePicture4). Eachlarvareceivesanestimated10,000feedingsduringthis stage.Larvalweightincreases5.5timesduringthefirstday

Picture4Workerscaringforlarvae (PhotobyZachHuang)

andapprox.1,500timesin6days.

Theprocessoffeedingandgrowingtakesplacewhilethecellsareuncapped;thelarvaespintheir cocoonsandchangeintopupaeafterworkershavecappedtheircells(Winston1987).Larvalstage durationsvary:5.5daysforqueens,6daysforworkers,and6.5daysfordrones.Regardlessof whetherthelarvaismaleorfemale,itmoultsfivetimesduringitslarvalstage(Stone2005). Pupa Thepupalstageisastageofmassivereorganizationof tissues:theadulttissuesdevelopfromtheimaginaldiscs carriedbythelarva.Organsalsoundergoacomplete transformation;whilethebodychangesfromthewormlike larvalbodyshapetotheadultbodyshapewiththreedistinct bodyregions(seePicture5).Thepupalstagelastsabout89 daysforworkersanddrones,and45daysforqueens.Itis followedbythefinalmoulttotheadultstage(Winston1987).
Picture5Workerandqueenpupae; notethelargersizeofthequeen pupabelow(PhotobyZachHuang)

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 Adult Asstatedabove,adulthoneybeesareeitherqueens,workersordrones.Themajorityofhoneybees thatoneseesoutsideofahiveareworkers.Atypicalcolonyinmidsummerconsistsofupto20,000 30,000workers,500to1,000drones,andonequeen.

2.2

Theforageofhoneybees

Theforageofhoneybeesconsistsoffourmainconstituents,namelypollen,nectar,waterand propolis: Pollen Pollenistheplantproteinsourceforthelarvaeandyoungbees,providingnitrogen,phosphorus, aminoacids,andvitaminsessentialfordevelopmentofthebees.Polleniscollectedinthefurof thebeeandstoredfortransportinpollenbaskets(corbicula)ontheworkers'rearlegs(tibia).As arule,honeybeesdonotcollectpollenandnectaronthesametrip.Forclarity,itneedstobe stressedthatalthoughnectarforagersaimfornectar,theyalsodowipethepollengrainsthat adheretotheirbodyintothecorbiculae,anddeposittheirpollenpelletsinthehive,inthecells ofthecomb,aroundthedevelopingbrood(seePicture6). Nectar Nectar,obtainedfromfloralnectaries,sometimesdeepinsideflowers,orfromextrafloral nectaries,providesanalmostpurecarbohydratesourceforallhoneybeelifestages.Each workerfillsitshoneysacorcropofitsdigestivetrackbyusingitsproboscis,increasingitsweight byuptoonehalf.Thenectarissuckedfromtheplantandtheworkerwilldiluteitwithsaliva containingsecretionsfromseveralglandssuchasthehypopharyngealglandthataddenzymes: invertase,diastase,andglucoseoxidase(Stone2005). Uponarrivalatthehive,theworkerregurgitatesthecontentsofthehoneysactotheyounger workerswithinthehive.Usuallythereceivingworkerwillmanipulatethissmallnectarloadinits mouthparts.Theworkerunfoldsandrefoldsitsproboscisexposingthenectartotheair circulatingwithinthehive.Duringthisprocess,moreglandularsecretionsareaddedbythe worker. Asthenectarisexposedtotheairofthehive,itslowlythickensanditisthensuspendedfrom theuppersurfaceofacellwall.Hereitwillcontinuetolosemoisture(dryout)andwhenthe moisturecontenthasbeenreducedtoaround18.6%,thehoneyisripeandthecelliscappedby theworkers.Pollenandhoneyarestoredinthecombconcentricallyaroundthecellscontaining thebrood(eggs,larvaeandpupae),firstthepollenandoutwardsthehoney(seePicture6). Duringastronghoneyflow(i.e.muchnectarisavailable)honeyisalsostoredfurtheraway fromthebroodnest.Thebeekeepercanutilizethisstoragebehaviourbyplacingaspecial honeysuperontopofthehive,ofwhichthecombsthenbecomefilledwithhoneyonlyandno

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 pollenorbrood.Thelattermakesaneasyandcleanhoneyharvestpossible,withoutany disruptionofthebroodnestofthecolony(seealsoSection3.5).
sealedhoney pollen sealedhoney

opencellswithlarvae

cappedbrood

queencellunderconstruction

openhoney opencellswithlarvae pollen

Picture6Abroodcombofhoneybees (PhotobyBramCornelissen)

Besidesgenuinenectar,honeybeesalsocollecttheexudatesthataphids(orplantlice)and otherscaleinsectsleaveonplants,calledhoneydew.Theseinsectsexploitthephloemofthe plantstoobtainsugarsandaminoacidsastheirproteinsource,butexcretethesugarsurplus. Water Honeybeescollectonlylittleamountsofwater,becausetheirmusclesproducemuchwater whenburningsugars,andthenectarcollectedcontainsalotofmoisturetoo.However,theneed forwaterrisesinhotweather:wateristhencollectedandspreadinsidethebroodcomb,tocool thebrood.Honeybeebroodneedsaconstanttemperatureofaround35C. Propolis Anothersubstancebroughtintothehiveispropolisorbeeglue.Thisisaplantresinusedto coverthehiveswallsandcombs,inordertoclosecracksandholes.Ithasantibacterialand antifungalproperties(seealsoSection3.5). Asstatedbefore,foragingforthementionedresourcesisaimedatfulfillingthedemandofthewhole colony,consistingofabout30,000workersandanalmostequalnumberoflarvaeandpupae.Itis obviousthatforagingforsomanycannotbeeffectiveifeveryforagerjuststartsattheentranceof thenestforagingonitsown.Therefore,theforagingistosomeextentorganized.Through trophallaxis(i.e.theexchangeoffood)andthecommunicationbythebeedance(waggledanceand

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 rounddance),thereexistsawarenessofneedsandresourcesamongtheworkersinacolony.Again, withinthetaskforager,beesspecialize,althoughthisspecializationisnotstrictnorforever.About onethirdoftheworkerbeesofacolonyisactiveasforager.

10,000Foragers:scouts,nectarforagers,pollenforagers,watercollectors
Scouts Duringmorningtime,asmallnumber(afewhundred)ofexperiencedforagerbeesflyouteveryday, andsurveythewideareaaroundthecolony,insearchforgoodresources.Thesebeesarescouts, whichbringtothecolonywhattheyfoundandstartadvertisingthisthroughthedancelanguage. Recruitsthatobservethedanceslearnabouttheresource(s):itsquality,itsdistanceanditsprecise direction.Onlythoseresourcesadvertisedthatarereallyneeded,willresultinarecruitfollowingup andgoingtoforage.Forexample,ifascouthasfoundwater,butthereisnoneedforwater,no foragerwillfollowupitsdance.Manyfeedbacksignalsinthecolonyguaranteethatpotential foragersareawareoftheactualneedsofthecolony.Incasethescoutswerenotabletofindany properresourcesatall,noforagerswilldepart.Inthisway,thecolonyavoidswastingenergywith ineffectiveforaging. Pollenforagers Ifthereisaneedforpollen,arecruitwillbetriggeredbyadanceinwhichascoutadvertisedapollen resource.Subsequently,itwillleavethehivetofindthatspecificresource.Itwillcollectpollenand learnhowtohandlethatspecificflowerspeciesmosteffectively.Itwillspecializeonthatspecific flowerspecies.Duringhandlingofthefloweranditsanthers,theforagerwillcollectthepollengrains onitsbodyandwipethesetoitscorbicula.Pollenthathasbeencollectedinthecorbiculaeisafood sourceforthecolony,buthasnovaluefortheplantanymore.Onlypollengrainsthatarescattered onthebodyandinthefurofthebeecanbedepositedonastigmaofanotherplantandresultin fertilization. Uponreturntothehive,thepollenforagerwillalsodancewhileshowingitsharvest,torecruitmore foragersforitssuccessfulresource.Ofcourse,manyotherreturningforagersalsodancefortheir resource,andsincethedanceincludesinformationontheprofitabilityofaresource,newrecruits canchooseforthemostprofitabletarget.Forinstance,aflowerpatchnearthehivewillbemore stronglyadvertisedthanaremotepatch.Pollenforagersbecomeverymuchdevotedtotheirflower species,andshowstrongflowerconstancy(Grteretal.2011).Pollenisgenerallyharvesteduptoa rangeof6kilometres.Theannualneedforpollenofanaverage(tenframe)colonyhasbeen measuredat1318kg(BrodschneiderandCrailsheim2010),whileacolonymaycollectatotalof10 26kgperyear.

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 Nectarforagers Foragingfornectarisoneofthetoughesttasksinthecolony,whichtakesalotofenergyfromthe forager(seeRortaisetal.2005).Theannualneedfornectarbyacolonyisestimatedas125kgina temperateclimate(Seeley1995).Ofcourse,foragingfornectarisonlyviableifthegaininenergy fromtheharvestednectarisgreaterthantheenergyconsumptionofthetrip.Dependingonthe sugarconcentration,theamountofnectarperflowerandtheabundanceofthespecificflowering plant,beescanflyuptomorethan14kmfornectar(BeekmanandRatnieks2000).Althoughnectar foragerssolelyaimatcollectingnectar,contactwiththeanthersand/orstigmataofaplantisnotor

cannotbeavoided.Therefore,nectarforagerscontributetopollenexchangebetweenplantsaswell. Generally,pollenforagersaremoreimportantforpollinationthannectarforagersare(Free1972), butinsomeplantspecies,suchasalfalfa,sunflower,plum,peach,apricot,andsweetcherry,nectar collectorsareveryimportantpollinatorstoo(Jay1986).Indioeciousplantslikewillows,onlynectar foragerswillvisitbothmaleandfemaleplantsandthereforeareimportantforpollination.This wouldalsobethecaseinmonoeciousplantswithseparatemaleandfemaleflowers,likecourgette. Althoughinapplethepollenforagersaremoreefficientpollinators thanthenectarforagers(Dagetal.2005),thelatterdocontribute significantlytopollination,sinceappleflowersproduceahighsugar nectar(>45%;Sternetal.2001).Nevertheless,itwasshownthat pollinationofappletrees(cultivarDelicious)byhoneybeeswas improvedifthecolonieswereplacedintheorchardsequentially insteadofsimultaneously.Namely,inexperiencedforagershadnot yetlearnedhowtoforageoptimallyfornectar:onlyaftersome time,beeslearntogetaroundtheanthers.Thissideworkinghelps themtoforagemoreefficiently(seePicture7),butitreducesthe changesonpollination(Sternetal.2001). Watercollectors Intemperateregions,ahoneybeecolonycollectsaround25kgofwateronanannualbasis(Seeley 1995).Becausethewatercollectediscarriedinthehoneystomach,awaterforagercannottakealot ofnectaronitstrip(i.e.thishastobestoredinthehoneystomachtoo).Therefore,watercollectors onlyflyshortdistancesofupto3kilometres.Whenthesituationarisesthat,forseveraldays,the weatherisnotsuitableforforaging,theneedforwaterinthehiveincreases.Namely,wateristhen necessarytodilutethefeedforthelarvae.Inaddition,asmentionedearlier,inhotweathertheneed forwaterraisesaswell:wateristhenutilizedtocoolthebroodnest(Nicolson2009).
Picture7Ahoneybee side workingonanappleflower (PhotofromSternetal.2001)

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2.3

Pollinationbyhoneybees

Pollinationisthetransferofpollengrains,themalesexcellsofaflower,fromtheantherwherethey areproducedtothereceptivesurface,orstigma,ofthefemaleorganofaflower.Fertilisationoccurs whenthepollengrainsonthestigmagerminateandgrowdownthestem(style)ofthestigmato fertilizetheovule.Honeybeesareamongtheinsectsthattransferpollenbetweenflowersand betweenplants,andareoftenregardedasbeingthemostimportantpollinators.Hence,theword pollinationishabituallyusedtodescribetheserviceofprovidinghoneybeestopollinatecrop plants. Honeybeesaresuitableforpollinationforseveralreasons.Theirhairybodieseasilytrappollenand carryitbetweenflowers(seePicture8).Sincehoneybeesrequirelargequantitiesofnectarand pollentoraisetheiryoung,theyvisitflowersregularlyinlargenumberstoobtainthesefoods.In doingso,individualhoneybeesshowflowerconstancy,whichalsocontributestothepollination efficiency. Thebodysizeofhoneybeesenablesthemtopollinate flowersofmanydifferentshapesandsizes.Thenagain, sometimesnopollinationoccursdespitenectarorpollen removal,forinstancebecausestigmasarenottouched.The pollinationpotentialofhoneybeesisalsohighbecausethey canbemanagedtodeveloplargepopulations.Thenumber andsizesofcoloniescanfurtherbeincreasedbybeekeepers accordingtotheneeds,andthecoloniescanbemovedtothe mostdesirablelocationforpollinationpurposes(Jaycox1985).
Picture8 Ahoneybeecollectingpollenof aDactylisglomerata,awindpollinated species(PhotobyBramCornelissen)

Notallcropsneedpollinationbyinsectpollinatorssuchashoneybeesthough:somecanproduce fruitwithoutfertilisationoftheflower.Inaddition,someflowersareselfpollinated,whichmeans thatpollenistransferredfromtheanthertothestigmaofthesameflowerortootherflowersofthe sameindividual.Althoughthistransfercanbeachievedbywindorrain,insectpollinatorsarethe mosteffectiveintransferringpollen.

2.4

Thefateofpollenasmanagedbyhoneybees

Honeybeesandplantshaveconflictinginterestsduringpollination:honeybeesaimatthehighest possiblerewardperflower,whiletheplantaimsatthehighestpossiblenumberofsuccessful transportationsofpollentootherflowersand/orplants,againstthelowestpossiblecostsofenergy (nectar)andnutrients(lostpollengrains)).Theevolvedcompromiseisacceptabletobothparties.

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 However,conflictinginterestsmaystillbepresent,aswasillustratedbythelearningbehaviourof foragersonapple(Sternetal.2001)althoughinthiscaseofamenbredcultivar,nonatural selectionisgoingon.Ontheotherhand,thementionedflowerconstancy(Grteretal.2011) benefitsthebees(moreefficienthandlingoftheflowersthroughspecialization)aswellastheplant (nopollenwastedonforeignstigmata). Whenvisitingaflowerforpollen,beesactivelyusetheirlegs,hairsandmandiblestomanipulatethe anthersinordertocatchthepollenontheirbodies,andtoimmediatelytransportitintotheir corbiculae.Despitetheirefficiencyincleaningthefurfrompollengrains,manygrainsarefoundon thebeesbodiesduringforaging,andthenumberswerethehighestonbeesthatalsocarriedlarge pollenloadsinthecorbiculae(Free1993).Typicalfiguresamountto1,000to10,000grainsperbee. Ifabeecarriespollenpelletsof25mgtogether,whichwouldcorrespondwith375,000to16.8 millionpollengrains(seeChapter5),thefractionfoundonthebeesbodymightbe0.06to27pro milleofthetotalpollenloadofabee. Polleninthepelletsinthecorbiculaemayremainviableforseveralhours(Free1993),butarelost forpollination.Pollenonthebeebodyisalsoabletogerminateafterseveralhours(Free,1993),but moreprobablymaybedepositedtoanearbyflowerofthesameplantthanlateronaflowerof anotherplant.Pollenlongevitymayvaryconsiderablybetweenplantspecies.Forinstance,itwas shownthatafter3hours,maizepollenwasstillviableandresultedinkernelformation.Thesame wasfoundforRaphanussativusafter5days,andpollengrainsofBrassicacampestrisstill germinatedfor50%after6days(DafniandFirmage2000).Inthesecases,itisnotclearifandhow muchlongerthepollengrainswouldhaveremainedviable.

