Combination of Methoprene and Controlled Aeration to Manage Insects in Stored Wheat

12 Pages·2016·0.37 MB·English

insects
Article
Combination of Methoprene and Controlled Aeration
to Manage Insects in Stored Wheat
SamuelS.Liu1,2,FrankH.Arthur3,DouglasVanGundy4andThomasW.Phillips1,5,*
1 DepartmentofEntomologyandPlantPathology,OklahomaStateUniversity,Stillwater,OK74078,USA;
[email protected]
2 FoodForensicsDeptartment,CertifiedLaboratories,200ExpressSt.,Plainview,NY11803,USA
3 USDA,AgriculturalResearchService,CenterforGrainandAnimalHealthResearch,1515CollegeAvenue,
Manhattan,KS66502,USA;[email protected]
4 CentralLifeSciences,12111FordRd.,Dallas,TX75234,USA;[email protected]
5 DepartmentofEntomology,KansasStateUniversity,123W.WatersHall,Manhattan,KS66506,USA
* Correspondence:[email protected];Tel.:[phone]
AcademicEditors:NickolasG.Kavallieratos,ChristosG.AthanassiouandOrlandoCampolo
Received:11May2016;Accepted:9June2016;Published:17June2016
Abstract: A commercial formulation of the insect growth regulator methoprene was applied to
wheatstoredinsmallbinseitheraloneorincombinationwithcontrolledaerationofthebins, to
lower grain temperature for insect pest management of stored wheat. Grain temperatures were
monitored and modified by a computer-controlled thermocouple system that also activated the
aeration system at programmed set-points to move cool ambient air through the grain mass to
lowergraintemperature. Resultsfromsamplinginsectpopulationsinexperimentalstoragebins
alongwithlaboratorymortalitybioassaysofinsectsplacedonwheattakenfromthebinsoverthe
courseofthestorageperiodshowedthatmethoprenewasveryeffectiveincontrollinginfestation
bytheexternally-feedingstoredgraininsectsPlodiainterpunctella(Hübner),theIndianmealmoth
Tribolium castaneum(Herbst),theredflourbeetle,Cryptolestesferrugineus(Stephens),therustygrain
beetle, andalsofortheinternal-feedingpestRhyzoperthadominica(Fauvel), thelessergrainborer.
Methoprene did not give good control of the internal-feeding pest Sitophilus oryzae (L.), the rice
weevil. Aerationalonewassomewhateffectiveinsuppressinginsectpopulationdevelopment,while
methoprenealoneorwhencombinedwithaerationgreatlyenhancedinsectcontrol. Commercial
graingradingforindustryqualitystandardsattheendofthestorageperiodconfirmedtheimpactof
insectsuppressiononmaintaininghighqualityofthestoredwheat. Thisfieldexperimentshowsthat
methoprenecombinedwithaerationtocoolgraincanbeeffectiveforpestmanagementofstored
wheatinthesouthernplainsoftheUnitedStatesofAmerica.
Keywords: Plodiainterpunctella;Triboliumcastaneum;Cryptolestesferrugineus;Rhyzoperthadominica;
Sitophilusoryzae;storedproducts;insectgrowthregulator
1. Introduction
Theinsectgrowthregulators(IGRs)areinsecticidesthatmimicinsectmoltinghormonesand
interferewithnormalimmatureinsectdevelopment[1–3]. IGRsincludejuvenilehormoneanalogues
(JHAs)whichareconsideredbiopesticidesbecausetheygenerallyhaveverylowmammaliantoxicity
andcanhavelimitedimpactsonnon-targetarthropodspecies[4].IGRsinterferewithinsectmetabolism
inamannerthatdisruptsnormalgrowthanddevelopment. Usually,theinsectdiesbeforeitreaches
full maturity. Insect growth regulators are typically selective for insects and can either inhibit the
synthesisofchitinrequiredforformingnewcuticleateachmolt,ordisruptorreplacetheproduction
ofjuvenilehormonesthatregulatethemoltingprocess[5–7].
Insects2016,7,25;doi:10.3390/insects7020025 www.mdpi.com/journal/insects
Insects2016,7,25 2of12
MethopreneisasyntheticJHAthatinterruptsinsectgrowthanddevelopmentandcancontrola
numberofstoredproductpests[1,8–11]. TheformulationDiacon®(CentralLifeSciences,Schaumberg,
IL,USA)wasregisteredduringthe1980sintheUSAbutitwasnotwidelyused[10].Alatercommercial
formulation,DiaconII®(formulatedat288mg/mLactiveingredient,AI),whichcontainsthes-isomer
ofmethoprene,wasregisteredasagrainprotectantintheUSin2002andcanbeappliedatratesof1.0,
2.5or5.0ppmonvariousgrainsandnuts[3].
Aerationwithambientairisanimportantcomponentofintegratedpestmanagementstrategies
tohelpmanageinsectinfestationinstoredgrainbycoolingthegrain[12–16]. Aerationslowsdown
thenormaldevelopmentofimmatureinsectsbyloweringtheambienttemperature. However,aeration
coolingwillrarelykillinsectsinstoredgrainasacutelylethaltemperaturesarenotattained[16,17].
Moststoredgraininsectpestspeciesdonotreproduceatgraintemperatureslowerthan15˝C[18,19].
Although aeration does not cause acute mortality, it is effective in controlling stored product pest
populationsbyreducingtemperaturesinthegrainmassandisthusrecommendedtocontrolstored
graininsects[4,20].
