Initial Exposure of Wax Foundation to Agrochemicals Causes Negligible Effects on the Growth and

13 Pages·2017·1.07 MB·English

insects
Article
Initial Exposure of Wax Foundation to Agrochemicals
Causes Negligible Effects on the Growth and Winter
Survival of Incipient Honey Bee
(Apis mellifera) Colonies
AlexandriaN.Payne†,ElizabethM.Walsh† andJulianaRangel*
DepartmentofEntomology,TexasA&MUniversity,2475TAMU,CollegeStation,TX77843-2475,USA;
[email protected](A.N.P.);[email protected](E.M.W.)
* Correspondence:[email protected];Tel.:[phone]
† PayneandWalshareco-firstauthors.
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:15July2018;Accepted:29August2018;Published:8January2019
Abstract: WidespreaduseofagrochemicalsintheU.S.hasledtonearlyuniversalcontamination
of beeswax in honey bee hives. The most commonly found agrochemicals in wax include
beekeeper-appliedmiticidescontainingtau-fluvalinate,coumaphos,oramitraz,andfield-applied
pesticides containing chlorothalonil or chlorpyrifos. Wax contaminated with these pesticides
negativelyaffectsthereproductivequalityofqueensanddrones. However,thesynergisticeffectsof
thesepesticidesonthegrowthandsurvivalofincipientcoloniesremainunderstudied.Weestablished
new colonies using frames with wax foundation that was pesticide free or contaminated with
field-relevantconcentrationsofamitrazalone,acombinationoftau-fluvalinateandcoumaphos,ora
combinationofchlorothalonilandchlorpyrifos. Colonygrowthwasassessedbyestimatingcomb
andbroodproduction,foodstorage,andadultbeepopulationduringacolony’sfirstseason. Wealso
measuredcolonyoverwinteringsurvival. Wefoundnosignificantdifferencesincolonygrowthor
survivorship between colonies established on pesticide-free vs. pesticide-laden wax foundation.
However,coloniesthathadVarroadestructorlevelsabove3%inthefallweremorelikelytodieover
winterthanthosewithlevelsbelowthisthreshold,indicatingthathighVarroainfestationinthefall
playedamoreimportantrolethaninitialpesticideexposureofwaxfoundationinthewintersurvival
ofnewlyestablishedcolonies.
Keywords: agrochemicals; Apis mellifera; honey bees; beeswax; colony growth; pesticides;
toxicology;miticides
1. Introduction
Honeybees(Apismellifera)provideover$15billionannuallytotheU.S.economybypollinating
approximately35%ofcommerciallygrowncropsthatdependdirectlyontheirpollinationservicesto
produceeconomicallyviableyields[1]. Becauseofpopulouscoloniesandeaseoftransportation,honey
beesarepreferredamongotherinsectspeciesforcommercialcroppollination[2–5]. Butdespitetheir
importancetoouragroecosystems,managedhoneybeepopulationscontinuetodeclineduetostresses
mainlycausedbypoornutrition,pestsandpathogens,queenfailure,andpesticideexposure[6,7].
In2012,approximately915millionacresofcommercialcroplandand88millionhouseholdsinthe
U.S.weretreatedwithpesticides[8]. Despitetheirpervasivepresenceintheenvironment,pesticide
riskassessmentsforinsectpollinatorsareprimarilybasedonapesticide’sacutelethalitytohoneybees
andthuspresentaninaccuratepictureofpollinator–pesticideinteractions. Theseassessmentsdonot
takeintoaccounttheeffectsofpesticidesonnativeandnon-honeybeepollinators,andtheydonot
Insects2019,10,19;doi:10.3390/insects10010019 www.mdpi.com/journal/insects
Insects2019,10,19 2of13
considersub-lethaleffectsorsynergisticandmulti-modalroutesofexposuretohoneybeeswhena
pesticide’sregistrationoritsrenewalisprocessed. Duetothecloseassociationbetweencommercial
cropsandhoneybees,aswellashighpesticideuseinagriculturalcroplands,theeffectsofpesticide
exposureonhoneybeehealtharelegitimatecausesforactiveresearch[9,10].
Problematicpesticidesforhoneybeehealthcangenerallybesplitintotwocategories: thosethat
are farmer applied to control agricultural pests, which are inadvertently brought into the hive by
exposedforagers,orthosethatareintentionallyintroducedtocoloniesbybeekeeperstocontrolthe
ectoparasiticmiteVarroadestructor. ThemiticidesmostwidelyusedforVarroacontrolinthelastthree
decadeshavehadasactiveingredientsthepyrethroidtau-fluvalinate(activeingredientinApistan®),
theorganophosphatecoumaphos(activeingredientinCheckmite+®),ortheformamidineamitraz
(active ingredient in Apivar®). Studies have shown that the majority of honey bee colonies in the
U.S.harborbeeswaxcontainingalarminglevelsofthesemiticides[11,12]. Mullinetal.[12]sampled
coloniesfromcommercialbeekeepingoperationsacrosstheU.S.andfound87differentpesticides
andtheirmetabolitesin259waxsamplesanalyzed. Themostcontaminatedwaxsamplecontained
39 pesticides and their metabolites. On average, each wax sample contained residues from eight
pesticides,withhalf(49.9%)ofallsamplescontainingoneormoresystemicinsecticides. Inparticular,
eventhoughthemiticidestau-fluvalinateandcoumaphosarepracticallynotusedanymoreforVarroa
controlinmostoftheU.S.[12],theywerepervasiveinwaxandwerefoundtogetherin77.7%ofallthe
bee,pollen,andwaxsamplesanalyzed. Therefore,wedecidedtotesttheeffectsoncolonygrowth
ofexposureofthewaxcombtothesetwomiticidescombinedintoonetreatment. Amitrazandits
metabolites,2,4-dimethylphenylformamide(DMPF)and2,4-dimethylaniline(DMA),wasthethird
most commonly found miticides found in wax, so we decided to test the effects of wax exposure
to amitraz (alone) on colony growth. The list of the five most ubiquitous pesticides found in wax
alsocontainedthefungicidechlorothalonilandtheorganophosphatechlorpyrifosasthetwomost
commonlyfoundagrochemicals. Wethereforedecidedtotesttheeffectsoncolonygrowthofcomb
exposuretoacombinationofthesetwonon-miticideproducts. Thehighprevalenceoftau-fluvalinate,
coumaphos, amitraz, chlorothalonil, andchlorpyrifoshasbeenconfirmedinmorerecentyearsby
anotherstudyconductedwiththreemigratorybeekeepingoperationsintheEasternU.S.[11].