Powderbox
Honeybeesthatleftthehivewerefoundtocarrymanypollengrainsofdifferentplantspecieson theirbodies(FreeandWilliams1972).Dependingonthepollensources,between4,000and13,000 grainswerealreadypresentatthestartoftheforagingtrip:8090%ofthesewereofthedominant cropvisited.Youngbees,aswellasdrones,alsocarriedequalnumbersofpollengrainsontheir bodies.Uponplacingcoloniesinsideagreenhouse,Paalhaaretal.(2008)wereabletoshowthatin hive(young)beesrapidlycarriedpollengrainsofthespecificgreenhousecropsontheirbodies,but only<1%oftheloadofforagingbees. Usingatwoentranceshive(withoneopeninginsideacagewithafewmangotrees,theother(main) entranceoutsidethecagetothemangoorchard),Dagetal.(2001)showedthatthetreesinsidethe cagecarried12%fruitsfertilizedbypollenfromoutsidesources.Thesepollengrainsmusthavebeen exchangedbetweenbeesinsidethehive,sincedifferentbeesforagedinsideandoutsidethecage.

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 Sotheairinsidethehiveappearstocontainpollenfromallsourcesvisitedbythebees.Duetothis powderboxeffect,individualbeesmaycarrysignificantamountsofpollenfromplantsnotactually visitedbythem.Thispossibilityofpollinationthroughexchangeinthepowderboxmayespecially besignificantforselfincompatibleanddioecioustrees. Althoughthenumberofpollengrainsexchangedinsidethehivemaynotbeveryhighcomparedto thenumberdirectlydepositedonforagingbees,itmayberelevantforpollentransportbetween plantsindifferentspotsthatarenotindirectflightconnectionofindividualbees.Thispathwaywith connectionflightsmightindicatethatthemaximumpollenflowdistanceactuallycanbetwicethe

maximumflightdistanceofthehoneybees.Becausethequantitativeimportanceofthiseffectisnot clearfromtheliterature,furtherresearchisneededtoquantifythepossibleroleofthepowderbox effect. 2

Pollenendingupinhoney
Alreadyinsidetheflower,somepollengrainsmayfallinthenectariesandsubsequentlymixwiththe nectar.Inhangingflowers,thiswillbelesscomparedtouprightcupshapedflowers.Inbothcases, mostofthefallenpollengrainswillbelostforpollination.Duetothebehaviourofthehoneybees, morepollengrainscanbebroughtintothenectarduringharvesting.Finally,onlyaverysmall fractionofthepollengrainsinnectarwillendupinthehoneyproduced(seeChapter5).Naturally, allofthesegrainswillbelostforpollination.

Honeyflows
Incertainperiods,whenveryrichorstronghoneyflowsareavailable,honeybeescaneasilyharvest muchhoneyandpollen.Thisbooststhedevelopmentofthecolony.Suchhoneyflowsallowthe beekeepertoharvesthoneybymeansoftheearliermentionedhoneysuper(Section2.2).In betweenhoneyflows,thebeesforageonseveralplantspeciesthatjustdeliverenoughresourcesto coverthepreservationofthecolony,ortheyrelyonstoredhoneyfromearlierhoneyflows. Mosthoneyflowsoccurduringspringandsomeduringsummerandthebeginningofautumn.The opportunisticnatureofhoneybeesresultsindifferentlimitsforasource(e.g.nectarorpollen)in ordertobeattractiveforthecolony.Figure4showsthethresholdsugarconcentrationsforwhich honeybeesweredancingfromMaythroughJuly(researchofLindauer(1949),fromMichener (1974)).WhileinJuly,0.2Msucrose(about7%w/v)oftheresourcewassufficienttoinducedancing byaforagerbee,itwasfarfromsufficientinMay,whenthetenfoldconcentrationwasneeded (almost70%w/v).
2

Whetherornotofprovensignificance,professionalbeekeeperssupplyingpollinationservicestoseedproducingcompanies aresometimesrequestedtotakeawaitingperiod(i.e.keepingthebeehiveclosed)of24hoursbeforeutilizingthehivefor pollinatingthecompaniesseedcrops(J.Calis,personalcommunication).

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 NotethatintheFigurebelow,severalspringhoneyflowcropsarenamed:redclover(Trifolium pratensis),snowberry(Symphoricarposspp.),rape(oilseedrapeorrapeseed,Brassicanapusor Brassicarapa),charlock(Sinapisarvensis),andsummerandwinterlime(Tiliaspp.).Asimilarlistof honeyflowscanbemadeforTheNetherlands,startinginspringwithwillows(Salixspp.),and dandelion(Taraxacumofficinalis)inApril,fruittrees(cherry,plums,apple),horsechestnut(Aesculus

hippocastanum)andoilseedrape(Brassicanapus)inMay,raspberryandblackberry(Rubusspp.)and limetrees(Tiliassp.)inJune,andfinallyheather(Callunavulgaris)inAugust.InsomeplacesinThe Netherlands,theremaybealsostronghoneyflowsofwhiteclover(Trifoliumrepens)andseaaster (Astertripolium)duringsummer,blacklocust(Robiniapseudoaccacia)inJune,andtheneophyte Himalayanbalsam(Impatiensglandulifera)inAugust.Duringastronghoneyflow,honeybee foragersmaybelesspronetovisitotherfloweringplantspecies.

Figure4Seasonalvariationintheminimalconcentrationofsugarneededtostimulatedancing(Lindauer(1949)from
Michener(1974)).1Molarsugarisabout34%w/vsugarconcentration(Molweightsucroseis342)

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3
3.1 Historyinbrief

BeekeepinginTheNetherlands

Beekeepingisoneoftheoldestknownformsoffoodproduction.Someoftheearliestknown evidencesofbeekeepingisfromarockpainting,datingtoaround13,000BC.Beekeepingwas particularlywelldevelopedinEgyptandwasdiscussedbytheRomanwritersVirgil,GaiusJulius Hyginus,VarroandColumella(Romanov2005).Traditionallybeekeepingwasdoneforthebees honeyharvest,althoughnowadayscroppollinationservicescanoftenprovideagreaterpartofa beekeepersincome.Forthemostpart,beekeepingisahobbyactivityinTheNetherlands,carried outbyroughly8,000beekeepers(Blacquireetal.2009).

3.2

Dutchbeekeepers:hobbyistsandprofessionals

Hobbyists
ThemajorityofDutchbeekeepersarenoncommercialhobbyiststhataimtoharvestsomehoney eachyearandpossiblyafewotherproducts,mostlyforfamilyandfriends,andattimesforselling theseproductstoshopsoratmarkets.Motivesforkeepinghoneybeesarebroaderthough,namely devotiontonature,spendingtimeoutdoors,passionfortheoldcraft,andfellowshipwithinan association(Blacquireetal.2009).Noncommercialbeekeepersregularlyhaveonlyafewbee colonies(averageoffive),buttheylivealloverthecountryandhenceprovideanationwide coverageofnaturalgreenareaswiththeircolonies,whichisanimportantfactorintheroleofthe honeybeeaspollinator.Overall,hobbyistbeekeepersseldommovetheirhives,excepttheonesthat providepollinationservicesinthefruitsector(seealsoSection3.4). Lately,Dutchsocietyingeneralshowssomemoreinterestinbeesandbeekeeping(seealsoBox1). Forexample,thelargestbeekeepingassociationinTheNetherlands(NBV,seebelow)haswelcomed manynewmembersinthepastfewyearsandtheirbeginnercourseshaveneverbeenvisitedso well.However,onthelongrun,thenumberofactivebeekeepersinTheNetherlandsisexpectedto furtherdecrease.Thisnegativetrendcould,amongstotherreasons,becausedbythefactthat peopleareincreasinglyoccupiedinmodernsocietyand,onthewhole,makeavailablelessandless timeforhobbyactivities.

Professionals
ProfessionalorcommercialbeekeepersactiveinTheNetherlandsaremainlyinvolvedinpollination activitiesintheagriculturalsector.Thesebeekeepersregularlymovetheirhives(every4weekson

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 average),andtheirmainworkingareasareindoors(greenhousesandpolytunnels).Commercial beekeepingonlyfortheproductionofhoneyorotherbeeproductsisvirtuallynonexistentinThe Netherlands,althoughsomeprofessionalbeekeepersdoselltheirharvestedhoney.Moreon commercialbeekeepinginSection3.4. Box1Anewdevelopment:urbanbeekeeping Duringthepastdecadewithinthemovementforasmallscale, traditionalartisanalandbiologicalapproachtobeekeepingthe trendurbanbeekeepinghasdevelopedincitieslikeParis,Berlin, London,TokyoandNewYork,butalsoincitiesinTheNetherlands, wheremoreandmorepeoplearetakingupbeekeeping.Beesare beingkepteverywherefromsmallsuburbanbackyardstohighrise rooftopsandbalconies.
(PhotobyDavidGleason)

Besidesthefactthatthebeespollinatethecitiesgreenareas,urbanbeekeepinghastheadditional positiveeffectthatthisnaturalformofhoneyproductionisbroughtclosertotheconsumeragain: peopleareabletoexperienceabeekeeperatworkintheirownneighbourhood.

3.3

Dutchbeekeepingassociations

Oftheapproximately8,000beekeepersinTheNetherlands,about6,900areorganisedinthe followingassociations: NederlandseBijenhoudersvereniging(NBV) DutchBeekeepersAssociation www.bijenhouders.nl AlgemeneNederlandseImkersvereniging(ANI) GeneralDutchAssociationofBeekeepers www.anibijen.nl ImkersbondABTB BeekeepersAssociationABTB www.imkersbondabtb.nl Below,ageneraloutlineofeachassociationisgiven.Moreinformationcanbefoundonthe respectivewebsitesoftheassociations. 400members 500members 6,000members

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 TheNederlandseBijenhoudersvereniging(NBV)wasestablishedon1January2006,asaresult ofamergerofseveralsmallerassociations.Theobjectiveoftheassociationistoendorse

beekeepinginitsbroadestsense.Theassociationiscommittedtothepromotionofbeekeeping, toincreasetheavailabilityofhoneyplantsforhoneybeesandotherpollinatinginsects,andto providesupporttobothhobbyistandprofessionalbeekeepers.Furthermore,theNBV representsDutchbeekeeperstowardsthenationalgovernmentanditmaintainscontactswith organizationsofbeekeepersinneighbouringcountries.TheNBValsorepresentsThe NetherlandsatApimondia,theInternationalFederationofBeekeepersAssociations. TheNBVhasanationalcoverage,whilstthetwootherassociationstendtohavearegional focus,ontheVeluweandBetuweareasrespectively.Since2007,theNBVpublishesitsown monthlymagazineBijenhouden(Beekeeping). TheAlgemeneNederlandseImkersvereniging(ANI)isanorganizationdedicatedtobeekeeping inallitsaspects,andpromotesanddefendstheinterestsofbeekeepers.Theassociationwas foundedin1934andhasbranchesinseveralplaces. TheImkersbondABTBwasfoundedin1947withtheaimtorepresenttheinterestsofits membersbothcommercialandnoncommercialinthefieldofbeekeeping.Withinthe association,acommissiononpollinationisactive,whichaimstopromoteunderstandingand knowledgeofpollinationamongstbeekeepersandgrowers,itoffersguidanceonpollination matters,anditprovidesbeecoloniesviabeekeeperstogrowersforpollinationservices. TheAlgemeneNederlandseImkersverenigingandtheImkersbondABTBpublishthemagazine Mijnbijen(Mybees)(7timesperyear).

3.4

Utilizationofhoneybeesforpollinationofcrops

InTheNetherlands,honeybeesarebeingutilizedinfruitorchards,inthehorticultureindustryand forseedproduction.Pollinationinclosedgreenhousesandpolytunnels(i.e.vegetablesandseed production 3 )ismainlycoveredbyprofessionalbeekeepers.However,muchcollaborationtakes placebetweenprofessionalandhobbyistbeekeepers,inordertoguaranteethesupplyofbee coloniesintheseedproductionsector(CalisandBoot2009).

For(hybrid)seedproduction,honeybeesareutilizedtomaintainparentplantsofcertaincropvarietiesandtoproduce hybridseeds.Nowadays,thispredominantlyhappensingreenhousesorpolytunnels,duetotheunreliableclimateinThe Netherlands.

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 Traditionally,pollinationinthefruitsectorismostlycoveredbynoncommercial,hobbyist beekeepers.Thislattergroupreceivesafeeof4060Europercolony(2040thousandhoneybees), per3weeksorless(ABTB2011a). Itisestimatedthataround32,000honeybeecoloniesareutilizedforpollinationinTheNetherlands byapproximately1,700beekeepers(NCB2011).Theannualvalueofpollinationfeesisroughly estimatedat4millionEuroforoutdoorcultivation,and7millionEuroforindoorcultivation (Blacquireetal.2009). InTheNetherlands,aroundtenbeekeepingcompaniesareprofessionallyinvolvedinthepollination ofcropswithhoneybeesandmakealivingoutofit.Thesecommerciallyoperatingbeekeepers, mostlypartoftheseedproductioncompaniesthemselves,oftenhavemorethanonehundred colonies.InordertoprovideaninsightinprofessionalDutchbeekeepingcompanies,themain featuresofoneofthesecompaniesaresummarizedinBox2. Theprovisionofhoneybeesforpollinationofcropplantsisaspecializedpractice,notjustasideline ofhoneyproduction,andinmanyways,theworkofsuchbeekeepersrequiresadifferentapproach towardsthehandlingofbeecolonies.Forexample,thedeclineofcoloniesthatareusedfor pollinationneedstobecompensatedforbeforehand,bycontinuouslycreatingnew(pollination) coloniesfortheupcomingseason.Asaresult,thecoloniesareusuallyrelativelyyoungandintheir growing,ormaturingphase.Thisisoneaspectinkeepingbeeshealthyandingoodcondition,which isessentialinordertobeabletoprovidealargeforceofworkerbeesneededtodothejobof transferringpollen(Jaycox1985),andforthebeestobeabletocovergreaterdistancesifneeded.

Duetothefurtherprofessionalizationinagricultureandhorticulture,hobbyistbeekeepersappearto belessandlessinterestedinprovidingtheircoloniesforpollination(Blacquireetal.2009).This providesextraopportunitiesforcommercialbeekeeperstofillthatgapanditmaywellleadto growthinthatsector.

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 Box2MainfeaturesaDutchbeekeepingcompany Beesareutilizedfor: 1. Seedproduction(predominantly) 2. 3. Foraselectionofcropplants,thefollowingtable(Table1)indicatestheminimumandoptimal ingreenhousesandpolytunnels cabbage(red,white,Brusselssprout),carrot,union,leek,rocketseeds flowerseeds seedsarehighlyvaluable,sohighbeedensity:easily20coloniesperhectare betweenAprilandtheendofJuly ingreenhouses courgette,eggplant,bellpepper numberofcoloniesvaries yearround inorchards pear,cherry,apple,blueberry,raspberry approximatelythreecoloniesperhectare,butvariesgreatly duringfloweringseason Fulltimebusiness,twostaff Approximately1,000colonies Onaverage50to150coloniesdeployedatatime Typically,coloniesareutilized23timesperyearforaperiodofapprox.4weeks Compositionofcoloniesvariesgreatly,becausecoloniesarecombinedandsplitupregularly Apiaries(beeyards)onapprox.15differentlocationsinthecentralandwesternpartofThe Netherlands ThewholeofTheNetherlandscanbeserviced,aswellassomepartsofGermany

Cultivationofvegetables

Cultivationoffruits

numberofhoneybeecolonies,requiredinthefield,perhectare.Thenumberofcoloniesneededper cropvaries,duetoseveralvariables,suchasthenumberofflowersofaplant(someplantshave manyflowers,othershavenot)andthenumberofactualbeevisitsnecessaryforfertilization(some plantsneedmorevisitsthanothersduetotheplantsspecificmorphology).