Residualdegradationofinsecticidesusedasgrainprotectants,especiallyolderorganophosphates,
increaseswithtemperature[14]. Althoughmethopreneisrelativelystableatdifferenttemperatures
theresidualactivityandefficacycouldbeevenfurtherenhancedinaeration-cooledgraincompared
tonon-aeratedgrainwhenstoredunderhotambientconditionsinthefield[21]. Variouslaboratory
studies that evaluated efficacy of IGRs as aerosols, contact surface treatments, or layer treatments
(e.g.,topofgraincomparedtootherapplications)tocontrolstoredproductinsectsandsomerecent
studieshaveevaluatedefficacyofmethopreneunderfieldconditions[3,21–24]. However,thereareno
scientificfieldstudiesthatresearchedtheefficacyofIGRscombinedwithaerationcoolingofstored
grain. Theobjectivesofthiscurrentstudywereto: (1)evaluateefficacyofmethopreneforinsectpest
controlwhenappliedasatop-layerorascompletebintreatmenttostoredwheat,eitherorwithout
aerationcoolingover40weeksofstorage;(2)evaluatetheimpactsofmethoprene-aerationtreatments
ongrainqualityoverthestorageperiod;and(3)determinethestabilityofmethopreneresiduesunder
thesesametreatmentandstorageconditions.
2. MaterialsandMethods
2.1. MethopreneApplication,GrainStorageandQualityEvaluations
ThemethopreneusedinthefieldtrialswastheformercommercialproductDiaconII®Emulsifiable
Concentrate (EC) (288 mg AI/mL, Central Sciences International, Schaumburg, IL, USA). The
concentratedinsecticidewasdilutedinwateraccordingtomanufacturer’sinstructionsandapplied
tothegrainat19.0L/t,whichwastoresultinacalculateddosageof1.0ppmbyweightappliedto
grain. Thesprayerusedforthetestwasa“LittleGus”fieldsprayer,originallysoldbyGustfasonInc.,
Plano,TX(nowapartofBayerCorporation,PittsburgPA,USA).Sixteencorrugatedroundsteelbins
withastoragevolumeofapproximately17.6m3,adequatetohold13.6MTofwheat,wereusedforthe
fieldtrial,whichwasdoneattheStoredProductResearchandEducationCenterofOklahomaState
University,Stillwater,OK,USA.Allbinswereequippedwitharaisedventilatedfloorandanaeration
faninthe“push”orientation,suchthatambientoutsideaircouldbedrawnintothebin,upthrough
thefloorandintothegrainmassduringacoolingcycle. Thelidsatthetopopeningsoftheaerated
binswereproppeduptoanopeningofabout3cmatthesideoppositethehingestoallowcoolingair
fromaerationtoescape. Graintemperatureswereautomaticallymonitoredandrecordedhourlyand
theaerationfanswereturnedonfortheaeration-treatedbinsusinganaerationcontrolsystemfrom
OPISystemsInc. (Calgary,Alberta,Canada). Fansforthedesignatedaerationbinsweresettoactivate
whentheoutsideairtemperatureat3mabovegroundwas5˝Clowerthanthegraintemperatureat
30cmbelowthegrainsurface. Grainforthisexperimentwasnewlyharvestedhardredwinterwheat,
Triticumaestvium,purchasedfromalocalproduceranddelivereddirectlyfromthefieldatthetimeof
harvestwithnoapplicationofanychemicalbeforeloadingintoourbinson21July. Thefollowingfour
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treatmentswererandomlyassignedtoeachoffourbins: (1)aerationonlywithnomethopreneasour
experimentalcontrol;(2)methopreneat1.0ppmappliedtotheentiregrainmasswithnoaeration;
(3) methoprene at 1.0 ppm applied just to the top 50 cm of the grain mass, with no aeration; and
(4)acombinationof1.0ppmmethopreneappliedtotheentiregrainmasspluscontrolledaeration.
Atrulyuntreatedexperimentalcontrol,forwhichtherewouldbenoaerationandnoapplicationof
methoprene,wasnotusedforthisexperimentduetoconcernswithover-infestationofvaluablewheat
insuchatruecontrol,ourpreferenceforadequateandbalancedreplication(n=4),andourdesireto
compareefficacyofmethopreneundercoolgrain(aerated)andwarmgrain(non-aerated)conditions.
Thegrainflowratewhileloadingintobinswascalibrated(kg/min)whentheaeration-onlybins
wereloaded,andthisvaluewasusedtocalculateappropriateapplicationofthemethoprenesolution
toreachthetargetapplicationrateof1ppm. Individualvolumesofsolutionwerepreparedforeach
bin involving methoprene. Methoprene spray treatments were applied directly to grain as it was
loadedintobinsbysprayingthedilutedproductontograinasitwasdumpedfromatruckintothe
collectionhopperatthebottomofa15-cmscrewaugerconveyorthatelevatedthegrainintothebins
atthetop-centercappedopeningontheroof.
Officialgraingradingandmethopreneresidueanalyseswerecarriedoutatboththebeginning
andtheendofthisstudy. Samples(each>1.0kg)fromeachbinweresenttoacommercialservice
laboratory(EnidGrainInspectionCompany,Enid,OK,USA)forgrading,andtoCentralLifeSciences
toanalyzeformethopreneresiduesviaestablishedmethods[25].
2.2. InsectIntroductionstoBins
Triboliumcastaneum(Herbst)(Coleoptera:Tenebrionidae),theredflourbeetle,(Rhyzoperthadominica
(Coleoptera: Bostrichidae),thelessergrainborer,andCryptolestesferrugineus(Stephens)(Coleoptera:
Laemophloeidae), the rusty grain beetle, were added to bins at various times after the bins were
loadedwiththewheat. Theinsectswereobtainedfromstockpesticide-susceptiblelaboratorycultures
maintained at Oklahoma State University, (OSU) Stillwater, OK, USA. T. castaneum was reared on
400 cm3ofwholehardredwinterwheatflourplus2.5cm3yeastin0.95Lglasscontainers. R.dominica
was reared on 400 g sound wheat containing 4% yeast in each container, while C. ferrugineus was
reared on 400 cm3 rolled oats with 5 cm3 whole hard red winter wheat flour and 2.5 cm3 yeast in
eachcontainer. Allspeciesweremaintainedingrowthchambersat28–30˝Cand65˘5%r.h. One
hundredmixed-sexadultsofeachofthesethreespecieswereaddedtoeachofthe12binsat7,14,
21and28daysafterthewheatwasloadedintothebins. Insectsamplesfromprobetrapsandalso
insectcountsfromdirectgrainsamplesweremadeatday28. OneWB-IIprobetrap(TreceInc.,Adair,
Oklahoma,USA)wasinsertedintothetop30cmofgrainineachbinandkeptthereforsevendays.