Previousstudieshavelookedattheeffectsofacutedosesofpesticidesonimmatureandadult
honeybeesattheindividuallevel. Someofthesestudiesarecontroversial,however,becausethey
have examined bees exposed to unrealistically high pesticide dosages—sometimes multiple folds
higherthanpesticideexposurefoundinthefield[13–15]. Furthermore,veryfewofthesestudieshave
lookedattheoveralleffectsofpesticideexposureatthecolonylevel. Morerecently,researchershave
attemptedtomeasurethe“exposome”,oracolony’stotalexposuretopesticides,throughroutesthat
varydependingontheenvironmentinwhichcoloniesaremanaged[10,11].
In this study, we explored whether pesticide residues in wax at field-relevant concentrations
affectthegrowthandsurvivalofnewlyestablishedcolonies,usingthehighestconcentrationsofthese
pesticidesinwaxsamplesfoundinthefield[12]. Wedidsobycoatingframesofplasticfoundation
with pesticide-free beeswax and then spraying them with a pesticide-free diluent, or with diluent
containing field-relevant doses of amitraz, tau-fluvalinate and coumaphos, or chlorothalonil and
chlorpyrifos. Wecomparedtheamountofcombbuilt,broodproduced,andfoodstored,aswellasthe
adultpopulationandseasonalcolonysurvivalofcoloniesestablishedonpesticide-freewaxfoundation
comparedtothoseestablishedonpesticide-ladenwaxfoundation. Determiningwhetherpesticide
residuesinwaxfoundationaffectthehealthofincipientcolonieswillprovidevaluableinformationto
beekeepersinorderforthemtobettermanagehealthierandmoreproductivehives.
2. MaterialsandMethods
2.1. StudySiteandColonyEstablishment
ThisstudywasconductedatanapiarylocatedinBryan,TX,USA(30.66290012N,−96.47334016E)
from May 2017 to May 2018. All colonies were established following a previously described
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protocol[16,17]. Atotalof30packagesconsistingof3lbsofworkerbees,orapproximately10,476
individuals[18],werecreatedfromlarger,unrelatedsourcecoloniesprovidedbyETzzzBzzzApiaryof
CollegeStation,TX,USA.Eachpackageofbeeswasshakenintoastandardpackagebox(15cm×25cm
× 35 cm), as described by Seeley and Visscher [19], along with a mated and caged Italian queen.
AllqueenswerereceivedinMay2017fromOlivarezHoneyBees,Inc.(Orland,CA,USA).Thepackages
werefedadlibitumwitha50:50w/vsucrosesolution,allowingthebeestoreachthenaturalinclination
ofwaxproductionfornewcolonyestablishment[20]. Threedayslater,eachpackagewasinstalled
byshakingthebeesintoafive-frame“nucleus”hivealongwiththecagedqueen. Thiswasdoneon
18May2017,whichmarkedthecolonyestablishmentdateandthefirstdayofthestudy. Eachnucleus
hivewascomprisedoffiveframesofalternatingfullorpartialPlasticellfoundation(PiercoBeekeeping
Equipment®, Riverside, CA, USA) coated with a layer of molten beeswax (see Section 2.2 below).
Frames with full foundation allowed bees to construct only worker comb (and consequently the
productionofonlyworkerbrood),whileframeswithpartialfoundationallowedbeestobuildeither
workerordronecombdependingonthecolony’sneeds. Thecagedqueenswerereleasedfromtheir
cagestwodaysafterestablishingthehivesonceitwasevidenttheyhadbeenacceptedbytheirworkers.
2.2. PesticideTreatmentofWaxFoundation
EachPlasticellfoundationframewascoatedwithalayerofpesticide-free,cosmeticgradebeeswax
pellets(KosterKeunenInc.,Watertown,CT,USA)previouslymeltedinawaterbath. Oncethemolten
wax layer had dried, a frame was sprayed using separate all-purpose sprayers containing either
acetoneonly(controlgroup)oracetonemixedwithpesticidesatthehighconcentrationsfoundinwax
samplesbyMullinetal.[12]. Becauseweusedthehighestconcentrationspreviouslyfoundinwax
samples[12],ourstudyrepresentsa“worstcase”scenarioofpesticideexposureofbeeswaxfoundation
anddoesnotaccountforotherroutesofadditionalpesticideexposure. Framesinthetreatmentgroups
weresprayedwitheither(1)4.3mgamitraz(“amitraz”group),(2)amixof20.4mgfluvalinateand
9.2 mg coumaphos (“F+C” group), or (3) a mix of 5.4 mg chlorothalonil and 0.09 mg chlorpyrifos
(“C+C”group),witheachpesticidegroupdissolvedin100mLofacetone. Fullandpartialfoundation
framesweresprayedonbothsideswithatotalof10mLor5mLofsolution,respectively.