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 Table1Theminimumandoptimalnumberofhoneybeecoloniesrequiredpercrop,perhectare,in thefield(ABTB2011b) Crop Apple,pear Cherry,sourcherry Peach,plum,mirabelleplum Whitecurrant,redcurrant,gooseberry Blueberry Strawberry Blackberry,raspberry Pickles,courgette Bluepoppy,caraway,rapeseed,radish,mustardseed,fieldbean, eveningprimrose Ornamentalplants,suchasberryshrubs Withingreenhousesorpolytunnels,thenumberofrequiredcoloniesandthesizeofthecolonies dependonthecropandonthesizeofthecompartmentinwhichtheyareplaced(CalisandBoot 2009).Thefollowingguideline(Table2)providesaninsightinthenumberofcoloniesneeded indoorsforaselectionofcrops.Itshouldbenotedthathoneybeesgenerallyhavetheopportunity toflyoutsidethegreenhouseorpolytunnelinwhichtheyarelocated. Table2Averagenumberofsquaremetresperindoorcropthatcanbecoveredbyonehoneybee colony(PPO2004) Crop Eggplant,bellpepper Melon,pickle,courgette Strawberry,raspberry,blackberry Onecolonyper 5,000m2 1,500m2 1,000m2 Numberofcoloniesrequired minimum optimal 2 3 4 6 5 510 4 68 5 515 2 24 4 46 2 3 410 3 4

3.5
Honey

Honeyandotherbeeproducts

Honeyisthemostrecognizableproductmadebyhoneybees.Itisproduced fromnectarandinthecourseofthisproductionprocessduetothemany interactionsbetweenbeeswithinthehivetraceamountsofpollenendupin honey.Inaddition,airbornepollen,notspecificallycollectedbybees,canalso endupinhoney.

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 Honeyisusedasanenergysourceforadultbeesandbrood.Incoldweather,orwhenfreshfood sourcesarescarce,beesusestoredhoneyastheirsourceofenergy.Beekeeperscanharvestthis honeyfromthehoneycomb,bymeansofahoneyextractor. Ahoneyextractorisamechanicaldevicethatextractsthehoneyfromthehoneycombwithout destroyingthecomb.Theseextractorsworkbycentrifugalforce:adrumorcontainerholdsaframe basketthatspins,flingingthehoneyout.Withthismethod,thewaxcombstaysintactwithinthe frameandthebeebreadstaysput:thebeescanreuseit.Analternativemethodistoputa honeycombintoahoneypresstosqueezeoutthehoney.Thismethodisseldomappliedin commercialhoneyproduction,becausethecombcannotbeusedagain. InTheNetherlands,theproductionofhoneyisnotarelevantmarketsectoranymore.Nowadays, honeyispredominantlyharvestedbyhobbyistbeekeepersandsoldonasmallscaleaslocalproduce inorganicfoodstores,ruralfarmshopsandonlocalfruitmarkets.In2009,theestimatedtotal homemadehoneyproductioninTheNetherlandswas1,422tons(NCB2011).Thisaccountsfor approximately8%ofthehoneyontheDutchmarket.Theremaining92%isimportedfromamongst othersChinaandArgentina;thetwolargesthoneyproducingcountriesintheworld(seeTable3). Table3Top10honeyproducingcountriesintheworld(2009)(FAOSTAT2009) Country Productionintonnes 1. China 407,367 2. Argentina 83,121 3. Turkey 82,003 4. Ukraine 74,000 5. USA 65,366 6. Mexico 56,071 7. Russia 53,598 Note: Chinaaloneproduces 8. India 43,865 morehoneythantherest 9. Ethiopia 40,688 ofthetop6combined. 10. Brazil 38,765

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Beeswax
Besideshoney,beeswaxiswonaswell.Honeybeesusebeeswaxtobuild honeycombcellsinwhichtheiryoungareraisedandhoneyandpollenare stored.Hence,traceamountsofpollencanbefoundinbeeswax.When beekeepersextractthehoney,theycutoffthewaxcapsfromeachhoneycomb cell.Itscolourvariesfromnearlywhitetobrownish,butmostoftenashadeof yellow,dependingonpurityandthetypeofflowersvisitedbythebees. Waxfromthebroodcombofthebeehivetendstobedarkerthanwaxfromthehoneycomb,since impuritiesaccumulatemorequicklyinthebroodcomb.Duetotheseimpurities,beeswaxhastobe rendereddownbeforefurtheruse.Theleftoversarecalledslumgum. Theextractedbeeswaxcanbesoldorforasmallfeesuppliedtocompaniesthatreusethewaxto makehoneycombfoundations.Purifiedandbleachedbeeswaxisusedintheproductionoffood, cosmetics,pharmaceuticals,candlesandwoodpolish.

Propolis
Propolisisapolyphenolrichresinoussubstancehoneybeescollectfrom treebuds(especiallypoplars),sapflows,orotherbotanicalsources.Itis usedasasealanttocoverunwantedopenspaces,cellsofthecomband theinnerwallsofthenest.Furthermore,propolishasanantibacterialand antifungalfunctionwithinthebeehive.Itscolourvariesdependingonits botanicalsource,themostcommonbeingdarkbrown. Propolisisstickyatandabove20Celsius;atlowertemperaturesitbecomeshardandverybrittle. Typicalnortherntemperatepropolishasapproximately50constituents,primarilyresinsand vegetablebalsams(50%),waxes(30%),essentialoils(10%),andpollen(5%).Propoliscanbe harvestedandprocessedintotincturesandointments,althoughthisnotoftenhappensinThe Netherlands.

Royaljelly
Royaljellyisusedbyhoneybeesinthenutritionoftheirlarvae,aswellasadult queens.Royaljellyissecretedfromthehypopharyngealglandsofworkerbees, andisfedtoallbeelarvaeinthecolony,whethertheyaredestinedtobecome drones(males),workers(sterilefemales)orqueens(fertilefemales).Between queencellsandworkercellsthebalancebetweensugarandproteincomponents differ,aswellassomespecificdeterminingcomponentsthathavebeenidentified recently(Kamakura2011).

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 Royaljellycouldcontaintraceamountsofpollenandissometimesharvested(fromthequeencells

only)andaddedtofoods(e.g.honey),becauseofitsallegedpositiveeffectstohealth.Royaljellycan alsobefoundinsomebeautyproducts.

Beepollen
Beepollenisplantpollencarriedbybeestothehivewhereitisgathered byplacingbrushesatthehive'sentranceknockingthepollenoffthebees astheyenter.Beepollenisusedinnaturopathicmedicinetraditionsand asanutritionalsupplement,althoughexposuremaytriggerallergicor anaphylacticreactionsinsensitivepeople.BeepollenavailableinThe NetherlandsismainlyimportedfromSpain,butalsofromSouthAmerica.

Beevenom
Apitoxin,orhoneybeevenom,isabittercolourlessliquid.Theactive portionofthevenomisacomplexmixtureofproteins,whichcauseslocal inflammationandactsasananticoagulant.Thevenomisproducedinthe abdomenofworkerbeesfromamixtureofacidicandbasicsecretions.It couldcontaintraceamountsofpollen.Beevenomtherapyisusedby someasatreatmentforrheumatismandjointdiseases,duetoitsanti coagulantandantiinflammatoryproperties. Beevenomisalsousedtodesensitizepeopleallergictoinsectstingsandcanbedeliveredinthe formofbeevenombalmaswell,althoughthismaybelesspotentthanusinglivebeestings.

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4
4.1 Introduction

Foragingandpollentransport

ThisChapterpresentsresearchdataonforagingrangesanddistancesofhoneybees.This informationcanbeusedtoestimatetheminimaldistancethatshouldbemaintainedbetween beehivesandcropswhencontaminationofhoneywithpollenfromthesecropsorcrosspollination shouldbeavoided.Itshouldbestressedthatflightdistancesorflightactivitypatternsingeneralis onlyoneaspectinpollentransportandpollinationphenomena. Thedistanceoverwhichpollenaretransporteddependsnotonlyofthepotentialflyingcapacityof honeybees,butitisdependentonthecomplexforagingbehaviourofbeesandthefateofpollen relatedtotheactivitypatterns.Theforagingbehaviourdependsona)thetimeoftheyear,b)the conditionofthecolony,c)thedistributionoffoodpatchesinthelandscapesurroundingthebeehive, andd)onthevariationinactivitybetweenindividualbees.Thequantificationofpollentransportin termsofamountsinspaceandtimeisthereforemuchmorecomplexthanonlymeasuringflying distances.Thelocalconfigurationofcrops,thenaturalenvironment,thepositionofthebeehiveand thenutritionalstateofthecolonymaytriggerthebehaviourandcommunicationpatternsamong individualbehaviourofbeesfromthecolony.Pollencarryoverfrombeestoandfromflowersand amongbeesthemselvesisanadditionalfactorinpollenflows. Itisknownthathoneybeesandothersocialinsectsstronglybenefitfromthecommunication betweenindividualstolocatefavourablefoodsources.Bythesocalledwaggledance,whichis performedinsidethebeehive,directionanddistanceofnectarandpollensourcesiseffectively communicated.Therecruitmentofpartofthehoneybeestoexplorenewsourcesfurtherawayfrom thenestsallowsthemtocollectfoodatconsiderabledistances.Thewaggledancebehaviourisin particulareffectivetooptimizethecolonysabilitytoexploitthemostfavourableforagingpatchesin theenvironment(BeekmanandLew2007). Theabilitytodetectandexploitpollenandnectarsourcesmakebeesveryeffectiveaspollinators andcollectorsofnectarandpollen,astheycancoverlargeareasfrombeehivesplacedatasingle spot.However,thisabilityalsoallowsbeestoswitcheasilytofindthemostrewardingplace.For farmerswhowanttooptimizepollinationoftheircropsthisswitchingtootherplantmaybe unprofitable.Onegeneralconcernintheuseofbeesforpollinationisthathoneybeesmay effectivelytransportunwantedpollentypesintothebeehiveortocropsthatshouldbevoidofcross pollination.Theonlywaytopreventforagingofhoneybeesonunwantedpollenistokeepbeehives atasafedistancefromthoseplaces.Tominimizeriskforundesiredpollenflowsbyhoneybees,

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 knowledgeonforagingbehaviourcanprovidecuesaboutkeyfactors.Foragingdistancesand coveredareaaroundbeehivesaresuchkeyfactors.Thefinalresultingundesiredcrosspollinationor contaminationofunwantedpolleninhoneyhowever,dependsonmanyotherfactors.

Eventhoughitislongknownthatbeescancollectfoodatseveralkilometresfromthehives,itisonly fromrecentstudiesunderwhatcircumstancestheytravelthatfarandtowhatextenttheydoso.In practise,suchstudiesaremorecomplicatedthanexpected.Thisismainlybecausebeesforageina heterogeneouslandscapewherethequalityofdifferentpatchesforfoodvariesthroughoutthe season.Beeswillforagenearbywhengoodsourcesarelocallyabundant,inordertosaveenergyand toreducemortalityrisk.However,whenhighqualitysourcesarefurtheraway,flyingoverlarge(r) distancesmayberewarding.Itisobviousforexample,thatbeeswillmoreeasilyflylargedistances toexploitafloweringcropofoilseedrape,whichisofhighfoodvalue,thantoforageondistant floweringpotatothatisoflowvalue.Thepatchinessandqualityofpatchesintheagricultural landscapeislikelytoaffecttheestimatesofrangesandflyingdistancesinspecificstudies. Therefore,datafromliteratureonsuchestimatesareinterpretedinthecontextofthe environmentalsetting.Atleastthesegivecuesaboutthechanceoflargedistanceforagingandthe magnitudesthatshouldbedealtwithwhentakingmanagementdecisions.InSection4.2,asummary ofstudiesisgivenwhichshowsthatresultsfromdifferentstudiesareveryvariable,butdistances coveredbythemajorityofthebeesareintherangeof1to2km.However,insomecases,thiscan increaseto5oreven10km.

Methodsofestimatingforagingdistances
Thefollowingmethodsareavailabletoestimateforagingdistancesofhoneybees: Trainingofbeesforaparticularrichfoodsource,situatedatdifferentdistancesfromthecolony. Theoreticalestimatesfromoptimalforagingtheoryandoptimalenergyuse. Optimalforagingtheorypredictsthatbeesshouldminimizeflighttimeandenergyexpenditure whilemaximisingtheenergyitgainsfromthefoodcollected. Theoreticalmodels,predictingforagerdistributionsforCentralPlaceForagers(deVriesand Biesmeijer1998;DukasandEdelsteinKeshet1998;Cresswell,OsborneandGoulson2000, VisscherandSeeley1982),differbetweenthoseforspeciesconsideredsocialforagers,e.g. honeybeesthatcommunicateinformationaboutresourcelocationtonestmates,andthose relyingonindividualexplorationtofindresources,e.g.bumblebees.However,notethatthe extraenergyandtimeforflyinglargerdistancesisrelativeandoftencompensatedbythegainof findingagoodpatch. Interpretingwaggledanceinformationfrombeehives.Atypicalexampleforthisapproachisthe paperofVisscherandSeeley(1982),whoextensivelyanalysedforagingdistributionsina

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 complexlandscapeduringvarioustimeframesshowingthecomplexityofthecoloniesforaging behaviouranddistancesupto10km. Allmethodsusedsofarhavebiasandlimitationsforobtainingsufficientandaccurateestimateson therangeofvariabilityinbeeflightpatterns.Spatialprocessesinecologyareanemergingfieldthat islikelytoresultinbettertechnologiesforanddatafromsuchstudiesoninsectmovements. Measurementsontransmissionofmarkedpollenloads. Bymarkandobservationstudiedinindividuals(Osbornetal.1999). Trackingbyharmonicradar(Osbornetal.1999). Evidencefromgeneflowmeasurements.

4.2

Keystudies

BeekmanandRatnieks(2000)studiedlongrangeforagingbyhoneybeesbydecodingwaggledance informationfromhoneybeesforagingatlargebloomingheatherfieldsthesecanbeveryattractive forbeesinEnglandandconcludedthatthemediandistanceforagedwas6.1km,andthemean5.5 km.Only10%ofthebeesforagedwithin0.5kmofthehivewhereas50%wentmorethan6km,25% morethan7.5kmand10%morethan9.5kmfromthehive.Thisstudyshowsthatbeesareableto coverlargedistancesintheparticularcase.Earlierstudiesshowedsmallerdistances(averageabout 1km).Theyassumethatsuchdistancesareonlyfoundinsituationswherefoodqualityperpatch variesmuchandpatchesarelarge.Onlyinsuchcaseslargedistancetravellingcanberewarding. Alsobydecodingwaggledances,VisscherandSeeley(1982)showedthathoneybeesregularlyfly severalkilometresfromthehive(seeFigure5).Intheirstudy,themostcommondistancewas600 800m.Themeanwas2.3kmandtherangeenclosing95%ofthecolony'sforagingactivityhada radiusof6km.