Capturedadultinsectswerethenidentifiedtospeciesandcounted. Grainsamplesof1.0–2.0kgwere
collected from each bin using a 1.5-m-long brass open-handle probe, referred to as a “grain trier”
(SeedburoEquipmentCo.,DesPlaines,IL,USA).Graintriersamplesweretakenfromthetopofthe
grainineachbinandthenweighedandsiftedforidentificationandcountingofadultinsects. Trap
andgrainsampleswerecollectedfromall16binsat4,8,12,16,20and40weeksafterthebinswere
loadedatthestartofthestudy.
2.3. LaboratoryBioassays
Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae), the Indian Meal moth, T. castaneum,
R.dominica,andSitophilusoryzae,(L.)(Coleoptera: Curculionidae)thericeweevilwereusedforthe
laboratorybioassayoftreatedgrainfrombins. Thecoloniesofthesefourinsectspecieswererearedin
growthchambers. P.interpunctellawasrearedin0.95Literglassjarsonadietof37%cornmeal,25%
eggcrumbles,25%chickstarter,and13%glycerin(v:v)at28˝C,65˘5%r.h.,anda16h/8hlight/dark
cycle. T.castaneumandR.dominicawasrearedasdescribedforthefieldstudy. Sitophilusoryzaewas
rearedon400cm3soundwheatplus2.5cm3ofyeastinthesamecontainersandrearingconditionsas
previouslydescribedfortheotherspeciesrearedonwheat.
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Grainusedforlaboratorybioassayswastakendirectlyfromthefieldstudygrainbinsatthree
differenttimesaftertreatment: onedayafterloading(21July)andthenafter20(9December)weeks
and40weeks(28April)ofstorage. P.interpunctellaeggsthatwere0–24-h-oldwerecollectedfrom
rearingjarscontaining1–3-d-oldmoths. TwentyP.interpunctellaeggsattachedtodouble-sidedsticky
tapeonblackpaperwereplacedinaeachof120-mLjarscontaining20gofsoundwheatkernelsplus
20gcrushedwheatfromeachbin,andmaintainedat28˝Cand65˘5%r.h. witha16h/8hlight/dark
photoperiodinagrowthchamber. Egghatchwascheckedafter1week,andcountsofnormaladults
weremadeafter6,7and8week. FiftyT.castaneum,R.dominica,andS.oryzaeadultswereseparately
placedinto240-mLjarseachcontaining100gwheat(5gcrushedforT.castaneum)fromeachbinand
maintainedat28˝Cand65˘5%r.h. undera16h/8hlight/darkphotoperiodinagrowthchamber.
After1weektheparentadultswereremovedfromeachjarandplacedinindividuallycleanPetri
dishesforarecoveryperiodof24h,andthencountedtodeterminemortality. After6weekstheF1
progenyfromthegrainweresiftedandcounted.
2.4. StatisticalAnalysis
DatasetswereanalyzedseparatelyforeachspeciesortreatmentusingtheGeneralLinearModels
procedure(SASInstitute,Cary,NC,USA).Alldataweresubjectedtoanalysisofvariance(ANOVA)
followedbyaprotectedleastsignificantdifference(LSD)testforcomparisonofmeans. Datawere
transformedbysquarerootofthenumberplus0.5priortotheANOVAanalysis,whilenon-transformed
meansandstandarderrors(SE)arereportedbelow. Significantbiologicaldifferenceswereconsidered
atthep<0.05level.
3. Results
3.1. TreatmentEffectsontheStoredWheat
Controlledaerationappliedtothesmallbinsofwheatfortwoofthetreatments,eightbinstotal,
causedadistinctcoolingofgrainwhencomparedtograintemperaturesfortheeightbinswithoutany
aeration(Figure1). Grainintheaeratedbinsaveragedabout6degreeslowerthangraininunaerated
binsafteroneweekofcooling,andachievedabouta10degreedifferencethatwasmaintainedforthe
courseofthestudy. Attheendofthe22ndweek,on16December,thetemperaturesinaeratedbins
wInsaesctsa 2b0o1u6,t 73, 2˝5C whiletemperaturesinunaeratedbinswereabout14˝C. 5 of 12
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3.2. Insect Populations in Bins
Tables 1–6 report the mean numbers of insects in samples taken from the bins at various time
intervals during the 40-week study. Probe traps samples (Tables 1, 3 and 5) report numbers trapped
during one weeks in the top of each bin, whole grain trier samples are the numbers of insects in an
approximately 1-kg sample of grain taken from each bin. Insect numbers overall were higher in
samples taken during the warmer times of week 4, week 8 and week 12, after which the numbers
were lower as temperatures reduced insect activity. In cases for which there were statistically
significant differences among the treatments the highest insect counts were in the bins treated with
aeration only.