2.3. ColonyGrowthMeasurements
Wefollowedthegrowthofnewcoloniesovertimebymeasuringanumberofvariablesfrom
18 May (day 0) to 17 October 2017 (day 146 after colony establishment). These included the total
combinedamountofnewlyconstructedworkeranddronecomb,thetotalcombinedamountofsealed
workeranddronebroodproduced,thetotalcombinedamountofhoney,nectar,andpollenstored
(food storage), and the estimated adult bee population. We used a gridded, wooden frame that
consistedof136separate1in×1insquarestocountthetotalareaoccupiedbyeachvariableonboth
sidesofeachframe, asdescribedpreviously[16,21]. Wealsocountedthenumberofadultbeesin
20 uniformly spaced 1 in ×1 in squares on both sides of each frame and used this to estimate the
totaladultpopulationsizeofeachcolony,asdonepreviously[16,21]. Allvariablesweremeasured
everythreeweeksforatotalofsevensamplingperiodsaftertheestablishmentdateexceptforadult
populationsize,whichwasestimatedeverysixweeksforatotaloffoursamplingperiods. Additional
framesweresprayedandaddedthroughouttheexperimenttoanyhiveinwhichcombproduction
extendedtoallexistingframestoensurethateachcolonyalwayshadspacetoexpand.
Inaddition,weperformedVarroamitecountsonsurvivingcoloniesinSeptember2017andagain
in May 2018 using the standard powder sugar shake method [22,23]. Briefly, one half cup of bees
(approximately300individuals)werecollectedfromthebroodnestareaandplacedinamasonjar
withalidconsistingof2mm×2mmhardwarecloth. Twotablespoonsofpowdersugarwereadded
throughthemeshandthejarwasshakenfor60stocoatthebeeswithsugar.Thejarwasflippedupside
downandshakenfor60soverawhitesurfacetocollectfallenphoreticmites,whichwerecountedand
extrapolatedtotheapproximatenumberofphoreticmitesper100bees. Allcoloniesweretreatedfor
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VarroainfestationwithvaporizedoxalicacidatthelegallabeldoserateinNovember2017andwere
nottreatedagainuntilaftertheoverwinteringsurvivalassessmentwasmadethefollowingyear.
ColonysurvivorshipwasrecordedeverythreeweeksfromMaytoOctober2017andagainin
May2018(oneyearaftercolonyestablishment)foratotalofeightsamplingperiods. Acolonywas
removedfromthestudyifitwasdeemeddeadorabscondedonanygivensamplingday.
2.4. StatisticalAnalyses
To test the effects of pesticide residues in the wax-coated foundation on colony growth,
we performed a repeated measures analysis of variance (ANOVA) test for each colony growth
parameter[24]. Becausethemeasurementsofgrowthweretakenfromthesamecoloniesovertime,
themodelwasbuilttotestthemaineffectsoncolonygrowthofthetreatmentgroup,thesampling
day,andtheirinteraction. Forallvariablesmeasured,theinteractioneffectsoftreatmentgroupand
samplingdaywerenotsignificantlydifferent. Therefore,wedidnotconductpair-wisetestsofmean
valuesforanycolonygrowthvariable[24].
To test the effects of pesticide treatment on overall colony survivorship, we conducted a
non-parametricKaplan–Meiersurvivalanalysis[25]. Varroainfestationratesandwintersurvivorship
were analyzed using t-tests through the standard least squares model platform. All tests were
performed using the statistical software JMP® 12.0 (SAS Inc., Cary, NC, USA). We set the level of
statisticalsignificanceatα=0.05foralltestsandreportedalldescriptivestatisticsasmeans±standard
errorsofthemean(S.E.M.).
3. Results
We did not find any significant differences in growth between colonies established on
pesticide-freefoundationandthoseestablishedonpesticide-ladenfoundation. All30ofthenewly
establishedcoloniesgrewsteadilyandinasimilarpatternthroughoutthesamplingperiod,having
builtanoverallaverage13,907±1872cm2ofcombinedworkeranddronecombby17October,thelast
daywemeasuredcolonygrowth. Therewerenodifferencesingrowthbetweenanyofthetreatment
groupsandthecontrolgroup(F =0.49,p=0.69;Figure1a).
3,21
A similar pattern of brood production was observed in all colonies, with the highest overall
averageofbroodproducedbeing3881±611cm2inSeptember. Nodifferencesbetweenthetreatment
groupsandthecontrolgroupwereobserved,however(F =0.34,p=0.80;Figure1b). Foodstorage
3,20
wasapproximatelyevenacrosscolonies,withcolonieshavingstoredanaverageof5320±1032cm2of
honey,nectar,andpollenbythelastdayofdatacollection. Therewerenodifferencesinfoodstorage
betweenthetreatmentgroupsandthecontrolgroup(F =0.28,p=0.84;Figure1c). Weobserved
3,22
adropintheadultpopulationsizeestimates18daysaftercolonyestablishment,whichwaslikely
causedbythebeesfromtheoriginalpackagesdyingoffandthefirstbatchesofnewbroodnothaving
emerged yet. After this dip, there was a steady and similar pattern of population growth for all
colonies,reachinganoverallaverageof19,410±2611adultbeespercolonybythelastsamplingdayin
October. Similartothepreviousmeasuresofcolonygrowth,therewasnodifferenceinpopulationsize
betweenanyofthetreatmentgroupsandthecontrolgroup(F =0.34,p=0.79;Figure2). Therewas
3,21
asignificanteffectofthesamplingdayonallmeasuresofcolonygrowth(p<0.0001),buttherewasno
interactioneffectbetweenthetreatmentgroupsandthesamplingday(seeTable1forallstatistical
values).