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Figure5Distributionofforagingactivitiesasanalysed bywaggledancedecoding(VisscherandSeeley1982)

AlsomuchshorterdistanceswerefoundinastudybyWaddingtonetal.(1994),wheretheforaging rangewas7451,413m.Inpatchylandscapes,wherefoodrichnessvaries,temporallyandspatially recruitmentofforagersthatexplorelargerdistancescanbeprofitable(dependingonscarcityand patchinessofpollennectardensity). Ramsayetal.(1999)placedbeehivesatadistanceof800mfromaGMoilseedsrapefield.Over50% ofthebeeshadGMpollenintheirpollenloads,showingthatthisdistanceiseasilycoveredbythe majorityofthebees.Fromthisandotherstudies,theyconcludethatpolleniseasilycollectedfrom thisfavouredcropatrangesupto2km. SteffanDewenterandKuhn(2003)observedanddecodedoverathousandhoneybeewaggle dancesfromcoloniesinsimpleandcomplexlandscapesindifferentseasons.Overall,themean distancewasabout1.5kmandrangedfrom60mto10km. Williams(2001)reviewedtheroleofbeesinpollenandgeneflowfromGMplants.Referringto maximumflightrangesupto10kmforhoneybees,thereisampleevidencethatbyfarmostpollen aredepositedonnearbyplantsduringforagingorbroughttothecolonywithintherangeofafew hundredmeters.Typicallymeandistancesarearound300m.Becauseoftheskeweddistribution (frequencyisexponentiallydecreasingwithincreasingdistance,seeFigure3)offlightdistancesand

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 pollentransport,occasionaltransportofpollenoverlargedistancesispossible.Forexample,the majorityofbeesmayforagewithinarangeof5001000meterbutasmallfraction(thatishardto quantify)mayforageatadistanceof5kmormore. Ofcourse,thisaffectsthefractionofpollentransportedoversuchdistancesbutforsomesettings thatcanberelevant.Inparticular,whenattractivepatchessuchasfloweringoilseedrapeiswithin reachofthecolonyandothergoodfoodsourcesnearbyarescarce.

4.3
Apple

Cropspecificinformation

Themajorroleofinsectsincludinghoneybeesinpollinatingfruittreeshaslongbeenrecognized (Free1993)Therefore,mostappleandotherfruitorchardsapplyhoneybeepollinationwith coloniestoimprovefruitsetting.Viceversa,forbeekeepersandhoneybeesappleandotherfruit treesareanimportantsourceforpollenandnectarinparticularinspringtime. Eventhoughmanyotherwildbees,bumblebeesandsyrphidscanplayanimportantroleinavaried landscape,pollinationandpollentransportfromandtoorchardswillbelargelyhoneybeemediated whenbeehivesareappliedatalargescalefororchards.Asappleflowersareapreferredforaging targetbeesfromacolonylocatedintheorchardswillmainlystaywithintheorchard(Free1993)or forageatsmallerdistancesinadjacentorchardswhenavailable.Becauseofmassivefloweringand abundantfoodthebeestendtostaywheretheyare.Thestrategyofplacementandnumberof beehivesperorchardsuggestthathoneybeesrestricttheirforagingrangetoasmallareawhen beingactiveinfloweringorchards. Theresearchershavenotfoundanyexperimentaldataabouttheattractivenessoforchardsfor distantcolonies,althoughitseemslikelythatfloweringorchardscouldattractbeesfromalarge distancebecauseoftheabundanthighqualityfood.However,atthesametimeoftheyearother flowersmaybeequallyattractivewhenavailablesuchaswillowsanddandelion.Thoughmuch informationisavailableforoptimizingapplefruitsettingandnecessarycrosspollination,itis remarkablethatnoinformationcouldbefoundonlongdistanceapplepollentransport.

Oilseedrape
Oilseedrapeisoneofthemostpreferredcropsforhoneybeesandpossiblyforotherpollinating insectsaswell.Whenmassflowering,itattractspollinatinginsectsfromoverlargedistances.Dueto theimportanceofthiscropandsuitabilityforexperimentalstudies,severalinvestigationshavebeen doneontheforagingactivityandpollentransportsfromoilseedrapeandsimilarcruciferouscrops

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 andweeds.MostrecentstudiesofRader(2011)andChiffletetal.(2011)haveshownthatthese cropsattractbeeseasilyfromdistancesofatleast500mto1,000m. Becauseofthesignificantroleofhoneybeesandbumblebeesinthepollinationandpotential unwantedcrosspollinationbetweenfields,orrelatedBrassicaspeciesoutsidethefield,much researchhavebeendoneonpollinationtransportandgeneflowpatterns(e.g.SmithKleefsmanet al.(2005),Cresswelletal.(2002),DamgaardandKjellsson(2005),LuytenandDeJong(2011)and manyarticlestheseauthorsarereferringto). Theresultsfromempiricaldataormodellingdataallpointtotheconclusionthatpollentransportby flightandprobabilityofcrosspollinationexponentiallydecreaseswithdistancefromthepollen source.Therefore,eventhoughhoneybeesorbumblebeesmaycoverlargedistancesunder particularcircumstances,themajoractivityandresultingpollinationoccurswithinadistanceofa fewhundredmeters(BeckieandHall2008).Ontheotherhand,potentialcrosspollinationcanoccur overlargedistancesupto3or4kilometres,eventhoughtheprobabilityofsuchapollinationisin theorderof0.01to0.001%.Dependingonthespatialarrangementoffieldsandthesizeofthebee populationandthenumberofflowerstobefertilized,thisstillmayresultinasignificantabsolute numberofcrosspollinatedflowerseventhoughthismaybeaverysmallfractionofthetotalflower population.

Potato
GenerallyPotatoisconsideredasalessrelevantcropforhoneybeesbecausemanycultivarsdonot floweratallorhavealessabundantfloweringwithverylittlepollenandnonectarbeingavailable forinsects(suchashoneybees).SanfordandHannemann(1981)alreadyshowedthathoneybees havenorealcuetovisitpotatoflowers.Onlyafewbumblebeespeciestendtoexploitpotato flowers.Potatoapparentlyisunattractiveforhoneybeesandhencenoinformationcouldbefound forthedistanceoverwhichpotatopollenistransported.

Sugarbeet
Althoughbeesareknowntovisitfloweringsugarbeets(Free1993),itiscertainlynotapreferredora rewardingcropforhoneybees.Nodatacouldbefoundonhoneybeeforagingpatternsorflight distancesinrelationtothiscrop.Normally,cultivatedsugarbeetiskeptvegetative,sotheonly possiblevisitedandpollinatedflowersareofthefewboltingplantsthatoccurinfields,and productionfieldsofbeetseeds(thelatterwillbeinsidegreenhousesorpolytunnels).Moreover, honeybeesgenerallydonotvisitscatteredindividualplants,butspecializeonrichpatchesofhighly rewardingplants.

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Maize
Maizeisamajorcropinmanycountriesthataresuitableforgrowing.Althoughitisawind pollinatedcrop,whichisnotdependentoninsectpollination,itproduceslargeamountsofpollen. Thoughmaizepollenisnotpreferredasafoodsource,thereisaccumulatingevidencethathoney beesregularlyfeedonmaizepollenwhenitisabundantandnomorerewardingcropsorflowersare available(Kelleretal.2005).Thenutritionalvalueofmaizepollenislowduetoitslowprotein content(Hrcherletal.2010). Duringitsfloweringtimeinlatesummermaizepollenmaybeasubstantialpart(80%)ofthefood collectionwhenmaizeisabundantandfewothersourcesareavailable(Odoux2004).Alsoinamixed landscapemaizepollenmaycanbeoneofthehighrankingfoodsourceswhenavailable(Keller 2005),inparticularwhenbeehivesarelocatednearfloweringmaizefields.Thereareno experimentaldataonhowfarbeeswillflytoforageonmaizewithandwithoutotherfoodsources nearby,butitseemsnotlikelythattheyflylongerdistancesforalesspreferredpollentypesuchas maizecomparedto,forexample,oilseedrape.Itshouldbenotedthatoilseedrapeisalsovery attractiveforitsnectar.

4.4

Otherpollinatinginsects

AccordingtoOsborneetal.(1999),whostudiedflightdistancesofbumblebees,theabilityto optimizeflightdistancesunderdifferentcircumstancesisessentialforpredictingcolonysuccess. Foragersweremassmarkedastheyleftthecolonyandpollenfromforagersreturningtothe colonieswasanalysed.Inthisway,thespatialdistributionofforagingcouldbemappedandforaging distancesandforageavailabilityforthebumblebeescouldbeestimated.Thebumblebeesforaged atleastupto1.5kmfromtheircolonies,andtheproportionofforagersflyingtoonefielddeclined, approximatelylinearly,withradialdistance. Otherfieldstudieshaveshownthatsomebumblebeespecies(includingB.terrestris)forageatleast severalhundredmetres(Dramstad1996;Osborneetal.1999);andevenkilometresfromthenest (2.2kminKreyeretal.2004).WaltherHellwigandFrankl(2000)found25%ofresightingsbetween 1500and1750mfromthecolony. Chiffletetal.(2011)studiedthepresenceofpollenonmanydifferentinsectsatvariousdistances fromaGMcropplothavingmarkedpollen.Manypotentialpollinatinginsectshadviablepollenwith thematadistanceof500m,butat1,000mtheincidencewaslow(05%)andonlyonesolitarybee specieswasfoundat1,500m.Theyconcludethatadistanceof1kmforisolationisnotenoughto avoidpollendispersaloutsidethisrange.

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 Thoughhoneybeesandbumblebeesareveryeffectivepollinatorsandcarrymorepollenper individual,manyotherinsectscancarrypollenwiththemandeasilyovercomelargedistances.For

examplesyrphidandstratomyidfliesthatalsohaveahighcapacitytotransportpollenwerefoundto bemoremobileandtransportpollenveryquicklyoverseveralhundredsofmeters(Raderetal. 2011).However,theyalsonotethatthereisgenerallackofdataconcerningthetransportofpollen bysuchspeciesoverlargedistances.

4.5

Concludingremarks

Thereismuchevidencethathoneybeescancoverandhencetransportpollenoverlargedistances upto10kmormore.However,inmanycases,coloniesareputinplacenearnectarandpollen rewardingplacesandwherefoodisabundantindividualstendtostayinafavouritesiteandafter collectingenoughfoodwillreturntothecolony.Thiscommonpatternwillleadtodominant pollinationpatternsthatoccurwithinarangeofafew100metersorevenless(e.g.whencolonies areplacedinafloweringorchard).Thewholeissueoftheimpactoflongrangeflightsonpollination overlargerdistancesdependsontoomanyfactorstodrawonegeneralconclusion.Inanextreme casescenario,twoisolatedbutattractivefieldsatalargedistancewithabeecolonyinbetweencan bevisitedbythesameindividualbeesatthesameday,takingmaximumpollenloadswiththem.In suchacase,significantpollentransportcouldoccuratadistanceoftwotimes10km. AsBeekmanandRatnieks(2000)haveshownforheatherfields,whichcanbeveryattractivefor bees,morethan50%ofthebeesofacolonycouldfocusonsuchdistantfields.However,no experimentalevidence,sufficientdataorfieldvalidatedmodelsareavailablethatcangiveclues aboutthefinalquantitativeimpactfordifferentcrops.Handlinglowprobabilitiesinavariable landscapecontextisextremelydifficultandmoreexperimentalandmodellingresearchisneededto getabetterunderstandonwhatisreallygoingon(BeckieandHall2008).Directmeasurementof labelledpollentransportandsubsequentpollinationhasnotbeenresearchedinanexperimental settingcoveringmorethanafewkilometres. Thespatialarrangementoffields,beecoloniesduringtheseason,thevariableflightactivityofbees makesitveryhardtodeterminearelationbetweendistanceandpollinationprobabilitiesthatis validformanydifferentconditions(SteffanDewenterandKuhn2003).Hence,settingdistance criteriaforpreventingundesirableoutcrossingalwaysincludesapoliticaldecisioninadditionto ecologicalarguments(Lezaun2011),especiallyaslongasnomorehardandconvincingdatais available.

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5
5.1 Introduction

Polleninhoney

Naturalhoneyalwayscontainspollengrains,andoftensporesoffungiaswell.Pollengrainscanend upinhoneythroughthreeroutes: 1. 2. 3. Whenanectarforagingbeehascollectedafullhoneystomach,itreturnstothehivetoturnoverits nectarloadtoayoungerworkerbee.Althoughaforagingbeegenerallygetsloadedwithpollenfrom theflowerantherswhilecollectingnectar(Free1993),itshoneystomachhardlyevercontainspollen grains.Inthehoneystomach,pollengrainsarerapidlyfilteredoutandareloadedintothereal stomach(ventriculus),bigpollengrainsfasterthansmallones.Ittakesabout10minutesforthe honeystomachtofilteroutallpollengrains,whichmeansthatthetraveltimebacktothehiveis important.Forsomeverygoodnectarsources,beesmayflyasfaras9kmorevenmore(Beekman andRatnieks2000),whichgivesenoughtimetofilteroutthegrainsduringflight,nectarcollected nearbymaystillcontainmorepollengrainsuponreturn.Inthehive,thereceivingworkerbeewill passonthecleannectartootherbeesimmediately(forinstancenursingbeesthatuseitforfeeding thelarvae)oritwillstarttostoreandprocessit.Hence,itisthestoringandprocessingofthenectar insidethehivethatisthemainsourceofpolleninthehoney. Whenabeekeeperextractsthehoneyfromthecombbycentrifugation(seealsoSection3.5),hardly anypollenapartfromthegrainsdissolvedinthehoneywillbeadded,despitethefactthatcells withbeebreadmaybeincludedinthehoneycombs.Thisbeebreadverystronglyadherestothe combandisnotextracted.Onlywhenthebeekeepersharvestshoneybypressingofthehoney comb,thepollenfromthecellscontainingbeebreadmaybemixedwiththehoney.However, pressingofhoneyisabandonedasawaytoextracthoney,apartfromruralbeekeepinginfor instanceAfrica. Toactasaconservedenergyreserve,thenectarhastobedehydrated(sothatitwillnotbe susceptibletofermentationanymore)andenzymesneedtobeadded.Processinganddehydration ofhoneyisdonebyregurgitatingthenectarseveraltimesandfanningwiththewingstoevaporate Pollenfromtheflowerthatisforagedonbythebeefallsintothenectarandisharvestedby thenectarforager; Pollenonbeesandintheairinsidethehivecontaminatethenectarduringprocessingby thebees(packing,drying,conserving);and Pollenpackedincellsinthesamecombasthehoneymaybemixedwiththehoneybythe beekeeperduringcentrifugationorpressingofthehoneyfromthecombs.

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 water.Onlywhenithasawatercontentbelow20%,thefullyloadedhoneycellcanbesealedwitha waxcap.Mostofthepollengrainsthatarefoundinhoneyhavegenerallyenteredunintended duringthefanning,sincetheairinsidethehiveisloadedwithpollengrainsofseveralplantspecies, reflectingtheavailabilityofcollectedpollenspeciesintheenvironment.Pollengrainsofwind pollinatedplantsmightbewellrepresentedinthefanningair,sincethesegenerallyarelesssticky andeasilyfloat.AhoneyfromSinderhoeve(nearHeelsum,TheNetherlands)in2007containedas muchas74%ofHypericumpollen,thoughthisplantdoesnotproducenectarandwasobviouslyjust visitedforitspollen(Blacquireetal.2008).