There were significant differences among treatments for numbers of T. castaneum adults in
probe traps at eight weeks, 12 weeks and 40 weeks (ANOVA, p < 0.05) with the lowest numbers
captured in bins completely treated with methoprene and under aeration (Table 1). Adults trapped
in the aeration-only bins averaged 1,017 and 1,975 at 4 weeks (August) and 8 weeks (September),
and these numbers are tremendously higher than expected given that only 400 adults had been
added to each bin during the first four weeks of storage. Thus, high numbers of T. castaneum likely
resulted from field immigrants to these bins. Nevertheless, by week 12 the population suppression
effect of the total methoprene treatment as a growth regulator was seen in comparison to the
methoprene top and aeration only treatments. T. castaneum adults collected in grain trier samples of
1 kg of wheat showed that there were significant differences in mean adult numbers among the
different treatments taken at the first three sampling periods, with the lowest beetle densities being
in treatments that had methoprene applied to the entire grain mass (Table 2). of 4, 8 and 12 weeks
but there were no significant changes in mean adult numbers among all treatments taken at the last
three sampling months (p > 0.2) (Table 2).
Table 1. Mean numbers (±SE) of adult T. castaneum captured by probe traps at different times after
bins were loaded with wheat that was either treated with aeration only, the top 50 cm-treated with
methoprene, methoprene treatment to all the wheat in a bin, or aeration plus complete treatment
with methoprene on all grain in the bin. Means within columns with the same upper case letter, for
which there was a significant (p ≤ 0.05) F-statistic, are not significantly different (least significant
difference (LSD) test, p < 0.05, SAS Institute, degrees of freedom for analysis within date for treatment
were 3, 12).
Treatments 4 Weeks 8 Weeks 12 Weeks 16 Weeks 20 Weeks 40 Weeks
Aer. Only 1017.5 ± 227.6 1974.7 ± 491.7 A 41.7 ± 16.9 A 22.0 ± 11.7 79.0 ± 50.5 2.5 ± 1.0 A
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Wheatsamplesfromthe12binstreatedwithmethoprenehadresiduesthatvariedfromabout
0.53 ppm in the top-treated bins to a range of 1.0 to 2.0 ppm in completely treated bins that were
measuredatthestartofthestudyjustafterbin-loading. Theaverageproportion(˘SEofthemeans)
of methoprene residue remaining at the end of the 40-week study was about 36.9% (˘3.8) for the
top-treated bins, 71.4% (˘8.9) for the methoprene only bins, and 68.7% (˘10.7) for the bins with
methopreneplusaeration. Althoughgraintemperatureswerewarmerinnon-aeratedbins,which
couldhavecausedsomedegradationoftheappliedmethoprene,therewerenosignificantdifferences
inmethopreneresiduesbetweenaeratedinnon-aeratedbins(ANOVA,p>0.05).
Gradingresultsshowedthatthebulkdensityofthewheatandtheofficialqualitygradeshadnot
decreasedafter10monthsofstorageforanyofthetreatmentsappliedtothesebins. Allthesampled
whatwasdeterminedtohaveaU.S.grade=1forallbinsattheendofthestudy. Ifwehadincludeda
controlsetofbinswithnoaerationandnomethoprenewesuspectthatthosebinswouldhavesuffered
areducedqualityscoretoinsect-associatedfactors. However,asexplainedabove,wewereunableto
includesuchacontrolinthisexperiment. Theaveragenumbersofinsect-damagedkernels(˘SEof
themeans)per100gram(IDK)was9.0(˘4.2)intheaerationonlytreatment,4.3(˘1.3)and3.3(˘1.3)
meanIDKinthetop-layermethopreneandmethoprenealonetreatments,respectively;buttherewere
only2.0IDK(˘1.0)onaverageinthemethoprenecombinedwithaerationtreatment.
3.2. InsectPopulationsinBins
Tables1–6reportthemeannumbersofinsectsinsamplestakenfromthebinsatvarioustime
intervalsduringthe40-weekstudy. Probetrapssamples(Tables1,3and5)reportnumberstrapped
during one weeks in the top of each bin, whole grain trier samples are the numbers of insects in
anapproximately1-kgsampleofgraintakenfromeachbin. Insectnumbersoverallwerehigherin
samplestakenduringthewarmertimesofweek4,week8andweek12,afterwhichthenumberswere
lowerastemperaturesreducedinsectactivity. Incasesforwhichtherewerestatisticallysignificant
differencesamongthetreatmentsthehighestinsectcountswereinthebinstreatedwithaerationonly.
Table1. Meannumbers(˘SE)ofadultT.castaneumcapturedbyprobetrapsatdifferenttimesafter
binswereloadedwithwheatthatwaseithertreatedwithaerationonly,thetop50cm-treatedwith
methoprene,methoprenetreatmenttoallthewheatinabin,oraerationpluscompletetreatmentwith
methopreneonallgraininthebin.Meanswithincolumnswiththesameuppercaseletter,forwhich
therewasasignificant(pď0.05)F-statistic,arenotsignificantlydifferent(leastsignificantdifference
(LSD)test,p<0.05,SASInstitute,degreesoffreedomforanalysiswithindatefortreatmentwere3,12).
Treatments 4Weeks 8Weeks 12Weeks 16Weeks 20Weeks 40Weeks
Aer.Only 1017.5˘227.6 1974.7˘491.7A 41.7˘16.9A 22.0˘11.7 79.0˘50.5 2.5˘1.0A
Meth.Top 660.1˘242.5 974.2˘473.7AB 153.0˘79.3A 46.0˘28.1 33.7˘30.4 1.5˘0.9B
Meth.Total 772.5˘383.4 340.2˘340.2B 24.2˘7.5B 5.0˘1.0 1.7˘1.4 0.2˘0.2B
Aer.+Meth. 250.2˘33.2 301.0˘73.3B 7.2˘10.1B 5.5˘1.3 0.0˘0.0 0.0˘0.0B
Fandp 2.1,0.16 5.1,0.02 4.2,0.03 2.4,0.12 1.6,0.25 3.3,0.05
Table2.Meannumbers(˘SE)ofadultT.castaneumcollectedingraintriersamplesatdifferenttimes
afterbinswereloadedwithwheatthatwaseithertreatedwithaerationonly,thetop50cm-treated
withmethoprene,methoprenetreatmenttoallthewheatinabin,oraerationpluscompletetreatment
withmethopreneonallgraininthebin.Meanswithincolumnswiththesameuppercaseletter,for
whichtherewasasignificant(p<0.05)F-statistic,arenotsignificantlydifferent(LSDtest,p<0.05,SAS
Institute,degreesoffreedomforanalysiswithindatefortreatmentwere3,12).