Theexperimentbeganwithsevencoloniesineachofthepesticidetreatmentgroupsandnine
coloniesinthecontrolgroup,foratotalof30colonies. Interestingly,inthetimeperiodbetweencolony
establishment(day0)andthefirstsamplingperiod(day18),twoofthesevenamitrazcolonies,twoof
thesevenC+Ccolonies,andoneofthesevenF+Ccolonieshaddiedpriortoanydatacollection. None
ofthecontrolcoloniesdiedduringthistimeperiod.
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FigureFi1g.uGrer 1o.w Gtrhowpathtt epranttser(pnsa n(pealsneal–s ca)–fco) rfonre nwewho hnoenyeyb ebeeec ocololonnieiess eessttaabblliisshheedd oonn 1188 MMaayy 2021071 7(d(adya y0).
Ofthe0)3. 0Otfo tthael c3o0 ltoontaiel sc,osloenvieens, wseevreene wstearbe leisshtaebdlisohnedw oanx wfoauxn fdouantidoantitorne atrteeadtewd iwthitahm aimtriatrza,zs, esveveennw  ith
chlorowthitahl ochnliolraonthdaclohnloilr apnydr icfholso(r“pCyr+ifCos” (t“rCea+tCm” etnreta),tmseevnet)n, swevitehn twauit-hfl utavua‐flliunvaatelinaantde acnodu mcoaupmhaopsh(o“sF +C”
(“F+C”  treatment),  and  nine  served  as  controls  that  were  established  on  pesticide‐free  wax 
treatment),andnineservedascontrolsthatwereestablishedonpesticide-freewaxfoundation.Colonies
foundation. Colonies were sampled every three weeks through 17 October 2017 (day 146) for a total 
weresampledeverythreeweeksthrough17October2017(day146)foratotalofsevensampling
of seven sampling periods. Data are presented as the mean ± S.E.M.
periods.Dataarepresentedasthemean±S.E.M.
Insects2019,10,19 6of13
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2 Figure2.FiEgsutrime 2a.t eEdstaimduatletdp oadpuullta tpioopnufloartioenac fhoro feathche 3o0f tnheew 30h onnewey hboeneeyco bloeen iceosloensiteasb leissthaebdlisohned1 8onM 1a8y M20a1y7 2(0d1a7y (d0a).yT 0h).e rTehweree rweesreev esenvceonl ocnolioensiiens tihn ethaem aimtraitzratzre atment
3 group,setrveeantminentth gerochuplo, rsoetvheanl oinn tihlea nchdlocrholtohraploynriilf oansdtr cehaltomrpeynrtifgorso turepat(m“Cen+t Cg”ro),uspe (v“eCn+Cin”)t,h seevtaeun -iflnu thvea ltianua‐ftleuavnaldincaoteu amndap chouomstarpehaotms terneattgmreonutp gr(o“uFp+ C(“”F)+,Ca”n)d, annidn einthe
4 nine in the control group. Each colony was started with an artificial swarm containing 3 lbs of adult bees, or approximately 10,476 individuals [18]. Population size 
controlgroup.Eachcolonywasstartedwithanartificialswarmcontaining3lbsofadultbees,orapproximately10,476individuals[18].Populationsizewasestimated
5 was estimated every six weeks through 17 October 2017 (day 146) for a total of four sampling periods. Data are presented as the mean ± S.E.M. 
everysixweeksthrough17October2017(day146)foratotaloffoursamplingperiods.Dataarepresentedasthemean±S.E.M.
6 Table1.TStaabtlies t1i.c Satlavtiastliuceals vfaolrurees pfoera treepdemateeda smueraessuarnesa alynsailsysoifs vofa vriaarniacnece(A (ANNOOVVAA))t teessttss ppeerrffoorrmmeedd fofro eraecahc ohf othfet hfoeufro vuarrivaabrleias bolfe csoolofncyo gloronwytghr aoswsetshseads sfreosmse ndefwro mnew
7 honeybeheonceoylo bneiee scoelostnaibesl iesshteabdliisnhe2d0 1in7 .2T01h7e. rTehwerae swaass iag nsiigfinciafincatnetf feeffcetcot fofs asammpplliinngg ddaayy ffoorr aalll lvvarairaibalbesle. sH.oHwoewveerv, tehre,rteh ewraes wneaisthneer iath seigrnaifsiciagnnti fiefcfaecntt oeff pfeecsttiocifdpe esticide
8 treatment group (i.e., amitraz alone, a mix of chlorothalonil and chlorpyrifos, or a mix of tau‐fluvalinate and coumaphos), nor an interaction effect between pesticide 
treatmentgroup(i.e.,amitrazalone,amixofchlorothalonilandchlorpyrifos,oramixoftau-fluvalinateandcoumaphos),noraninteractioneffectbetweenpesticide
9 treatment group and sampling day on any of the variables measured. Significant statistical values are shown in bold. 
treatmentgroupandsamplingdayonanyofthevariablesmeasured.Significantstatisticalvaluesareshowninbold.