Pollendeterminationinhoney
Pollendeterminationinhoneysisawaytodeterminetosomeextentthebotanicaloriginofthe honey.Suchpollenprofilesaregenerallymadebymicroscopicaldeterminationofthefamily,genus orspeciesofplantof500pollengrainsinthehoney.Adominantpollenspeciesisthensupposedto beanimportantdonorofnectar,andthuscandidatetobethenominatorofthehoney.However,it isnotalwaysthatstraightforward,sincedifferentpollentypeshaveverydifferentprobabilitiesto endupinhoney.Thisdependslargelyonpollensizeandmorphology,abundance,stickinessbutalso onflowershapeandarrangementoftheflowersininflorescences.Thesefactorstogetherdetermine towhichextentthebees,andeventuallythehoney,getcontaminatedwithairbornepollengrains. Qualitativeandquantitativepollendetermination Theabovementionedpollenprofilesofhoneysdoinformabouttherelativenumbersofdifferent pollenspeciesinhoney:thisisarelativemeasureonly.Sincepollenspeciesdonotshowupequally easyinhoneyamorequantitativeapproachtodeterminethebotanicaloriginofhoneyhasbeen adopted,inwhichthespecificpollentraitshavebeentakenintoaccount.Forinstance,ahoneymay carrythenameofchestnuthoneyonlyifmorethan90%ofthepolleninthehoneyoriginatesfrom chestnut.Nectarofchestnutisalreadyrichinpollengrains.Thegrainsarerelativelysmall,which retardstheremovalfromthenectarinthehoneystomachbytheproventriculus.Moreover,bees foragingonchestnutareoverloadedwiththeabundant(andsmallgrained)pollengrains.Some otherhoneyscanalreadybeattributedtoacertainspecieswithonly20%representingthespecific plantspecies.Byusingapollencoefficient(thenumberofpollengrainsper10gramsofmonofloral honeydividedby1000),amorerealisticshareofthecontributingplantspeciestohoneyscanbe obtained(Kerkvliet2011a,2011b). Althoughthiswayofdeterminingthehoneyspeciesonthepollenpresentinthehoneyiscalled quantitativepollendetermination(Crane1975)itisactuallynotquantitativebutjustrelative:it doesnotgiveanassessmentofthenumberofpollengrainsnortheweightofthepollenloadin honey.Whenpollenprofilesofhoneyneedtobeusedtopredictthepresenceandconcentrationof

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 pyrrolizidinealkaloids,seeKempfetal.2011)orpesticideresidues,realquantificationisneeded. Accordingtotheveryscarceliteraturefoundabouttheamountofpollenpresentinhoneysthe shareistypicallylowerthan0.5%(0.25%,Graham1992)or015,000grainsper1ghoney(Lieux 1972;PersanoOddoetal.1995).Itisnoteasytorecoverthepollengrainsfromhoneyandthen quantifythem.Therefore,inthesmalllaboratoryexperimentbelow,whichispartofthisreviewon polleninDutchhoneys,theresearcherschoseanoppositeapproach:sampleswerespikedof artificialhoney(nonaturalpollen)withknownamountsofdifferentpollenspecies.Subsequently, pollenintheseartificialhoneysweredeterminedintheusualway,bycountingthemmicroscopically inacountingchamber(Brker).Hence,acalibrationcurvewasmadetorelatethemicrogramsof pollentothecountedpollennumbersfoundinnaturalhoneys. InthisChapter,anoverviewofthepollenprofilesofasurveyofDutchhoneysfrom2008is presented,focussingonthepollenofRosaceae,Brassicaceaeandofmaize(Zeamays).Fromfiveof thesehoneysthenumberofpollengrainspresentpergramhoneywasdetermined.Finally,an experimentwasperformedtoestimatetheamountofpollenpresentinhoneysbyweight.Thiswas donebymakingcalibrationcurvesofknownquantitiesofpollenaddedtoanartificialhoneywithout pollenlinkedtothecountedpollennumbersinthesehoneys.

certaincompoundsassociatedwithit,forinstanceallergens,toxins,secondaryplantsubstances(e.g.

5.2

Materialsandmethods

PollendataofDutchhoneys
Duringspringandsummerof2008,bees@wurcarriedoutamonitoringprogramamong172Dutch beekeeperstogainsomeknowledgeabouttheprevalenceofseveraldiseasesandparasitesinthe apiaries.Thankstothechosensetupoftheprogramme,aproperdistributionofthecontributing beekeepersoverthecountrywasobtained.InJune,samplesofbeesweretakenfromfivehivesper apiary,whichwereanalysednotonlyfordiseasesbutalsoforheavymetals.Inadditiontothebee samples,thebeekeeperswereaskedtodeliveroneorafewpotsofhoneyharvestedthatspringand summer.These190honeyshavebeenanalysedforthepresenceoftheAmericanFoulbrood bacteriumPaenibacilluslarvae,butthepollenprofilewasdeterminedaswell. In2008and2009,fromtheobtainedhoneyspollen,extractsweremadeaccordingtoVanderHam etal.(1999):10gramofhoneywasdilutedwith20mldemiwater,thoroughlymixedandthen centrifugedfor10min.at2,200rpm.Theliquidwasdecantedandthepelletwassuspendedintwo stepsin10mldemiwater,andagaincentrifugedat1,000rpmfor10min.Afterdecanting,the resultingpellet(orpartofit)wassuspendedinadropletofdemiwaterandmixed.Thesuspension wasbroughtontoamicroscopyglassslideintwoaliquotsanddried.Adropletofglycerolgelatine

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 fuchsinewasaddedandthetwoaliquotswereclosedwithtwocoverslides.Theslideswere microscopicallyexaminedsystematically,until500pollengrainshadbeendetermined.The determinationwasdoneusingacollectionofreferencepreparationsandliteratureandwebsite collections,uptotheplantgenuslevel.

Dataofthehoneysamplesfromthe2008monitoringhavebeenusedtoshowthepresenceofpollen ofBrassicaceae,RosaceaeandmaizepolleninDutchhoneys.Inthisreport,especiallythepresence ofpollenfromRosaceae(becauseofthefocusonapple)andfromtheBrassicaceae(becauseof oilseedrapeBrassicanapus)isconsidered,aswellassamplescontainingpollengrainsofmaize,beet andpotato.Thepolleninhoneydataweresortedintofiveclassesofpresence(020%;2040%;40 60%;6080%and80100%)allocatedtothehomeaddressesofthebeekeepersbyGIS.

Pollencountingandcalibration
Pollencountsinfivehoneysfromthesurvey Aselectionoffivehoneysfromthe2008surveyknowntoincludepollenfromRosaceaeand Brassicaceae(seeTablesA1A3)wereusedtopreparenewpollensediments.2ghoneywastaken anddilutedwith4mlwater,mixedandcentrifuged,andwashedagainwith2times2mlwaterand centrifugedagain.Aftercentrifugation,thepollenpelletsweresuspendedinanendvolumeof10as wellas100l.Inbothaliquots,thenumberofpollengrainswascountedinduplicateinaBrker cytometercountingchamber.Foreachcounting,thenumberofpollengrainswascountedin20 countingchambers.ABrkercountingchamberhasavolumeof0.004l.Thefivehoneysused (numbered132,133,171,181,186)havebeenmarkedblueintheTablesA1A3givingthedata aboutthe2008honeysurvey(Annex1). Calibrationofpollennumbersusingartificialhoney Beefitsugarsyrup(sugarcontent70%)wasusedastheartificialhoney(withoutpollen contamination).Beefitiscommerciallyavailableasbeefeed.To18gramsofthisartificialhoney,in2 mlwater,standardquantitiesofpollenwereaddedinaconcentrationrange:50;5;0.5and0.05mg pollenpersample,endingupinthesameamountsin20gofhoney.Sugarcontentoftheartificial honeywasreducedto66%afteraddingthepollen,becausethepollenwassuspendedinwater.Bee collectedpollenpelletswereusedaspollensources.Pollenspecieswerechosenthatareoften foundinhoney.Thesepollenpelletswerecollectedfrombeesusingapollentrapatthehive entranceinthesummerof2010byMoosBlom.Thepollenpelletswerehomogenousandthepollen hadbeendeterminedontogenuslevel.Thepollensampleshadbeenstoredinplasticbagsina refrigeratorat20Csincethen.ThepollenspeciesselectedfortheexperimentareshowninTable4.

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 Table4Pollenspeciesusedinartificialhoney(picturesfromwww.paldat.org).Pollengrainshave beendeterminedtotheleveloftheplantfamilies.Thespeciesmentionedbetweenbracketsreferto thepollenspecimensinthepictures Pollenspecies code Aceraceae (Acercampestris) A size[m] 30x32 form tricolpate (pollengrain with3long (colpate) apertures) Brassicaeae (Brassicanapus) B 31x32 tricolpate picture

Fabaceae (Trifoliumrepens) K 24x26 tricolpate

Phacelia (P.tanacetifolium) P 20x22 heterocolpate (pollengrain withbothlong aswellas combinedlong +porous apertures) tricolporate (pollengrain with3colpi withonepore each) Mixtureofthe5species M

Rosaceae (Malussylvestris)

31x35

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 Thewashingandconcentrationofthepollenpelletsfromthisartificialhoneywasperformedaswith thenaturalhoneys(seeabove)usedforthecountingofpollengrains.Pollencountsweremadein duplicate.Fromeachsample,thenumberofpollengrainswascountedin20countingchambers, these20countswereaveragedandthenumberofpollengrainswascalculated.Subsequently,the numberofpollengrainswasrelatedtotheamount(g)ofpollenaddedtothesample. Watercontentofpollenpelletswasdeterminedbyweighingandreweighingafterdrying. Asbeesmayusesomenectartosticktogetherthepollengrainsintheircorbiculae,whichmight contributetothemassofthepollenpelletsuseditwasdecidedtodeterminethesugarcontentin thepollenpellets.Toestimatethesugar(nectarorigin)contentofthedrypallet,repeatedwashing (andreweighing)wasused. Itwasfoundthatpollenpelletshadawatercontentof9.7%(SD<0.1%),andthesugarcontentwas 0.01%.Thisindicatesthatthesampleshadalreadybeendesiccatingtosomeextent,sincefresh pollengenerallyhasawatercontentof2030%(Camposetal.2008).Inthiscase,usingfreshpollen pelletsdoesnotintroduceaverybigaberrationfromusingdryweights.Itcanbeconcludedthatthe sugarcontentisnegligible. Statistics Fornaturalhoneys,differencesbetweenpollencountsinnaturalhoneyweretested,usingatwo wayANOVAforthedifferentendvolumes(10or100l)andthedifferentnaturalhoneystested.A Sidakposthoctestwasusedtotestdifferencesbetweenmeans(interaction). Forartificialhoney,theaveragepollenpergramhoneywasdeterminedanditwastestedwhether

thecalibrationcurvecrossedtheorigin.Therefore,linearregressionmodelswereusedforallhoneys separately.4 Totestdifferencesinslopesofthecalibrationcurvesbetweenhoneys,theresearchers usedagenerallinearmodelwiththecountedpollen(#)persampleasafunctionofthepolleninput persample(covariable;grampollenpersampleofartificialhoney,andthedifferentartificialhoneys (fixedfactor). Forthistest,boththepolleninputasthecountedpollenwerelog10transformed,inordertoobtain anormaldistributionoftheresiduals.ASidakposthoctestontheinteractionbetweenpolleninput andhoneytypewasusedtotestdifferencesbetweenmeans(onlythedifferencesatthehighest concentrationswereused).
4

Theresearcherscouldnotlogtransformthedataforthefirstanalysis,asithastobetestedwhetherthecalibrationlines crossedtheorigin(notpossiblewithlogtransformationondatarangingbetween01).Residualswerenormallydistributed withouttransformationofthedata.Apointofargumentwouldbethatthewithouttransformation,thedataofthehighest concentrationhasarelativelylargeimpactonthecurveestimation.

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5.3

Results

Honeysurvey2008
Figure6to12showthelocationswhere,forexample,80100%(reddots)ofthepollengrainsinthe honeybelongedtotheindicatedbotanicalgroup.Whenmorethanonesampleperlocationwas available,thehighestvalueisshown.ThefulldataofthehoneysislistedinAnnex1inTablesA1to A3.Honeysfromthesurveythroughoutthecountrycarriedpollengrainsofatleast50plantfamilies, windpollinatedaswellasinsectpollinatedspecies.Somefamiliesareveryabundant,likeSalicaeae, Rosaceae,BrassicaceaeandFabaceae.

Figure6:Distributionandpercentage Brassicaceaepollenoutof500grainsinhoneyin TheNetherlands

Figure7:DistributionandpercentageRosaceae pollenoutof500grainsinhoneyinThe Netherlands

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Figure8:Distributionandpercentage Brassicaceaepollenoutof500grainsinspring harvestedhoneyinTheNetherlands

Figure9:DistributionandpercentageRosaceae polleninspringharvestedhoneyinThe Netherlands

Figure10:Distributionandpercentage Brassicaceaepollenoutof500grainsinsummer collectedhoneyinTheNetherlands

Figure11:DistributionandpercentageRosaceae pollenoutof500grainsinsummercollected honeyinTheNetherlands

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 Thefiguresclearlyshowthatbothhoneysrichin Brassicaceaepollen(>80%)andRosaceaepollen (6080%)arequiteabundant,especiallyin spring.Thecategoryof020%maybealittle rude,butrealzerosareveryrareforRosaceae andBrassicaceae(seeTablesinAnnex1).Spring honey(AprilthroughJune)includesthe floweringtimeofbothoilseedrape(Brassica napus)aswellasapple(Malusdomesticus). Summerhoneyandlatesummer/autumnhoney stillmaycontainalotofpollenfromboth BrassicaceaeandRosaceae,butthesamples withveryhighsharesarefew(seeTableA3). Figure12:Distributionofsamplespositivefor MaizepollenfoundincollectedDutchhoney, summer2008 Duringthesummerseason,apartfromLime (Tiliasp.)andHeather(Callunavulgaris),honeys generallybecomemoremultifloral(with individualsharesdeclining).

Maizepollenwasonlyfoundintwosamplesoutof~190,thepercentagemaizepollenwas1.5%and 4.2%(~500grainscounted).PollenofChenopodiaceaissometimespresentanditcannotfullybe excludedtobepartlyfrombeet,althoughhoneybeeforagingonbeetisveryimprobable. Solanaceaeismoreoftenrepresentedandpotatomightbeincludedinveryfewcases.Pollenof potatowasfoundinhoneyswhenbeescollectaphidderivedhoneydewfrompotatofields(vander Hametal.1999).

Numberofpollengrainsinfivehoneys
Naturalhoneysshoweddifferentpollencounts,rangingfromlessthan1,000/ghoneytomorethan 25,000/ghoney(Figure13)butwithintwoofthesehoneys(honey177and186)therewerealso differencesbetweenthetwoendvolumesinwhichthepollennumberswerecounted(suspendedin either10or100l)(honeyF4,10=239.0,P<0.001;polleninputF1,10=154.2,P<0.001;interaction F4,10=64.1,P<0.001;Figure13).Thenaturalhoneyswiththehighestpollencount,showedan additionalhigherpollencountforthe100lendvolume.Thismayresultfromtoohighnumbersof grainsinthecountingchambersatthe10lendvolumecausingsomegrainstobeoverlooked.