Treatments 4Weeks 8Weeks 12Weeks 16Weeks 20Weeks 40Weeks
Aer.Only 11.7˘227.6A 5.7˘2.2A 5.1˘1.8A 6.4˘4.7 3.4˘2.2 2.5˘1.0
Meth.Top 0.8˘0.4B 2.2˘0.7A 3.0˘1.1AB 1.5˘1.5 1.5˘0.8 0.0˘0.0
Meth.Total 1.05˘0.5B 0.8˘0.6B 1.0˘0.4BC 0.6˘0.6 0.2˘0.2 0.0˘0.0
Aer.+Meth. 0.6˘0.4B 1.9˘0.5B 0.4˘0.4C 0.6˘0.4 0.6˘0.3 0.0˘0.0
Fandp 23.5,<0.01 4.3,0.03 4.7,0.02 1.4,0.28 1.6,0.25 1.0,0.42
Insects2016,7,25 6of12
Table3. Meannumbers(˘SE)ofadultR.dominicacapturedbyprobetrapsatdifferenttimesafter
binswereloadedwithwheatthatwaseithertreatedwithaerationonly,thetop50cm-treatedwith
methoprene, methoprenetreatmenttoallthewheatinabin, oraerationpluscompletetreatment
withmethopreneonallgraininthebin.Meanswithincolumnswiththesameuppercaseletter,for
whichtherewasasignificant(p<0.05)F-statistic,arenotsignificantlydifferent(LSDtest,p<0.05,SAS
Institute,degreesoffreedomforanalysiswithindatefortreatmentwere3,12).
Treatments 4Weeks 8Weeks 12Weeks 16Weeks 20Weeks 40Weeks
Aer.Only 1.2˘1.0 1.7˘0.6 3.5˘1.0 1.2˘0.8 9.7˘8.7 0.2˘0.2B
Meth.Top 1.2˘1.0 1.5˘1.0 2.7˘1.8 0.0˘0.0 17.8˘9.8 0.0˘0.0B
Meth.Total 3.7˘0.5 0.2˘0.2 0.5˘0.5 0.5˘0.5 1.2˘0.5 0.8˘0.5AB
Aer.+Meth. 4.5˘1.8 0.2˘0.2 0.5˘0.3 0.5˘0.5 2.2˘0.7 2.0˘0.7A
Fandp 0.9,0.46 1.6,0.23 2.7,0.10 1.14,0.40 2.1,0.16 4.53,0.02
Table4.Meannumbers(˘SE)ofadultR.dominicacollectedingraintriersamplesatdifferenttimes
afterbinswereloadedwithwheatthatwaseithertreatedwithaerationonly,thetop50cm-treated
withmethoprene,methoprenetreatmenttoallthewheatinabin,oraerationpluscompletetreatment
withmethopreneonallgraininthebin.Meanswithincolumnswiththesameuppercaseletter,for
whichtherewasasignificant(p<0.05)F-statistic,arenotsignificantlydifferent(LSDtest,p<0.05,SAS
Institute,degreesoffreedomforanalysiswithindatefortreatmentwere3,12).
Treatments 4Weeks 8Weeks 12Weeks 16Weeks 20Weeks 40Weeks
Aer.Only 2.7˘0.7 2.8˘0.9AB 4.4˘1.1A 2.4˘1.0 3.8˘1.6A 2.5˘1.0
Meth.Top 0.6˘0.2 4.0˘1.4A 2.2˘1.5AB 2.0˘1.1 1.3˘0.6B 0.0˘0.0
Meth.Total 1.3˘0.4 1.0˘0.7B 0.8˘0.4B 0.2˘0.2 0.6˘0.4BC 0.0˘0.0
Aer.+Meth. 1.7˘0.5 0.9˘0.5B 1.1˘0.5AB 0.4˘0.2 0.0˘0.0C 0.0˘0.0
Fandp 2.2,0.14 2.9,0.08 2.9,0.08 2.47,0.11 15.6,<0.01 0.8,0.50
Table5.Meannumbers(˘SE)ofadultC.ferrugineuscapturedbyprobetrapsatdifferenttimesafter
binswereloadedwithwheatthatwaseithertreatedwithaerationonly,thetop50cm-treatedwith
methoprene,methoprenetreatmenttoallthewheatinabin,oraerationpluscompletetreatmentwith
methopreneonallgraininthebin.Meanswithincolumnswiththesameuppercaseletter,forwhich
therewasasignificant(p<0.05)F-statistic, arenotsignificantlydifferent(LSDtest, p<0.05, SAS
Institute,degreesoffreedomforanalysiswithindatefortreatmentwere3,12).
Treatments 4Weeks 8Weeks 12Weeks 16Weeks 20Weeks 40Weeks
Aer.Only 40.0˘13.4 34.0˘0.6 26.20˘9.6 24.2˘13.4A 5.2˘1.9A 43.5˘12.32A
Meth.Top 29.0˘10.8 20.0˘1.0 14.50˘11.2 7.0˘1.7A 7.2˘2.5A 11.5˘10.2B
Meth.Total 45.8˘8.5 28.7˘0.2 2.8˘1.0 1.5˘0.9B 0.0˘0.0B 0.0˘0.0B
Aer.+Meth. 43.5˘28.2 25.3˘7.0 5.2˘2.6 3.2˘2.1B 0.8˘0.8B 1.0˘0.4B
Fandp 0.2,0.87 0.2,0.90 2.4,0.12 4.1,0.03 6.8,<0.01 4.53,0.02
Table6.Meannumbers(˘SE)ofadultC.ferrugineuscollectedingraintriersamplesatdifferenttimes
afterbinswereloadedwithwheatthatwaseithertreatedwithaerationonly,thetop50cm-treated
withmethoprene,methoprenetreatmenttoallthewheatinabin,oraerationpluscompletetreatment
withmethopreneonallgraininthebin.Meanswithincolumnswiththesameuppercaseletter,for
whichtherewasasignificant(p<0.05)F-statistic,arenotsignificantlydifferent(LSDtest,p<0.05,SAS
Institute,degreesoffreedomforanalysiswithindatefortreatmentwere3,12).