Effect of Pesticide Treatment  Effect of Sampling Day  Interaction Effect 
Parameter Measured 
ParameterMeasured EffectofPestDicFindue  TrDeFadtemn. entF‐Value  p‐Value  DFnu EfDfeFcdteno. fSFa‐mVaplluien gDpa‐Vyalue  DFnu  DFden.  F‐Value  Inpt‐eVraalcuteio nEffect
AmouDntF onfu comb buDilFt den. 3  F-Val2u1e  p0.-4V9a lue 0.69 DFnu 7  D13F6d en. 131.1F8- Valu<e 0.0001p -Va2l1u e 136D  Fnu 1.36  DFde0n.1.5  F-Value p-Value
Amount of brood produced  3  20  0.34  0.80  7  136  68.00  < 0.0001  21  136  0.88  0.61 
Amountofcombbuilt 3 21 0.49 0.69 7 136 131.18 <0.0001 21 136 1.36 0.15
Amount of food stored  3  21  0.28  0.84  7  136  55.70  < 0.0001  21  136  0.34  1.00 
Amountofbroodproduced 3 20 0.34 0.80 7 136 68.00 <0.0001 21 136 0.88 0.61
Estimated adult population  3  21  0.34  0.79  4  79  48.77  < 0.0001  12  79  1.29  0.24 
Amountoffoodstored 3 21 0.28 0.84 7 136 55.70 <0.0001 21 136 0.34 1.00
10
Estimatedadultpopulation 3 21 0.34 0.79 4 79 48.77 <0.0001 12 79 1.29 0.24
Insects 2018, 9, x FOR PEER REVIEW   7 of 13 
11 This survivorship trend may merit further study, although no summer die off was significantly 
12 different between treatment groups. By the last sampling period (day 125), five of the initial seven 
13 colonies for all treatment groups and seven of the nine control colonies were still alive. Overwintering 
14 Isnuserctvsi2v0a1l9 ,w10a,s1 9low across all treatments, with only seven of the initial 30 colonies remaining ali7voef 1b3y 
15 18 May 2018 (day 378). The surviving group consisted of two colonies in the amitraz group, one 
16 colony in the C+C group, two colonies in the F+C group, and two colonies in the control group (Figure 
17 3). WTeh ifsosuunrdv invoo rdshififperternenceds mina yovmeerwritinfuterrtihnegr sstuurdvyiv,aall tbheotuwgehenn ocosulomnmiese rind itehoe ffcownatsrosli ggnriofiucpa natnlyd 
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19 colonWiesitfho rraelslpterecat ttmo eVntargrrooau mpsitaen idnsfeesvteantioonf thleevnelisn,e mcoonsttr oolf ctohleo ncioelsownieerse tshtiallt ahliavde .aO pvherowreitnicte rminitge 
20 siunrfevsitvaatliowna asbloovwe a3c%ro isns aSlelpttreematbmeer n2t0s1,7w (i6th5.2o%nl)y hsaedv ednieodf tbhye Minaityia 2l03108c. oTlhoen iceoslorenmieas itnhiantg daileivde hbayd 
21 1s8igMniafyic2a0n1tl8y( dmaoyr3e 7V8)a.rrTohae (smuervaniv i=n g5.g71ro mupitceos/n≈s1i0st0e dbeoefst)w thoacno ltohnoieses itnhatht ehaamd istruarzvgivreodu p(m,oenaenc o=l o2n.0y5 
22 imnitthees/C≈1+0C0 bgeroesu)p b,yt wMoayc o2l0o1n8i e(ts‐rinatitoh e= F2+.9C6; gpr =o u0.p0,0a7n5)d, itnwdoepceonlodneniets oifn ththee trceoantmtroenltg grorouupp(.F Siigxu orfe t3h)e. 
23 Wseevfeonu cnodlonnoiedsi ftfhearte nsucervsiivneodv tehrew fiunltle yrienagr souf rsvtuivdayl b(8e5tw.7%ee)n hcaodl oan piehsoirnettihce mcoitne tlreovleglr aotu opr abnedlotwho 3s%e iinn 
24 aSneyptoefmthbeerp 2e0s1ti7c,i dweitthre tahtem eexnctepgtrioounp osf (oχn2e= C1+.1C5 ,cpol=on0y.7 t6h;aFti ghuarde 53.)3.3 mites per ≈ 100 bees (Figure 4). 
1.0
0.8
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hi
s
or 0.6
v
vi
r
u
s
y
n 0.4 Amitraz
o
ol C+C
C
F+C
0.2 Control
0.0
0 50 100 150 200 250 300 350 400
Days after colony establishment
25  
Figure3. Survivorshipcurvesforcoloniesestablishedusingframeswithplasticfoundationcoated
26 Figure 3. Survivorship curves for colonies established using frames with plastic foundation coated 
withpesticide-freewax(Controlgroup)orwaxexposedtoeitheramitraz(“amitraz”group),amix
27 with pesticide‐free wax (Control group) or wax exposed to either amitraz (“amitraz” group), a mix of 
ofchlorothalonilandchlorpyrifos(“C+C”group),oramixoftau-fluvalinateandcoumaphos(“F+C”
28 chlorothalonil and chlorpyrifos (“C+C” group), or a mix of tau‐fluvalinate and coumaphos (“F+C” 
group).ColonysurvivalwasevaluatedeverythreeweeksfromMaytoOctober2017.Afinalevaluation
29 group). Colony survival was evaluated every three weeks from May to October 2017. A final 
ofsurvivorshipwasdoneoneyearfromtheestablishmentdateonMay2018.Acolonywasremoved
30 evaluation of survivorship was done one year from the establishment date on May 2018. A colony 
fromthestudyifitwasdeemeddeadorabscondedonagivensamplingday.