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Figure13Pollencountpergramnaturalhoneyfor10land100linput.ASidakposthocwasusedtotestfor
differencesbetweenmeans.Asterisksshowdifferencesbetweengroups(**P<0.01;***P<0.001)

Amountofpollenaddedversusnumbersdeterminedincountingchambers
Inallartificialhoneys,thecalibrationcurvescrossedtheorigin(PvaluesConstant>0.05,Table5,see alsoFigure15)).Theaveragenumberofaddedpollengrainscountedpergramaddedpollenvaried between1.5x1071.1x106forAceraceaeand2.3x1087.0x106forPhacelia(slopepollen input,Table5).Thecalibrationslopesdifferedbetweenallartificialhoneys(honeyF5,36=5.7,P=0.001; polleninputF1,36=1830.6,P<0.001;interactionF1,36=7.2,P<0.001;Figure14).Omittingthelowest inputconcentration,becausesomesamplesgavelowcounts(whichmayaffectthereliability),did notsignificantlyaltertheslopesnorthestatisticalsignificance. Table5Thetestresultsofthelinearregressionmodelsofthedifferentartificialhoneys,where showstheestimatedvalueforthenumberofpollenmeasuredat0gofpolleninput,andslopeshows theaveragenumberofpollengrainscountedpergrampollenadded Constant Polleninput(gpersample) Honey R2
Aceraceae 0.966 3.45*10
3 3 3 3

s.e.m.()
4 4 4 4

slope
6 6 6 6

s.e.m.(slope) 1.14*10 1.99*10 1.45*10 3.13*10 6.98*106 1.48*106

6 6 6 6

2.92*10 0.12 0.910 14.9*10

13.08 ,000 37.48 ,000 54.15 ,000 9.80 ,000 33.17 ,000 35.12 ,000

Brassicaeae 0.996 40.34*10 5.00*10 0.81 0.451 74.7*10 Rosaceae Fabaceae Phacelia Mixture 0.998 40.29*10 3.65*10 1.10 0.312 78.5*10 0.941 18.25*10 7.88*10 0.23 0.824 30.7*10
3 4 0.995 89.19*10 17.5*10 0.51 0.629 3 0.995 10.73*10

232*106

3.72*104

0.29 0.783 52.0*106

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Figure14Calibrationcurvesforthedifferentartificialhoneys(seelegend).Atthehighestinput(0.05),
allhoneysdifferedfromeachother(SidakposthoctestP<0.001forallcombinations)

Figure15Detailofcalibrationcurves(Figure14)forthedifferentartificialhoneys(seelegend)
toshowproximityofcalibrationcurvestoorigin.

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5.4

Discussion

Botanicaloriginofpolleninhoney Inthe2008honeysurvey,manyhoneyswithveryhighshareofBrassicaceaepollenwerefound throughoutthecountry,sometimesevenhigherthan80%.Brassicaceaeareespeciallyabundantin springhoney,correspondingtothefloweringtimesofoilseedrape(Brassicanapus)andcharlock (Sinapisarvensis).SincenotsomuchB.napusisgrowninthewesternpartofthecountry(provinces Utrecht,SouthHollandandpartsofNorthHolland)andferalpopulationsofitarerare(Luijtenand DeJong2010),mostoftheBrassicaceaepolleninhoneystheremayoriginatefromferalpopulations ofB.rapaandSinapisarvensis.Bothspeciescanbeveryabundantonruderalplaces(LuijtenandDe Jong2010).Theone>80%BrassicaceaehoneyinthenorthofNorthHolland(Figure6)maywellbe ofB.napus,sincethelocationcorrespondswithalargeproductionareaofB.napusin2008(Luijten andDeJong2010).MostoftheB.napusisgrownintheEasternpartofTheNetherlands,especially EasternGroningen,whileB.rapaandS.arvensisaremorerarethere(LuijtenandDeJong,2010). ThismightindicateagreaterchancethatBrassicaceaepolleninthesehoneysincludesB.napus. Similarly,toBrassicaceae,alsoRosaceaepollenaremoreabundantinspringhoneythaninsummer honeys.Inspringthesepollengrainsrepresenthawthorn,wildcherry,cultivatedfruittrees(apple, pear,cherry,plum)aswellasroseshrubs,insummerrosesandblackberries(Rubusfruticosus). Theonlytwofoundexamplesofpollenofmaize(Zeamays)inhoneyseemtoconflictwithreports thatstatethatmaizepollencanbeamajorpartofthepollenintakeofacolonyinFrance(Odouxet al.2004).IntheUSstateofIndiana,maizepollencomprisedover50%ofthepollencollectedbybees (byvolume)in10outof20samples(Krupkeetal.2012).Theresearchersdonotknowtowhich extenthoneybeesforageformaizepolleninTheNetherlands.Despiteasometimeshighintakeof maizepollenbybees,inhoneyonlyinfewcasesandwithlowpercentagesmaizepollenisfound.In ourcase,theoccurrenceofmaizepolleninhoneywasrareand,additionally,thesharewasverylow (max.4%).InGermanyinabout10%ofthehoneysmaizepollenwasdetected,butgenerallythe sharewasaround0.2%ofthepollengrainsonly(W.vonderOhe,personalcommunication).Alsoin France,theshareofmaizepolleninhoneywaslow:only10%ofthehoneyscontainedmaizepollen inlownumbers(PatriciaBeaune(LaboratoryFamilleMichaudApiculteurs(France))personal communication). PossibledifferencesbetweenforinstanceFranceandTheNetherlandsmaybethedifferentmaize varieties(withdifferingpollenproduction),aswellasthepoorhoneyflowduringmaizeflowering timeinTheNetherlands.Duringthetimemaizeflowers,Dutchbeekeepersrarelyharvesttheir honey,becauseduringthisperiodoftheseasonhoneyflowinTheNetherlandsispoor.Although maizepollenmayoftenbecollectedbybees,thelowhoneyflowpossiblyresultsinmaizepollen hardlyevershowingupinharvestedhoneybybeekeepers.Honeydewhoneycollectedonpotato

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 cropsmaycontainlownumbersofmaizeaswellaspotatopollen,blownintothestickyhoneydew bywind(J.D.Kerkvliet,personalcommunication),inrealflowerhoneysthesepollenspeciesarenot found. Somecareshouldbetakenwheninterpretingtheregionalimplicationsofthepollenprofiles,sinceit isnotabsolutelycertainthatthebeehiveshaveactuallybeenallthetimeonthespotwherethe researcherslocalizedthem.Theresearchersusedthehomeaddressesofthebeekeepers,andsome ofthemmayhavebeentravellingwiththeirbees,althoughthisisnotverycommonpractice. However,asmostofthefruitorchardsarepollinatedbyhoneybeecoloniesofhobbyistbeekeepers inTheNetherlands(Blacquireetal.2009),especiallyinspringasignificantpartofthehivesmay havebeenmoved.Therefore,thissurveycanonlybeusedasafirstindication,anditwouldbevery worthwhiletoinitiateamorefocusedsurvey. Quantificationofpollennumbers Inthefivehoneysfromthesurveyusedforabsolutequantificationofthenumbersofpollengrains, thenumbersvariedfromlessthan1,000to28,000pergramofhoney.Thesefiguresnicely correspondtodatafromtheliterature(mosthoneys:2,000to10,000grains;VanderHametal. 1999;VonderOheetal.2004;PersanoOddoandPiro2004),althoughahoneywith25,000grains

pergramofhoneyisconsideredasahoneywithahighpollennumber(VonderOheetal.2004).Ina ringtest(VonderOheetal.2004);itwasalsofoundthatthereproducibility(betweenlaboratories) ofpollencountingwasbetterinhoneyswithhighnumbersofpollenthaninhoneyscontaininglow numbersofpollen.Inourcase,inthehoneyswiththehighestnumbersofpollengrainsthecounts werehigherwhenthepollenpelletwasresuspendedin100lthanin10l,suggestingthatpollen grainrecoverywasbetterwithahigherdilution,i.e.withlowerdilutionthenumberofpollengrains wassystematicallyunderestimated.Thismaymoderatetheabsolutevalueofpollencounts. Therangesofabsolutenumbersofpollenofseveralunifloralhoneysareknown(PersanoOddoand Piro2004),soiftheresearchershavehoneysamongthefiveanalysedwithahighshareofoneofthe unifloralpollentypes,theresearcherscancomparetheabsolutenumbersbetweentheirhoneyand theunifloralhoney.ThespringhoneyNr.177has50%Brassicaceae,andcontained12,000grains pergram,whichisverymuchcomparabletorealunifloraloilseedrapehoneywith7,570grainsper gram(PersanoOddoandPiro2004).Theotherfourchosenhoneysdidnothaveahighshareofany oftheunifloraltypesandthereforenocomparisoncouldbemade. Calibrationofrecoveredpollennumberstotheweightaddedtoartificialhoney Thenumbersofpollengrainspergramofartificialhoneyvariedbetween74,000(Aceraceae)and 1.15million(Phacelia).Thismeansthatourartificialhoneysoutrangednaturalhoneysseveraltimes, sincethelattermaycontaingenerally10,000grainspergramatmost(VanderHametal.1999).

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 Numbersintherangeofmillionspergramcanonlybereachedsometimesforpressedhoneys. Thesenumbersof74,000through1.15millionhadbeenrecoveredfromanadditionof0,05gram pollenper10gramofhoney,i.e.0,005g/g(0,5%w/w).Lookingbackthehighestadditionwasout ofrangeofnaturalhoneys,howeveritwasbasedonthereported0,25%mentionedbyGraham (1992).Theotheradditionswereintherangeofnaturalhoneys.Moreover,insideandoutsidethe naturalrangetherelationshipwassimilarlylinear.The0.25%mentionedbyGrahamwouldindicate 37,500to570,000grainspergramofhoney.Inthatcase,thenumbersof015,000grainsper1g honeyreportedbyLieux(1972)andPersanoOddoetal.(1995)wouldbeatthelowerendofthe range. Despitebeingoutofthenaturalrange,ourestimatedlinearcalibrationcurvesshowedveryhigh valuesforthecoefficientofvariation 5 (whichmeansthatthemodelisaverygoodpredictorfor

futureoutcomes)anddidcrosstheorigin,whichmeanstheslopecanbeusedtorelatepollencounts topollenweights.Deducedfromthecalculatedslopes,theresearchersconcludethat1gramof polleninhoneycorrespondswith14.9millionpollengrains(Aceraceae)to232millionpollengrains (Phacelia).Tenthousandpollengrainsthenweigh43g(Phacelia)to670g(Aceraceae). Recalculatingthistotheexpectedpollenpercentageinhoney(10,000grainspergramhoney(Van derHametal.1999)),thismeansthatanormalhoneywouldcontain43gto670gpollen/g honey(or43mg/kgto670mg/kg).Thiscorrespondsto0.043to0.670.Thelatterisonly4times lowerthanthe0.25%ofGraham(1992).Ifhoney27(Figure12)withashareofmaizepollenof4.2% wasanormalhoneywith10,000grainspergram,themaizepollencontentofthehoneywouldbe only0.042X670=28g/g.However,theweightshareofthemaizepollenmightbehigherthan4%, sincemaizepollenisverybigandheavy:onepollengrainweighs250350g(Fonsecaetal.2003). AccordingtoVanderHametal.(1999),honeysofbothoilseedrapeandfruittreescarrypollen numbersuptoaround10,000pergramhoney,andneedtocarryatleast45%ofthenamegiving pollenspecies:4,500grainspergramhoney.FromTable5(slopecolumn)itcanbededucedthat1 gpollenofRosaceaerepresents78grainsresultinginaweightof0.0128gpergrain.4,500grains pergramhoneywouldweigh58g.Foroilseedrape,1gpollenis74.7grains,resultinginaweight pergrainof0.0134gpergrain,and4,500grainsweighing60g.Anotherapproachtoestimatethe weightofapollengrainwasusedbyKerkvliet(personalcommunication):bycalculatingthevolume ofapollengrainfromitsmeasureddiameter(4/3XXr3)andassumingaspecificgravityof1,0,this wouldresultinonemillionpollengrainsweighing: Phacelia:diameter=20m:4.2mg Acer:diameter=30m:14.1mg

Theseveryhighvaluesforthecoefficientofdeterminationdidholdorevenincreasedwhenthelinearregressionwasdone usingthelogtransformeddataforboththedependentinindependentvariable.Thismeansthatthehighvaluesforthe nontransformeddatawerenotonlyduetothefactthemostdatapoints(exceptforthehighestconcentration)clustered neartheorigin.

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 Thesefiguresverywellmatchwithourestimation:4.3and67mgrespectively.Althoughour approachappearsreliable,itisjustthefirststep;moreresearchinthisdirectionhastobedone.

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Conclusions

Thisreportpresentedanoverviewofrelevantinformationconcerningtherelationshipbetween honeybeesandpollendispersal,basedonaliteraturesurvey,adatabaseofpollencompositionsof Dutchhoneysandaconciselaboratoryexperiment.Themainconclusionsofthereport,clustered pertopic,arethefollowing:

BeekeepinginTheNetherlands
BothhobbyistandprofessionalbeekeeperspracticebeekeepinginTheNetherlands.Ofthe approximately8,000Dutchbeekeepers,about6,900areorganisedinthreebeekeeping associations,ofwhichtheDutchBeekeepersAssociation(NederlandseBijenhoudersvereniging NBV)isbyfarthelargestwith6,000members. Hobbyistbeekeepershaveanaverageoffivecolonies,professionalbeekeeperswelloverone hundred. InurbanareasinTheNetherlands,moreandmorepeoplearetakingupbeekeeping.Beesare beingkepteverywherefromsmallsuburbanbackyardstohighriserooftopsandbalconies.Yet, thisgrouprepresentsasmallpartofthetotalnumberofbeekeepersinTheNetherlands. Aroundtenbeekeepingcompaniesareprofessionallyinvolvedinthepollinationofcropswith honeybeesandmakealivingoutofit.Overall,itisestimatedthatsomethinglike32,000honey beecoloniesareutilizedforpollinationinTheNetherlandsbyapproximately1,700beekeepers. Hobbyistbeekeepersseldommovetheirhives(exceptwhenhiredforpollinationinfruit orchards),whilstprofessionalsregularlymovetheirhives(every4weeksonaverage). InTheNetherlands,honeybeesarebeingutilizedinfruitorchards,inthehorticultureindustry andforseedproduction.Professionalbeekeepersorbeekeepingcompaniescovernearlyall pollinationinclosedgreenhousesandpolytunnels.However,regularcollaborationtakesplace withhobbyistbeekeepers,inordertoguaranteethesupplyofbeecolonies.Pollinationinthe fruitsectorismostlycoveredbynoncommercial,hobbyistbeekeepers. Theannualvalueofpollinationfeesisroughlyestimatedat4millionEuroforoutdoor cultivation,and7millionEuroforcultivationingreenhousesandpolytunnels.However,the indirecteconomicvalueorbenefitofpollinationbyhoneybeesandotherinsectpollinatorsis consideredtobemuchhigher. Honeybeesarenotonlyutilizedforpollination.Severalproductsthatareproducedbybeescan beharvestedbythebeekeeper.Obviously,themostrecognizableproductmadebyhoneybees ishoney.Otherproductsincludebeeswax,propolis,royaljelly,beepollenandbeevenom.Allof thesebeeproducts(could)contain(traceamountsof)pollen. MosthoneyontheDutchmarket(92%)isimportedfromamongstothersChinaandArgentina; thetwolargesthoneyproducingcountriesintheworld.