Treatments 4Weeks 8Weeks 12Weeks 16Weeks 20Weeks 40Weeks
Aer.Only 6.4˘2.8A 17.7˘2.2A 12.0˘3.5A 22.4˘12.3A 31.0˘3.8A 7.0˘2.3A
Meth.Top 1.7˘0.9AB 1.5˘1.0B 0.6˘0.4B 2.9˘1.8AB 3.1˘1.2B 0.0˘0.0B
Meth.Total 1.0˘0.2AB 0.4˘0.4bB 0.0˘0.0B 0.0˘0.0B 0.0˘0.0C 0.0˘0.0B
Aer.+Meth. 1.0˘0.6B 1.5˘0.8B 0.2˘0.2B 1.5˘0.7B 0.0˘0.0C 0.0˘0.0B
Fandp 2.6,0.10 33.69,<0.01 23.0,<0.01 2.6,0.06 6.8,<0.01 27.2,<0.01
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ThereweresignificantdifferencesamongtreatmentsfornumbersofT.castaneumadultsinprobe
trapsateightweeks,12weeksand40weeks(ANOVA,p<0.05)withthelowestnumberscaptured
in bins completely treated with methoprene and under aeration (Table 1). Adults trapped in the
aeration-onlybinsaveraged1,017and1,975at4weeks(August)and8weeks(September),andthese
numbersaretremendouslyhigherthanexpectedgiventhatonly400adultshadbeenaddedtoeach
binduringthefirstfourweeksofstorage. Thus,highnumbersofT.castaneumlikelyresultedfrom
field immigrants to these bins. Nevertheless, by week 12 the population suppression effect of the
total methoprene treatment as a growth regulator was seen in comparison to the methoprene top
andaerationonlytreatments. T.castaneumadultscollectedingraintriersamplesof1kgofwheat
showedthatthereweresignificantdifferencesinmeanadultnumbersamongthedifferenttreatments
taken at the first three sampling periods, with the lowest beetle densities being in treatments that
hadmethopreneappliedtotheentiregrainmass(Table2). of4,8and12weeksbuttherewereno
significant changes in mean adult numbers among all treatments taken at the last three sampling
months(p>0.2)(Table2).
Numbers of R. dominica adults caught in probe traps showed that there were no significant
treatmenteffects(p>0.05)inthefirstfivesamplingdates,butthelastsampletimeof20weeks,atthe
endofthestudy,showedsignificantlymorebeetlesintheaerationplusmethoprenebins(Table3).
ThetrapcapturesforR.dominicawereverylowcomparedtothoseforT.castaneum,whichprobably
reflectsthedifferencesinmobilitybetweenadultsofthesetwospecies. Insectscounteddirectlyfrom
graintriersamplesgaveadirectmeasurementofinsectdensityatthetimeofsampling(Table4),and
infiveoutthesixsampletimesthereweresignificanttreatmenteffectswiththelowestinsectnumbers
foundinthemethopreneonlyandmethopreneplusaerationbins.
ThemeannumbersofC.ferrugineusadultsrecoveredbyprobetrapsandingraintriersamples
areshowninTables5and6,respectively. Therewerenosignificantdifferencesamongtreatmentsfor
meanadultnumbersinprobetrapsforthefirstthreesamplingperiods,butthereweresignificant
treatmenteffectsseeninthelastthreeperiodswithtotalmethoprenetreatmentshavingthelowest
numbersofC.ferrugineus. Nevertheless,thereweresignificantdifferencesinmeanadultnumbers
foralltreatmentsatthelastthreesamplingmonths(Table5). Graintriersamplesofgrainshowed
treatmenteffectsfornumbersofC.ferrugineusateachofthesixsamplingtimes(Table6),withthe
highestnumbersintheaerationonlybinsandsubstantiallylowernumbersinbinsthathadanyofthe
methoprenetreatments.
3.3. LaboratoryBioassaysofTreatedGrainfromBins
Therewasnomortalityofanyoftheparentadultbeetlesaddedtoexperimentalgrainthathad
been collected just after bin-loading, 20 weeks and 40 weeks after loading, whether on untreated
wheat (aeration only) or methoprene-treated wheat, following the one week of exposure during
controlledlaboratoryinfestations. Adultprogenyemergencefromthesesamplesexposedtolaboratory
infestationsofbeetlesarereportedinTable7. TherewasjustoneTcastaneumadultproducedinone
jar of wheat taken from an aeration + methoprene bin 40-weeks after treatment, while there were
no adults produced in any of the other samples that had been treated with methoprene. Results
weresimilarforR.dominicaexposedtowheattakenfrombinsonedayafterloading,forwhichthere
wereonlyafewbeetlesproducedinafewjars. At20weeksand40weekstherewerelownumber
ofR.dominicaproducedinthegrainthathadthemethoprenetoponlytreatment,whilenearlyzero
R.dominicaweregeneratedinwheatfrombinstreatedentirelywithmethoprene. Bioassaysusing
S.oryzaeweremarkedlydifferentthanthosewiththeothertwobeetlespeciesashundredsofbeetles
weregeneratedfromallsamplesofgraininthisexperiment. Significanttreatmenteffects(p<0.01)
forS.oryzaeprogenyproductionwerefoundonlyforthe20-weekoldgrain,althoughallmeanswere
quitehigh,rangingfrom430.5to681.3.