31 was removed from the study if it was deemed dead or absconded on a given sampling day. 
With respect to Varroa mite infestation levels, most of the colonies that had a phoretic mite
infestationabove3%inSeptember2017(65.2%)haddiedbyMay2018. Thecoloniesthatdiedhad
significantlymoreVarroa(mean=5.71mites/≈100bees)thanthosethathadsurvived(mean=2.05
mites/≈100bees)byMay2018(t-ratio=2.96;p=0.0075),independentofthetreatmentgroup. Sixof
thesevencoloniesthatsurvivedthefullyearofstudy(85.7%)hadaphoreticmitelevelatorbelow3%
inSeptember2017,withtheexceptionofoneC+Ccolonythathad5.33mitesper≈100bees(Figure4).
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Insects 2018, 9, x FOR PEER REVIEW   8 of 13 
14 18-Sep-17 18-May-18
s 12
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of 4
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e
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m 2
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2 3 4 5 6 1 2 5 6 7 1 2 3 4 6 1 2 3 4 6 7 8 9
A A A A A C C C C C C C C C C C C C C C C C C
C C C C C F F F F F
32 Colony ID  
33 FFiigguurree 44.. EEssttiimmaatteedd nnuummbbeerr ooff VVaarrrrooaa ddeessttrruuccttoorr mmiitteess ppeerr aapppprrooxxiimmaatteellyy 110000 bbeeeess ((pprriimmaarryy yy-‐aaxxiiss)) 
34 oobbttaaiinneedd ffrroomm eexxppeerriimmeennttaall ccoolloonnieiess uusisningg thteh epopwowdedre srusguagr asrhashkae kmeemtheotdh.o Tdh.eT xh‐aexxis- adxeinsodteesn oeatecsh 
35 ecaoclhoncyo laonndy tahned etxhpeereixmpeenritmal egnrtoaulpg riot ubpeliotnbgeeldo ntgoe. dExtpo.erEimxpeenrtiaml etrnetaatlmtreenattsm inecnltusdien cclouldoeniceos loinn itehse 
36 ianmthiteraazm girtroauzpg (rAou),p th(Ae )c,hthloerocthhlaolroonthila laonndi lcahnldorcphylroifropsy rmifoixs gmrioxupgr o(CuCp)(, CtChe), ttahue‐ftlauuv-aflluinvaatlein aanted 
37 acnodumcoaupmhoasp hmoisx mgrixougpro (uFpC)( FaCn)da tnhde tchoenctroonlt rgorlogurpo u(Cp).( CT)h.eT hfiersfit rVsatrVroaar rmoaitme icteoucnotu nwtaws adsodnoe noeno 1n8 
38 1S8epSetepmtebmerb 2er01270 (1g7re(gyr beayrsb)a arns)da tnhde stehceonsedc oconudnct owuanst dwoanse dono n1e8 oMnay18 20M18a y(b2la0c1k8 b(barlas)c kfobr aarlls )cofolornaiells 
39 ctohlaotn iweserthe aatlwiveer eoanl itvheaot ndtahtaet. dTahtee. hTohreizhoonrtizaol nbtlaalcbkl alcinkel ininediincdaitceast ethseth tehtrhersehsohlodl doof ftthhrreeee mmiitteess ppeerr 
40 aapppprrooxximimaatetelyly1 10000b beeeess( (i.ie.e.,.,3 3%%i innffeessttaattioionnl leevveel)l)a abboovveew whhiicchhi itt iiss ttyyppiiccaallllyy rreeccoommmmeennddeeddt toop peerrffoorrmm 
41 ssoommeet tyyppeeo offV Vaarrrrooaac coonnttrrool.l. 
4. Discussion
42 4. Discussion 
We sought to discover the effects of initial pesticide contamination in wax foundation
43 We sought to discover the effects of initial pesticide contamination in wax foundation on the 
on the growth and survival of newly established honey bee colonies. Our results showed
44 growth and survival of newly established honey bee colonies. Our results showed that initial 
that initial contamination of beeswax foundation with field-relevant concentrations of amitraz
45 contamination  of  beeswax  foundation  with  field‐relevant  concentrations  of  amitraz  alone,  a 
alone, a combination of tau-fluvalinate and coumaphos, or a combination of chlorpyrifos and
46 combination of tau‐fluvalinate and coumaphos, or a combination of chlorpyrifos and chlorothalonil 
chlorothalonilatfield-relevantconcentrationsdidnothavesignificanteffectsoncolonygrowthor
47 at field‐relevant concentrations did not have significant effects on colony growth or overwintering 
overwinteringsurvival.
48 survival. 