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Foragingandpollentransport
Honeybeesvisitflowersforpollen(theirprotein,fatandmineralsource)andnectar(energy source).Individualbeesspecializeanddonotforagefornectarandpollensimultaneouslyand devotetheirforagingtoonlyoneplantspeciesatatime. Foragingisnotonlyoptimizedattheindividualforagerlevel,butonthewholecolonylevel.This isachievedthroughcommunicationandtaskdivision. Onlyasmallpartofthepollenthatiscollectedonflowersisavailableforpollination:beesuse morethan99%asfood. Thedistanceuptowhichbeesforageforwater,pollenandnectarmayrangeupto2.5,10and 14kmrespectively.However,ifarichresourceisnearby,mostoftheforagingtripswillbemuch shorter(0.51km).Iffoodnearbythecolonyisscarceandveryrewardingpatchesarefurther away,largeflightdistancesarelikelytobecomefrequent. Insidethehiveofhoneybees,pollenisexchangedunintendedbetweenindividuals:thismay leadtopollentransportbetweendistantflowerpatchesthatarenotvisitedbythesame individualforager. Foroilseedrape,ithasbeenshownthata)beesmayflylongerdistancestoforageonsuch fields,andb)beestendtostayclosetotheirhiveifthehiveislocatednexttoanoilseedrape field. Appleisaveryattractivecropforhoneybeestoforageon.However,itisnotdocumentedwhich distanceshoneybeesflytoforageonapple,becauseitiscommonpracticetoplacecoloniesin (orverynear)appleorchardsinordertooptimizepollination. Honeybeesarenotattractedtothecroppotato,unlessaphidsfeedonthepotatoplants. However,farmersrarelytolerateaphidsthatareattractivetohoneybees,andevenifaphidsare present,aphiddensitiesoftenpeekbeforeflowering. Sugarbeetcropsarenotmeanttoflowerandarethereforenotattractivetohoneybees. Maize,althoughitisawindpollinatedplant,isalsovisitedbyhoneybees.However,nodatais availableaboutthedistancesbeescoverformaize.Inaddition,hardlyanyofthemaizepollen collectedmayresultinpollination,sincehoneybeesarenotatallinterestedinfemalemaize flowers. Thoughpotentialflightdistancesofhoneybeeshavebeenmeasuredandestimatedinanumber ofdetailedstudies,itisalmostimpossibletosetupageneralmodelthatpredictsactivityand pollinationpatternsforspecificcases,asthenumberofconditionsthatcaninfluencesuch patternsisalmostunlimited. Itshouldbekeptinmindthatbesideshoneybees,alsobumblebeesandmanyotherinsectsare involvedinpollentransport.Therefore,muchinformationonwhatthismeansatalandscape scaleisneededtoestimateitsimpactontheoutcrossingphenomena.

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Polleninhoney
Thepresenceofpolleninhoney Honeycontainssmallamountsofpollen.Thispollenhasenteredthehoneythroughthree pathways:directcontaminationofnectarattheflower,contaminationbybeesinsidethehive, andcontaminationbythebeekeeperduringhoneyharvestingandprocessing.Contaminationby thebeesduringprocessinginsidethehiveisthemostimportantrouteofpollenintohoney. Althoughthebotanicaloriginofthepollengrainsinhoneycanbeanindicationofthenectar sourceofthehoney,thisisnotstraightforwardduetothecontaminationpathsmentioned above.Blendingofcommercialhoneysmayalsobeanadditionalobstacletotraceability. About200honeysamples,fromasurveythroughoutthecountryinTheNetherlands(2008), carriedpollengrainsofatleast50plantfamilies,windpollinatedaswellasinsectpollinated species. Rosaceaepollen(includingapple),Brassicaceaepollen(includingoilseedrape)werevery abundant.Chenopodiaceae(towhichBetabelongs)andSolanaceae(towhichpotatoSolanum tuberosumbelongs)andPoaceae(grassfamily,includingmaize)werealsorepresentedinthe honeys.Maizepollenwasonlyencounteredtwice(outof200)andinverysmallquantities. Experimentalestimationonthepollencontentofhoneys Thenumbersofpollengrains,countedinfiveoftheDutchhoneysfromthesurvey, correspondedwellwiththosereportedinliterature,generallytoppingat10,000grainspergram honey. Thereliabilityofpollencountingdependsonthedilutionofthesuspensionusedincounting chambersandneedstobefurtherimproved. Astrongandsignificantrelationshipwasfoundbetweentheamountofpollen(grams)addedto anartificialhoney,andthecountednumberofgrainsinthehoney.Thehighlysignificant calibrationfactorswereusedtoestimaterealpollenconcentrations(g/g)ofrealhoney. Preliminaryresultsindicatethatonlytraceamountsofpollenarepresentinhoney,rangingfrom 43gto670gpollen/ghoney. Basedonthecalibrationcurves,oilseedrapehoneyandfruitblossomhoneywerecalculatedto containatleast60and58gofthespecificpollen(oilseedrape,appleandpear)pergramof honeyrespectively.

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Stone,D.(2005).AnIntroductiontoBeeBiology.UniversityLaboratoryHighSchool,Urbana, Illinois.PreparedfortheUIUCBeeSpaceProject. VanderHam,R.W.J.M.,J.P.Kaas,J.D.KerkvlietandA.Neve(1999).Pollenanalyse. Stuifmeelonderzoekvanhoningvoorimkers,scholenenlaboratoria.StichtingLandelijk ProefbedrijfvoorInsectenbestuivingenBijenhouderijAmbrosiushoeve.

Visscher,P.K.andT.D.Seeley(1982).Foragingstrategyofhoneybeecoloniesinatemperate deciduousforest.EcologicalSocietyofAmerica63:17901801. VonderOhe,W.,L.PersanoOddo,M.L.Piana,M.MorlotandP.Martin(2004).Harmonized methodsofmelissopalynology.Apidologie35:S18S25. Williams,I.H.(2001).BeemediatedpollenandgeneflowfromGMplants.Proc.8thPollination Symp.ActaHort.561. Winston,M.L.(1987).TheBiologyoftheHoneyBee.HarvardUniversityPress.Cambridge,MA. Zurbuchen,A.,L.Landertetal.(2010)."Maximumforagingrangesinsolitarybees:onlyfew individualshavethecapabilitytocoverlongforagingdistances."BiologicalConservation143(3): 669676.

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Listofinterviewedpersons

Boot,Willem.OwnerInbuzz,professionalbeekeepingcompany.8September2011. Dommerholt,Jan.ChairmanDutchBeekeepersAssociation(NBV).15July2011. Rietveld,Aat.VicechairmanDutchBeekeepersAssociation(NBV).15July2011. Vorstman,Jeroen.ManagerBijenhuis,DutchBeekeepersAssociation(NBV).15July2011.

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9
Annex1Pollendeterminationtables TableA1:listofpolleninspringhoneys,harvestedinMayJune2008 TableA2:listofpolleninsummerhoneys,harvestedinJulyAugust2008

Annexes

TableA3:listofpolleninautumnhoneys,harvestedinSeptemberOctober2008

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Annexpage0

Annex1.pollendeterminationtables
TableA1
Listofthespringhoneysfromthesurveyof2008.ThesehoneyswereharvestedinMayandJune. Thehoneysamplesarelistedwithanidentificationnumber.Ofeachhoneyabout500pollen grainsweredetermineduptofamilyleveltheexactnumberdeterminedpersampleisincolumn 2(Totaalaantalpollen).Thepollenfamiliesarelistedinthetopheaderofthetable. :thispollenwasoverrepresented,andnotincludedinthedeterminationofthe500grains. ThecolumnswiththepollenofBrassicaceae,Rosaceae,SolanaceaeandChenopodiaceaehave beenmarkedyellow. Thehoneysusedforthepollencounting(Chapter5)havebeenmarkedasbluecolouredrows. Gemiddeldeninthelowestrowmeans:average.Thisaverageisofthepositivesamplesonly (zerosarenotincluded).

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Annexpage1

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Annexpage2

38 41 42 528 543 511 520 500 504 511 523 523 509 508 503 569 526 539 532 565 T o t a a l a a n t a l p o lle n A c e ra c e a e 1 41 8 1 1 1 4 7 1 3 9

47 48 55 57 58 60 66 77 78 80 82 84 86 88 90 2 3 5 6 8 12 13 21 24 26 28 30 33 35 531 525 551 502 507 543 534 515 542 545 548 543 503 583 543 2 6 9 11 1 5 7 4 2 2 1 4 1 A p ic a c e a e A q u if o lia c e a e - h u ls t (ile x ) 12 24 5 1 4 15 2 2 2 3 2 3 5 5 1 2 1 2 3 1 2 2 2 6 5 5 12 10 43 5 1 2 5 7 1 3 24 4 2 6 1 1 1 1 5 B e t u la c e a e B o ra g in a c e a e B o ra g in a c e a e - v e rg e e t -m e -n ie t je B ra s s ic a c e a e C a m p a n u la c e a e 6 4 1 ? 127 2 26 2 21 421 278 26 62 30 430 319 340 169 333 156 215 63 A s t e ra c e a e B a ls a m in a c e a e B e rb e rid a c e a e - b e rb e ris

ameco|bees@wur
H o n in g m o n s t e rn u m m e r

100 401 349

325 40 286 57 10 432 402 5 199 219 383 14 28 159 1 4

17

1 4 1

2 1 2 5 2 1 2 1 10 13 10 1 2 41 57 54 1 9 12 82 75 5 48 9 10 10 14 22 32 6 51 2 5 10 9 2 1 1 1 19 6 3 8 1 116 5 345 8 1 3 38 33 6 2 1 4 1 8 21 4 4 1 1 15 3 3 1 5 1 1 5 1 5 1 2 8 1 7 21 15 10 107 12 3 1 1 7 2 31 4 26 299 3 40 25 4 1 13 405 336 36 2 3 43 25 5 27 15 6 11 105 1 7 1 3 1 20 161 21 4 6 1 5 30 86 3 30 136 3 14 4 1 1 1 1 1 1 35 2 7 7

C a p rif o lia c e a e C a ry o p h y lla c e a e C e la s t ra c e a e - k a rd in a a ls m u t s C h e n o p o d ia c e a e C is t a c e a e C u p re s s a c e a e - J u n ip e ru s c o m m u n is E la e a g n a c e a e E ric a c e a e Fabac eae Fagac eae F a g a c e a e - e ik F a g a c e a e - C a s t a n a s a t iv a G ro s s u la ria c e a e H ip p o c a s t a n e a c e a e H y a c in t a c e a e H y d ro p h y lla c e a e - p h a c e lia J unc ac eae L a m ia c e a e

Annexpage3

1 3

1 3

L ilia c e a e L o ra n t h a c e a e L y t h ra c e a e M a lv a c e a e M e n y a n th a c e a e - N y m p h a e a O le a c e a e 2 1 4 1 8 2 2 5 10 8 1 1 2 48 2 7 6 3 2 1 7 136 55 1 61 139 57 13 272 7 111 6 414 62 4 17 18 19 4 38 33 46 33 7 1 205 56 35 7 3 9 137 18 191 163 56 87 52 68 148 22 23 49 11 116 70 3 341 67 65 2 250 9 97 4 93 16 3 129 47 17 360 6 1 22 7 19 43 28 23 9 3 4 101 6 138 140 170 29 29 9 216 136 83 12 1 1 231 6 96 1 4 77 62 2 3 2 5 1 5 1 8 1 2 3 7 10 1 4 11

O le a c e a e -lig u s t e r v u lg a ris O n a g ra c e a e P a p a v e ra c e a P in e a c e a e 1 17 1 1 P la n t a g in a c e a e 2 3 1 1 P oac eae P o le m o n ia c e a e - G ilia c a p it a t a P o ly g o n a c e a e P o ly g o n a c e a e - ru m e x , z u rin g P rim u la c e a e R a n u n c u la c e a e R h a m n a c e a e R h a m n u s f ra n g u la Ros ac eae R u b ia c e a e -G a lliu m R utac eae

S a lic a c e a e S a x if ra g a S im a ro u b a c e a e - h e m e lb o o m S o la n a c e a e T a x u s b a c c a ta T ilia c e a e - lin d e V it a c e a e onbek end

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Annexpage4

C upres s ac eae - J uniperus c om m unis E laeagnac eae

B oraginac eae - v ergeet-m e-nietje

R ham nac eae R ham nus frangula

P oac eae P olem oniac eae - G ilia c apitata

C elas trac eae - k ardinaals m uts

P oly gonac eae - rum ex , z uring P rim ulac eae

91 94 98 101 105 108 110 114 115 117 118 119 120 121 123 128 129 130 137 142 144 146 149 156 159 166 172 174 177 181 185 186 189 194 197 202 Gemiddelden

0 533 624 580 502 507 545 503 520 564 519 533 523 526 502 555 502 651 506 536 609 640 554 531 581 554 519 548 560 539 505 512 501 501 517 564 528

11

5 9 5

5 12 2 3 4 3 17 1 9 1 9 13 94 15 3 3 14 9 1 164

296 412

1 1 6 6 1

351 208 17 39 1 331 7 72 51 453 70 62 11 36 19 282 27 168 212 36 38 36 19 3 250 18 426 280 31 9 33 107 47 18 87 151 3 6 1

12

3 19 145 13 13 15 111 21 114 9 5 50 300 73 1 13 21 80 40 3 1 19 48 38 1 85 26

4 206 4 12 1 256 7 37 1 12 5 3 9 177 1 11 38 2 5 10 1 1 5 17 25 6 2 1 17 7 47 8 17 1 17 9 7 13 17 11 3 7 12 73 12 2 24

11 8 22 13 1 75 28 4 29 60 7 3 22 1 2 40 33 5 5 28 86 38 16 3 5 15 8 212 1 6 42 27

1 2 5 15 1 6 3 3 1 1 1 1 1 410 27 1 1 21 3 7

1 3 1 3 3 3

7 1 3 3 3 10

1 18 3 23 9 7

7 1 3

90 212 79 65 7 140 14 13 87 72 161 32 64

67 108 30 9 29 7 3 45 67 5 249 100 30 11 13 10 68 1 90 54 8 87 243 198 98 175 7 7

56

276

1 3 3 9 10 7

4 1 10 1 7 5 1 3 4

43 1 2 1 5 3 5 1 5 24 3 1 5 5 3 3 1 3 3 3 11

3 18 8

1 1 38 16 19 3 1 1 5 1 9 1 23 3 1 10 9 453 168 84 34 4

1 9 5

5 3 1

13 1 89

3 20 23 1 3 1 3 7 7 3 9 5 8 7 5 5 6

1 1

1 215

1 7 5 3 5 7 1 17 3 11 1 3 1 2 3

2 5

10 3 14 1 1

380 18 13 1 17 1

13 164 336 7 54 34 88 119 14 22 66 10 305 11 208 33 1 50 11 102 1 227 61 39 2 16 1 13 18

62 6 1 98 226 56

1 4

3 1 15 9 11 75 17 41

1 1

19 19 3 64 3 1 10 1 1 13 1 1 5 19 15 4 8 13 7

1 131 232 164

3 1 1 1 2 2 1

15 75 3 7

26 17 99 145 13 15 150 59 46 15 65 5 54 17 103 1 73 9 5 3 115 121 74

2 3 10 5 135

6 27 2 56 10 4 19 1 3 1 1 28

16

4 77

18 104 70 7 69 32 8 7 1 12 35 8

ameco|bees@wur

Annexpage5

S alic ac eae S ax ifraga S im aroubac eae - hem elboom S olanac eae T ax us bac c ata T iliac eae - linde V itac eae onbek end 5 2 5 1 3 13 1 7 3 1 30 27 5 10 12 3 1 1 5 1 11 7 3 14 6 44 1 1 9 11 2 11 12 1 1 1 13 73 1 1 3 4 1 3 1 4 1 2 84 6 59 5 3 3 1 2 2 24 1 6 12 1 61 7 9 7 22 3 1 7 9

M eny anthac eae - N y m phaea

H y drophy llac eae - phac elia

F agac eae - C as tana s ativ a

O leac eae -ligus ter v ulgaris

A quifoliac eae - huls t (ilex )

B erberidac eae - berberis

H oningm ons ternum m er

H ippoc as taneac eae

T otaal aantal pollen

R ubiac eae -G allium R utac eae

C ary ophy llac eae

C henopodiac eae

G ros s ulariac eae

B ras s ic ac eae C am panulac eae

P lantaginac eae

R anunc ulac eae

B als am inac eae

F agac eae - eik

C aprifoliac eae

P oly gonac eae

B oraginac eae

H y ac intac eae

Liliac eae Loranthac eae

P apav erac ea

O nagrac eae

A s terac eae

B etulac eae

Ly thrac eae

J unc ac eae

Lam iac eae

M alv ac eae

A c erac eae

P ineac eae

A pic ac eae

R os ac eae

C is tac eae

E ric ac eae

F abac eae

F agac eae

O leac eae

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Annexpage6

TableA2
Listofthesummerhoneysfromthesurveyof2008.ThesehoneyswereharvestedinJulyand August. Thehoneysamplesarelistedwithanidentificationnumber.Ofeachhoney,about500pollen grainsweredetermineduptofamilylevel.Theexactnumberdeterminedpersampleisin column2(Totaalaantalpollen).Thepollenfamiliesarelistedinthetopheaderofthetable. :thispollenwasoverrepresented,andnotincludedinthedeterminationofthe500grains. ThecolumnswiththepollenofBrassicaceae,Rosaceae,SolanaceaeandChenopodiaceaehave beenmarkedyellow.Sample27and56showayellowmarkedcellforPoaceae:thesepollen grainsarefrommaize(Zeamays). Thehoneysusedforthepollencounting(Chapter5)havebeenmarkedasbluecolouredrows. Gemiddeldeninthelowestrowmeans:average.Thisaverageisofthepositivesamplesonly (zerosarenotincluded).