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Table7.Meannumbers(˘SE)ofF1progenyfrom50parentaladultsofeitherT.castaneum,R.dominica,
andS.oryzaeexposedon100gofwheatonedayaftertreatmentinbins,20weeksand40weeksafter
treatment.Meansforeachspecieswithinacolumnhavingthesameuppercaseletter,forwhichthere
wasasignificant(p<0.05)F-statistic,arenotsignificantlydifferent(LSDtest,p<0.05,SASInstitute,
degreesoffreedomforanalysiswithindatefortreatmentwere3,12).
Species Treatment 1Day 40Weeks 40Weeks
T.castaneum AerationOnly 248.5˘14.3A 133.2˘22.7A 278.5˘2.2A
MethopreneTopOnly 0.0˘0.0B 0.0˘0.0B 0.0˘0.0B
MethopreneTotal 0.0˘0.0B 0.0˘0.0B 0.0˘0.0B
Aeration+Methoprene 0.0˘0.0B 0.0˘0.0B 0.3˘0.2B
FandPvalues 1096,<0.01 105,<0.01 352,<0.01
R.dominica AerationOnly 696.7˘37.9A 482.0˘22.7B 514.7˘2.2A
MethopreneTopOnly 1.5˘1.2B 31.2˘25.6B 25.2˘8.4B
MethopreneTotal 0.0˘0.0B 0.7˘0.7B 0.2˘0.2C
Aeration+Methoprene 0.5˘0.5B 0.2˘0.2B 0.2˘0.2C
FandPvalues 926,<0.01 105,<0.01 352,<0.01
S.oryzae AerationOnly 680.5˘37.9 598.8˘22.7AB 724.2˘55.9
MethopreneTopOnly 825.7˘207.2 430.5˘53.9B 517.7˘81.6
MethopreneTotal 1260.0˘588.6 582.3˘34.3AB 487.0˘50.8
Aeration+Methoprene 464.5˘133.5 681.3˘61.2A 680.0˘100.2
Fandpvalues 1.0,0.42 5.4,<0.01 2.4,0.12
Our bioassays for effects of treated grain on the development of P. interpunctella following
inoculationofjarswith20eggsshowedthatadultemergencewasnearly100%,with19or20emerging
per jar, for wheat taken form the aeration-only bins at one day, 20 weeks or 40 weeks of storage
(Table8). Wheatfrombinsreceivingthetop-onlytreatmentofmethoprenehadonly3.9%emergence
fromgrainsampledat1-dayafterstorageandthenaslightincreasetoapproximately8%and22%at
thelongerstorageperiodsof20weeksand40weeks,respectively. Wheatfrombinstreatedentirely
withmethoprenehadalmostnoadultemergence,onlytwoadultsat40weeks,fromthegrainsampled
atanyofthethreetimeperiods.
Table8. Meanpercentage(˘SE)ofP.interpunctellaadultsemerging(fromadditionof20eggsper
jar)from40g-samplesofwheattakenfrombinsatonedayaftertreatment,20weeksand40weeks
post-treatment.Meanswithinacolumnwithsameletter,forwhichtherewasasignificant(p<0.05)
F-statistic, arenotsignificantlydifferent(LSDtest, p<0.05, SASInstitute, degreesoffreedomfor
analysiswithindatefortreatmentwere3,12).
Treatment 1Day 20Weeks 40Weeks
AerationOnly 98.7˘1.3A 98.8˘1.3A 98.8˘1.3A
MethopreneTopOnly 3.9˘2.5B 8.0˘5.1B 21.8˘10.6B
MethopreneTotal 0.0˘0.0C 0.0˘0.0C 0.0˘0.0C
Aeration+Methoprene 0.0˘0.0C 0.0˘0.0C 0.0˘0.0C
Fandpvalues 190.5,<0.01 79.4,<0.01 41.1,<0.01
4. Discussion
Theresearchreportedheremayrepresentthefirstcontrolledfieldtrialofmethopreneapplied
to bulk-stored grain over a long storage period. The work demonstrates the residual activity of
methopreneasanIGRduringgrainstorageandtheresultssuggestthatmethoprenecanreducepest
populationincreasesandassociatedgraindamageovertime. Datacollectedongraintemperatures
clearlyshowthatcontrolledaerationcanreducethetemperatureofstoredwheatbeginninginjust
daysfromthestartofstorageandthenachievea10-dgreedifferencebetweenaeratedandnon-aerated
binsforover20weeks. Lowergraintemperatureslikethoseachievedherecandoubleortriplethe
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egg-to-adultdevelopmenttimesofgrainpestscomparedtothoseofwarmerunaeratedgrain,resulting
insubstantialdecreasesintherateofpestpopulationgrowth[4]. Insectpopulationsmeasuredwith
probetrapsanddirectgrainsamplesweregenerallythehighestinouraeratedbins,butwedidnot
haveanon-methoprenewithno-aerationasatruecontrolforcomparison. Oneobjectiveforstudying
thecombinationofaerationcoolingwithmethopreneapplicationwastoseeifpestpopulationgrowth
and could be lower and IGR activity higher from grain in those combination bins compared to
methoprene-onlybins. However,numbersofthethreebeetlespeciesstudieddidnotdifferbetween
thesetwobintreatmentsineitherprobetrapsamplesordirectgrainsampleatanyofthesampling
periods. Similarly,IGRactivitytestedinlaboratoryassaysagainstfourspeciesshowednoimproved
activityfromaeration. Ourinsectdatathereforesuggestthatlevelsofmethopreneonaeratedand
non-aeratedwheatweresimilar. Thisconclusionisvalidatedbyourchemicalresidueanalysesthat
foundthepercentageofmethopreneremainingonnon-aeratedwheatwasnotstatisticallydifferent
fromthatonaeratedwheatattheendofthe40-weekstudy.