Initially,theneutralresponseofincipientcoloniestopesticide-ladenwaxfoundationcouldbe
49 Initially, the neutral response of incipient colonies to pesticide‐laden wax foundation could be 
consideredpositivenewsforthebeekeepingindustry,giventhatthemanystudiesshowingnegative
50 considered positive news for the beekeeping industry, given that the many studies showing negative 
effectsofpesticidecontaminationonbeehealthattheindividuallevelhaveelevatedconcernsabout
51 effects of pesticide contamination on bee health at the individual level have elevated concerns about 
pollinator–pesticideinteractionsinthebroodrearingenvironment. Thatsaid, itseemspotentially
52 pollinator–pesticide interactions in the brood rearing environment. That said, it seems potentially 
disastrous to take these results at face value and conclude definitively that pesticide exposure of
53 disastrous to take these results at face value and conclude definitively that pesticide exposure of bees 
bees through the beeswax appears to have no impact on colony health or productivity. A hive’s
54 through the beeswax appears to have no impact on colony health or productivity. A hive’s beeswax 
beeswaxmatrixishighlylipophilic,sotheactiveingredientsofpesticidesquicklyenterandstayin
55 matrix is highly lipophilic, so the active ingredients of pesticides quickly enter and stay in this matrix 
thismatrixyearsafteraproductisnolongerused[12]. Forinstance,recentstudiescontinuetoshow
56 years after a product is no longer used [12]. For instance, recent studies continue to show that 
thatbeekeeper-appliedmiticidesarefrequentlyfoundinwax[11,26],whichisasignificantcausefor
57 beekeeper‐applied miticides are frequently found in wax [11,26], which is a significant cause for 
concern,giventhatallstagesofbeedevelopment,inadditiontofoodstorage,occurinconjunction
58 concern, given that all stages of bee development, in addition to food storage, occur in conjunction 
withthebeeswaxmatrix. Thisleadstoconstantandvaryinglevelsofpesticideexposuretoallcolony
59 with the beeswax matrix. This leads to constant and varying levels of pesticide exposure to all colony 
membersovertime[27],especiallywhenthesamecombisusedformultipleseasonswithoutbeing
60 members over time [27], especially when the same comb is used for multiple seasons without being 
replaced. For instance, pesticide application to adult workers of varying ages and genotypes has
61 replaced. For instance, pesticide application to adult workers of varying ages and genotypes has been 
62 bseheonwsnh otwo ncatuoscea usysenesrygniestrigci satnicda nndegnaetigvaet ivefefeecfftes cotsno tnheth seusruvrivviavl aolfo flalarvrvaea eaanndd aadduullttss aanndd oonn tthhee 
63 pprroodduuccttioionno offc cyyttoocchhrroommeeP P445500d deetotoxxifiifcicaatitoionne ennzzyymmeess[ 2[288––3322].]. 
64 Furthermore, the presence of amitraz, tau‐fluvalinate, and coumaphos in the brood‐rearing wax 
65 environment has been shown to cause lower spermatozoa viability in queen spermathecae, lower
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Furthermore, the presence of amitraz, tau-fluvalinate, and coumaphos in the brood-rearing
wax environment has been shown to cause lower spermatozoa viability in queen spermathecae,
lowerqueenreproductivepotential,highersupersedurerates,andloweregg-layingrates[16,17,33–37].
These miticides also affect drone development, as they can reduce drone production [38] and
survival[39],aswellasspermatozoaproductionandviability[40–43].
Like other organophosphates, chlorpyrifos inhibits the production of acetylcholinesterase,
which prevents the processing of neurotransmitters at nerve synapses [44]. Although not much
isknownabouttheeffectsofchlorpyrifosonhoneybees,itcancausesubstantialsynergisticeffects
whencombinedwithotherpesticides,leadingtohighlarvalmortality[45]. Moreover,arecentstudy
inwhichwaxwascontaminatedwithfield-relevantconcentrationsofchlorpyrifosincombination
withchlorothalonilshoweddecreasedspermatozoaviabilityinsexuallymaturedrones[43]. Themost
recent data published has estimated that, conservatively, the U.S. used over 5 million pounds of
productswithchlorpyrifosastheactiveingredientfrom2000–2010. Thisisadeclineinchlorpyrifos
use,asmorethan10millionpoundsperyearwasusedinthe1990’s[8].
Chlorothalonilisanon-systemicfungicideknowntoreduceintracellularlevelsoftheantioxidant
molecule glutathione, causing poor enzymatic activity and, eventually, fungal cell death [46].
Chlorothalonilwasshowntocausehighlarvalmortalitywhenlarvaewerefedbeebreadcontaminated
withthisfungicide[32].Therateofmortalitywasevenhigherwhenitwasmixedathighconcentrations
with tau-fluvalinate, coumaphos, or chlorpyrifos, although this was not true at more diluted
concentrations of the fungicide [32]. Chlorothalonil is also considered a “marker” that activates
honeybeepollen-entombingbehaviors[9,47]. Entombingissuspectedtobeaprotectivebehavior
consisting of storing pollen in sunken or capped wax cells, and it is associated with high colony
mortality. It is estimated that over 8 million pounds of products with chlorothalonil as the active
ingredienthavebeenusedinU.S.agricultureeveryyearsince1992,withusereachingover11million
poundsin1997. Theseestimatesdonotaccountfornon-agriculturaluse,whichwouldincreaseits
usetoapproximately15millionpoundsperyearsincethe1990’s[8]. Despitetheirpotentialharmto
colonyhealth,bothchlorothalonilandchlorpyrifosarelicensedforuseonhoneybee-pollinatedcrops,
whichexplainstheirhighprevalenceinhoneybeecolonies[11,12].
OurstudydidnotfindasignificanteffectofanypesticidetreatmentgrouponVarroamitelevels.
This is not surprising in regards to tau-fluvalinate and coumaphos, as both are ineffective against
Varroa[48–52]. However,itisintriguingthatamitraz-coatedfoundationdidnotcauselowerVarroa
levels in our study, given that it is still an effective treatment for Varroa control in the U.S., and
miteresistancetothisproducthasonlybeenreportedinafewregionswithinNorthAmerica[9,51].