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Annexpage7

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Annexpage8

1 4 7 10 11 22 23 25 27 29 31 32 36 565 505 503 573 528 524 548 517 643 509 468 542 573 T o ta al aa ntal po lle n

ameco|bees@wur
39 40 44 45 54 56 61 67 71 75 76 83 85 89 92 93 95 99 100 102 103 104 106 107 109 H on ing m on s ternu m m er 7 5 A c e ra c e ae 5 101 1 1 A pic ac ea e A qu ifo liac e ae - hu ls t (ile x ) 2 7 11 1 11 10 42 1 5 3 1 A s terac ea e B als am in ac ea e B erbe rid ac ea e - be rb eris 16 1 3 8 3 6 1 36 25 7 6 1 3 7 3 63 1 3 5 2 B etula c e ae B orag ina c e ae B orag ina c e ae - v erge et-m e -n ietje 65 28 13 26 7 5 48 82 55 421 85 14 6 6 B ra s s ic a c e ae C am p an ula c e ae C ap rifolia c e ae C ary o ph y lla c e a e C ela s tra c e ae - k ardin aa ls m u ts 8 4 1 4 2 1 1 2 14 2 1 21 4 6 6 18 1 1 1 1 30 1 11 13 1 7 1 3 5 2 375 1 2 1 503 548 514 516 506 519 503 534 508 503 701 531 550 687 590 509 503 548 500 614 549 501 529 504 533 1

378 27 11 43 16 9 17 36 32 13 100 28 82 10 22 18 15 12 13 15 1 1 13 4 30 1 1 5 4 1 17 2 2 2 2 1 9 3 7 1 1 1 1 155 144 378 102 400 272 27 400 72 64 11 14 202 88 71 54 128 241 50 21 125 134 111 13 3 2 1 305 343 24 2 2 20 7 21 7 4 7 17 1 1 1 7 293 6 2 1 3 19 18 15 5 20 1 5 18 3 4 31 9 1 1 8 6 3 3 7 2 57 18 8 1 5 7 1 1 1 1 1 2 6 6 2 3 1 3 4 3 5 8 1 3 4 6 7 1 3 5 3 10 24 1 1 24 1 65 10 11 1 35 33 9 9 7 73 5 12 5 32 13 5 9 7 1 2 1 5 12 2 1 1 22 1 179 222 30 18 25 31 19 10 14 7 101 37 6 8 9 118 5 11 57 7 5 20 11 17 8 4 6 6 16 10 17 5 30 10 6 22 2 1 23 5 1 2 2 18 4 286 188 4 37 182 13 44 13 23 18 10 15 1 7 3 16 2 7 1 19 1 1 1 4 1 4 27 1 5 3 8 12 9 17 2 20 11 25 10 6 3 5 7 7 1 17

41 114 403 108 342 459 270 264 17 71 213 185 74 3 75 64 1 3 1 355 256 160 1 147 300 268 394 4 17

C he no po dia c e a e C is tac ea e E ric ac ea e E uc aly ptus

56 52 35 14 122 15 36 73 228 3 11 255 267 1 266 357 420 5 371 38 3 8 7 4 11 14 6 1 6 3 2 2 1

F a ba c e ae F a ga c e ae F a ga c e ae - e ik F a ga c e ae - C as ta na s ativ a H ipp oc as ta ne ac e ae H y d ro ph y lla c e a e - p ha c elia Irid ac ea e L am iac ea e L ilia c e ae L is ta c e ae L oran th ac e ae L y thrac ea e M a lv ac ea e O le ac ea e 1 8 5 11 9 30 33 9 O le ac ea e -lig us te r v u lga ris O n ag ra c e ae 3 P ine ac ea e P lan ta gin ac ea e 2 P oa c e ae P ole m on iac ea e - G ilia c a pitata 126 P oly go na c e ae P oly go na c e ae - rum e x , z uring R an un c u lac ea e R ha m na c e ae R ha m nu s fran gu la 11 119 2 8 1 12 18 263 1 41 7 28 2 24 83 72 21 8 58 44 3 251

Annexpage9
3

15 125 4 41 4 83 2 72 180 2 35 2 97 4 85 10 1 15 17 5 10 18 9 24 25 5 15

R os ac ea e R ub iac ea e -G allium R utac ea e 1 158 47 21 47 13 83 36 200 6 14 6 1 3 3 11 9 3 1 3 12 38 S alic ac ea e S ax ifrag a S im arou ba c e ae - h em e lbo om S ola na c e ae T a x u s ba c c ata T iliac ea e - lind e V iola c e ae o nb e k e nd d iv e rs en

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Annexpage10

B o ra g in a c ea e - v erg e e t-m e -n ietje

R h a m na c e ae R h a m nu s fra ng u la

P o le m o n iac e a e - G ilia c ap ita ta

C e la s tra c e ae - k a rdin a als m uts

P o ly go n a c e a e - rum e x , z u rin g

S im a rou b a c e a e - he m e lb o o m

F a g ac e a e - C as ta n a s a tiv a

H y d ro ph y lla c e ae - p h ac e lia

O le a c ea e -lig u s ter v u lga ris

A q u ifolia c e a e - hu ls t (ile x )

B e rb e rid a c ea e - b erb e ris

H o n ing m o ns te rn um m e r

H ip p oc a s tan e a c e a e

T o ta al a a nta l p olle n

R u b iac e a e -G a lliu m

o n be k e nd d iv e rs e n

C a ry o ph y lla c e ae

C h e n op o d ia c e ae

C a m p an u lac e a e

T ilia c e ae - lind e

P la n tag in ac e a e

R a n u nc u lac e a e

B a ls am in ac e a e

T a x us b a c c a ta

F a g ac e a e - e ik

C a p rifolia c e a e

P o ly go n a c e a e

B ra s s ic a c ea e

B o ra g in a c ea e

L o ran th a c e a e

O na g ra c e a e

S o la na c e ae

111 112 113 116 122 124 125 127 132 133 135 138 140 143 145 147 151 152

516 566 502 536 518 539 535 520 540 42 515 529 4 543 534 509 525 532 540 352 6

5 33 220 11 5 3 1 1

3 3 45 141 5

2 3 8

1 3 9 3 1 111 3 143 1

61 38 7 13 60 31 23

56

335 152 3 396 188 21 50 9 66 1

40 159 6 14

1 16 5 2 3 7 1 1 1 1 3 17 1 3 10 3 48 3 1 1 3 5 1 1 1 1 1 9 20 5 2 13 7 5 1 1 3 1 1 1 1 1 3 10 3 1 8 7 7 13 1 3 3 13 4 1 1 1 81

69 1 1 15 1

1 17

21 3 7 3 3 23 128 134 3 1 381 63 129 3 15 256 12 295 16 1 11 7 19 32 11 76 13 45 11 132 12 131 1 47 9 253 3 5 45 3 7 73 5 15 164 37 7 70 1 174 3 180 6 39 75 42 27 4 7 168 1 9 52 214 12 78 25

14 7 1 89 53 51 78 5 21 11 137 78 8 6 7 21 1 47 76 195 11 102 45 88 5 46 64 27 9 3 56 47 13 27 67 51 42

14 4 1

12 180 2 96 16

125 13 1 7 13 9 1 1 27 1 3 1 1 1 21 1 14 7 8 1 1 7 6 4 23 6 1 23 9 7 11 1 11 3 1 9 1

7 zomer, aug zomer, juli zomer, juli 1 zomer, juli 3 zomer, aug 4 zomer, juli 3 zomer, aug 4 zomer, aug 33 11 2 5 2 3 14 3 71 68

105

8 6 3 1 8 2 8 3

1 1 24 3 3

1 3 1 3 11 10 1 7 2 1 3 3 11 5 1 9 7 7 3 5 1 1

237 1 15 15

33 110 15 47 75 1 29 5 39 17 40 23

3 3

11 3 1

3 1 30

25 54 40 132 2 16 1 26 11 170 66

17 5 7 5 3

19 160 111 8 8 80 7 424 263 158 3 403 1 6 45 19 13 31

3 12

5 44 1

3 1 1 1 1 1 1 5

21 1 27 28

5 6 1 15 1 1 1

zomer zomer zomer zomer, juli zomer zomer zomer, juli zomer, juli

153 521 3 154 563 1 155 502 158 534 160 541 161 534 162 538 170 505 171 562 173 561 190 583 1 192 558 195 500 200 509 201 514 203 559 204 519 1 205 523 1 206 503 207 514 ameco|bees@wur 208 539 Gemiddelden 7 533

231 12 85 148 82 9 62 13 120 190 33 7 102 150 15 16 11 46 5 79 25 47

13 16 14 1 15

3 9 3 7 3 5 5 39 3

61

1 5 7

1 3 5 1

96 36

3 1 5 11 1 8 5 469 5 27 1 470 74 1 7 130 3 7 1

1 3 92

1 4 13

5 1 7

17 178 6 3 11 7 193 243 29 133 91 108 5 73 3 143 24 40 1 117 5 99 80 42 11 10 215 12 11 39 106 3 18 20 91 138

21

12 2 8 1 13 16 19 3 13 3 5 9 6

21 20 6 144 21 19 1 39 54 43 1 162 29 5 3 32 5 11 11 72 30 194 5 1 11 47 1 433 440 7 1 11 14 26 7 1

26

1 8

3 1 18

17

75 10 1

6 3 1 9

9 5 3 10

8 2 6 27

1 1 5 2

5 1 7

1 1 3 2 2 3 3

4 7 3 13 22 7 8

1 90 4 63 25 162

1 2 6 2 7 14 12 10

Annexpage11
20 45

14

7 7 18

9 zomer, juli zomer, juli 1 zomer, juli 64 zomer 1 zomer, aug 15 zomer, juli zomer, juli 9 zomer 4 zomer, juli 8 zomer, juli 8 zomer 7 zomer, aug 1 11 zomer 3 zomer, aug zomer, juli 1 zomer, juli 2 zomer, aug zomer, juli zomer, aug 34 zomer, juli 30 zomer, aug 15 12

h o nin g s o o rt

A s te ra c e a e

B e tu lac e a e

E u c a ly p tu s

L y thra c e ae

S a lic a c e ae

M a lv a c ea e

L a m ia c ea e

A c e ra c e a e

A p ic ac e a e

P in e ac e a e

C is ta c e a e

E ric a c e a e

Ros ac eae

V io lac e a e

F a b ac e a e

F a g ac e a e

L is ta c e ae

O le a c ea e

S a x ifrag a

R u ta c ea e

Irid ac e a e

P o a c ea e

L ilia c ea e

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Annexpage12

TableA3
Listoftheautumnhoneysfromthesurveyof2008.ThesehoneyswereharvestedinSeptember October. Thehoneysamplesarelistedwithanidentificationnumber.Ofeachhoneyabout500pollen grainsweredetermineduptofamilyleveltheexactnumberdeterminedpersampleisincolumn 2(Totaalaantalpollen).Thepollenfamiliesarelistedinthetopheaderofthetable. :thispollenwasoverrepresented,andnotincludedinthedeterminationofthe500grains. ThecolumnswiththepollenofBrassicaceae,Rosaceae,SolanaceaeandChenopodiaceaehave beenmarkedyellow. Gemiddeldeninthelowestrowmeans:average.Thisaverageisofthepositivesamplesonly (zerosarenotincluded).

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Annexpage13

ameco|bees@wur

Annexpage14


9 34 37 43 52 69 70 72 73 74 79 81 87 136 148 150 Gemiddelden H o n in g m o n s t e rn u m m e r

ameco|bees@wur

528 503 T o t a a l a a n t a l p o lle n A c e ra c e a e A p ic a c e a e A s te r a c e a e B a ls a m in a c e a e B e t u la c e a e 3 9 251 1 34 20 1 B ra s s ic a c e a e C a m p a n u la c e a e C a p rif o lia c e a e C a ry o p h y lla c e a e C a rn a c e a e C is t a c e a e E ric a c e a e 15 105 Fabaceae F a g a c e a e - e ik 193

B o ra g in a c e a e - v e rg e e t -m e -n ie t je

79 2 1 56 5 2 44 5 225 1 569 1 40 3 2 518 35 1 87 35 235 69 4 1 531 437 2 2 5 49 28 536 1 243 17 18 139 1 61 533 10 15 7 128 126 1 1 1 559 60 35 102 4 52 305 537 8 57 1 62 170 33 23 48 10 517 1 419 7 56 5 3 2 635 1 8 11 48 3 41 6 52 26 229 549 1 1 41 365 68 12 555 18 6 59 23 240 161 3 6 11 500 3 462 7 13 7 1 503 14 25 2 2 8 86 2 4 230 19 516 7 3 2 4 10 3 124 72 1 1 242 1 7 537 1 15 68 121 5 31 43 6 2 1 3 35 149 108 1 69 3 10 76 3 54 43 5 1 225 61 7 1

Annexpage15

F a g a c e a e - C a s t a n a s a t iv a G ro s s u la ria c e a e 3 10 1 8 2 6 H ip p o c a s t a n e a c e a e H y d ro p h y lla c e a e - p h a c e lia I rid a c e a e L a m ia c e a e L ilia c e a e L y t h ra c e a e M a lv a c e a e M y rt a c e a e O le a c e a e

2 2 8 4 1 19 6 24 21 5 7 26 3 11 10 5 1 6 6 2 5 17 8 3 13 68 2 20 9 4 7 7 4 6 14 1 1 2

1 3 1

1 2

3 9 5 87 3 2 3 191 58 13 27 5 8 1 7

O le a c e a e -lig u s t e r v u lg a ris P in e a c e a e P la n t a g in a c e a e P oaceae 6 17 3 1 1 8 3 320 21 2 3 1 1 8 63 9 28 23 3 2 1 2 11 17 24 70 139 28 P o le m o n ia c e a e - G ilia c a p it a t a P o ly g o n a c e a e P o ly g o n a c e a e - ru m e x , z u rin g R h a m n a c e a e R h a m n u s f ra n g u la Rosac eae R u ta c e a e S a lic a c e a e S a x if ra g a S im a ro u b a c e a e - h e m e lb o o m S o la n a c e a e

T ilia c e a e - lin d e onbekend

ameco|bees@wur

Annexpage16