WhencomparingthelevelofVarroatakeninSeptember2017withthesurvivorshipofcoloniesinMay
2018,colonieswithmiteloadsabovethethresholdofthreemitesper100bees[6]hadasignificantly
higherchanceofdeathaftertheoverwinteringperiodcomparedtocoloniesthatwerebelowthismite
threshold. OurresultsarethuscongruentwiththenotionthathighVarroamiteinfestations,andlikely
highVarroa-vectoredvirusloads,continuetobethemainculpritofcolonymortality[6,53,54]. Italso
reinforcestherecommendationthatbeekeepersshouldperformsometypeofVarroacontrolinlate
summerorearlyfallwhentheircoloniesexhibitaphoreticmitelevelabovethe3%threshold,andthey
shouldbeawareofcolonieswithhighmiteinfestationthatcouldpotentiallycausedriftofphoretic
mitestoothernearbycolonies.
In the future, similar studies should track other variables of colony health. For instance,
measuring the production of propolis, which is known as a way for colonies to “self-medicate”
againstpathogens[55,56],couldyieldinterestinginformationonhowcoloniesdealwithpesticide
exposure. Othervariablescouldincludemeasuringbroodproductionpatternsandhowtheyrelateto
queenquality[16,57],theidentificationofpollenentombingbehavior,measuringVarroa-vectoredvirus
levels,measuringthecreationofburrcombinuncontaminatedareasofthehive,ordeterminingother
routesofpesticideexposureinthehive[47,58].Forinstance,itispossiblethatourcolonieswerefurther
exposedtoadditionalpesticidesviaforagingactivities. However,thisrouteofpesticideexposurewas
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likelyminimalduetotheapiarylocationandtheabsenceofanyentombedpollen,whichseemsto
indicatethatpesticidecontaminationofpollen,atleastwithchlorothalonil,wasprobablylow[47].
Furthermore, the area surrounding our apiary site was predominantly cattle pastures as opposed
to commercial cropland, so there was likely few pesticides applied to agroecosystems around our
apiary. Interestingly, althoughitwascostprohibitivetoconductpesticideresidueanalysisonour
comb samples, we noticed a large amount of burr comb constructed throughout the course of the
study,whichcouldhavebeendoneasanattemptbythebeestoavoidthepesticide-ladenfoundation,
althoughpesticidescanmovedowntheconcentrationgradientinbeeswaxtocontaminatepreviously
cleanbeeswax[59]. Anotherstudyshowedthatpesticidecontaminatedbeeswaxhadsub-lethaleffects
onworkerbeesbycausingaprematureshiftintemporalpolyethism[59]. Ifthishappenedinour
colonies,itdidnotcausenoticeableeffectsonapopulationlevel. Whilenoneofthesevariableswould
explaincolonyhealthstatusaselegantlyastheestimatesofcolonygrowthandoverwinteringsurvival
would,theycouldpotentiallyshedfurtherlightonhownewcoloniesreacttocontaminatedcomb.
5. Conclusions
Ultimately,itwouldbeirresponsibletouseourresultstoconcludethatinitialcontaminationof
waxfoundationwiththefivepesticidesweexaminedhasnoimpactoncolonyhealth. However,itis
possibletoconcludethatnewcoloniesareindeedabletoflourishinapesticide-ladenwaxenvironment
ifthesepesticidesoccuratfield-relevantconcentrationsandnoadditionalpesticidesareintroduced
intothehive. Furthermore,ourresultsindicatethathighVarroainfestationlevels—andpotentially
highviruslevels—arearecipeforcolonydemise.
Studies on the toxicological effects of pesticides on honey bees are commonly done using
individual bees and unrealistic pesticide exposure routes or dosages. To date, there still exists a
literaturegapinourknowledgeaboutthesynergisticeffectsofpesticidecontaminationatthecolony
levelusingrealisticroutesofexposureandfield-relevantconcentrationsoftheproductstested. While
theeconomicandtemporalcostsofconductingsuchcolony-levelassessmentsarehigh,thebenefitsof
obtainingthesedatawillincreaseourunderstandingofhowpesticideexposureaffectsoverallhoney
beehealth.
AuthorContributions:Conceptualization,J.R.andE.W.;Fundingacquisition,J.R.andE.W.;Investigation,A.P.,
E.W.andJ.R.;Datacuration,A.P.andE.W.;Methodology,A.P.,E.W.andJ.R.;Formalanalysis,A.P.;Resources,J.R.;
Supervision,J.R.;Writing—originaldraft,A.P.,E.W.andJ.R.;Writing—review&editing,A.P.,E.W.andJ.R.
Funding:ThisresearchwasfundedbyProjectApism.award(TexasAgriLifeResearchSponsoredResearchProject
M1701575),aUSDA-NIFAaward(2015-67013-23170)andTexasA&MUniversity’sHatchProject(TEX09557).
Acknowledgments:TheauthorswouldliketoacknowledgethegenerosityofTomMcDonald,whokindlyletus
toputcoloniesonhisproperty,aswellasE.T.Ash,whogaveusbeekeepingadviceandguidance. Wewould
alsoliketothankJakalynneGosnellforhelpingwithdatacollectionandPierreLauforprovidingknowledgeand
statisticalsupport.
ConflictsofInterest: Theauthorsdeclarenoconflictofinterest. Thefundershadnoroleinthedesignofthe
study;inthecollection,analyses,orinterpretationofdata;inthewritingofthemanuscript,andinthedecisionto
publishtheresults.
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