Insectnumbersinprobetrapsandthosecollectedfromdirectgrainsamplesprovidedinformation
onvariationofinsectpopulationsovertimeandamongthebinswithdifferentexperimentaltreatments
relatedtomethopreneandaeration. Grainprobetrapsarerelativesamplingtoolsthatarebiasedby
mobilityoftheinsectsbeingtrapped,comparedtograinsamplesthatgiveaone-timemeasureofinsects
presentinthatgrainsampleatthetimeofcollection.Insectswithmobileadultsthatmoveanddisperse
activelythroughagrainmass,suchasT.castaneumandC.ferrugineusstudiedhere,willbecaughtat
highernumbersinprobetrapscomparedtoalessmobilespecieslikeR.dominica[26]. Ourexperiment
foundthatnumbersofTcastaneumandC.ferrugineusfrombothprobetrapsandgrainsampleswere
significantlyreducedinmostbinswithmethopreneduringoneormoresamplingperiodsofthisstudy
(Tables1,2,5and6). ThenumbersofR.dominicaadultshowever,werequitelowinbothprobetrap
samplesandgrainsamples,butitwasthegrainsamplesthatshowedsignificantmethopreneeffects
duringseveralweeks(Tables3and4)Interestingly,thenumbersofTcastaneuminprobetrapswere
muchhigherthanexpectedatthefirstsamplingtime,fourweeksafterbin-filling. Eachofour16bins
had100adultsofthethreebeetlespeciesaddedweekly,foratotalof400adultsaddedbyweek4,
butprobetrapnumberintheaerated-onlybinsaveragedover1000beetles,suggestingthanseveral
thousandadultT.castaneumwereineachbinatweek4. Thesehighnumberswereunlikelytohave
resultedfromreproductioninthat4-weekperiod;thewarmgraintemperaturesof30–32˝Cinthose
non-methoprenebinsduringthattimelikelydidnotcontributetogenerating1000’sofnewadults
basedondevelopmentratesofthosespecies[13]. Binswerecleanandfreeofgrainbeforeloading,
soresidualT.castaneumarenotimplicatedincontributingtothesehighnumbers. Morelikelyisthe
possibilitythatferalimmigrantT.castaneumadultsenteredthebinsviaopeningsusedinaeration
andventilation(e.g.,thatpartiallyopenedtoploadingport),andperhapsstimulatedbythenaturally
producedaggregationpheromonesemittedbytheresidentbeetles[27].
OurlaboratorybioassaysdemonstratedthatactivityofmethopreneasanIGRwasretainedfor
upto40weeksinbinsofwheattreatedat1ppmjustonthetop50cmorwiththeentiregrainmass
treatedat1ppm(Tables7and8). Allgrainsamplesusedforthelaboratoryassayswerecollected
withagrainsamplertoadepthof1.5m,soitisunderstandablethatthesamplesfromthetop-treated
methoprenebinshadlessresidualmethoprenethanthosefromcompletedtreatedbins. Bioassayswith
P.interpuntella,T.castaneumandR.dominicahadloworcompletelynoprogenyproducedinwheat
frombinswitheithertop-treatedorcompletelytreatedmethopreneapplication,andtop-treatedwheat
hadintermediatebutverylownumbersofadultprogenyforP.interpuctellaandR.dominica. The
partialeffectivenessagainstR.dominicainbioassaysdoneat20and40weeksislikelyduetofemales
ofthisspecieslayingeggsinthegrainmassoutsideofkernels,whereneonatelarvaemayreceivean
effectivedoseofmethoprene,followedbygoodsurvivalfromunaffectedlarvaethatboreintoakernel
tocompletedevelopmentwheretheyescapethemethoprene. Otherstudiesalsoreportedmethoprene
control of R. dominica on wheat and on rice [9,10]. Implications for sub-optimal applications of a
“top-dressing”tograinwitharesidualinsecticidewerealsosuggestedinlaboratorystudiesthatused
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thedesiccantdiatomaceousearth,DE[28]. Inthatstudy,R.dominicawereabletopenetratethrougha
toplayerofwheattreatedwithdiatomaceousearth(DE)andovipositintheuntreatedwheatbelowthe
treatedlayer,eventhoughsomeadultfemaleslaterdiedfromtheexposuretoDE.ProgenyofS.oryzae
wereplentifulafterexposureoftreatedwheatto50adultsforoneweekinourbioassays,andshowed
noexpectedtreatment-relatedeffectsintheselaboratorybioassays. TheseresultswithS.oryzaecanbe
explainedbythefactthatmethopreneisnotacutelytoxictoadultinsectsandthisspeciesdepositsits
eggsandcompletesfulldevelopinsideakernelofwheatwherenomethopreneispresenttoimpair
development. ThissituationthatrendersmethopreneineffectiveagainstSitophilusoryzaewasalso
demonstratedby[29].
TheworkreportedherecontributestothegrowingnumberofstudiesonIGRsforstoredproduct
insectpests. WheatintheUSAisusuallystoredfor12-36weeksinfarmbins[30]andourstudyclearly
showsthatmethopreneappliedtowheatat1.0ppmcanprovideprotectionfrominsectinfestation
foratleast40weeksbasedontheinsectnumbersresultingintheinfestedbins,effectiveIGRactivity
for40weeksinthetreatedgrain,methopreneresidueanalysesandlowergraindamageofthetreated
wheat. Methoprene does not, however, control all grain insect pests, so alternative or additional
pestcontrolmethodsmaybeneeded[24],anditsuseformaangingresistancetoottherinsecticides
should be approached with caution. Methoprene is a synthetic mimic of insect juvenile hormone
andthereforecouldbeofinterestforresistancemanagementsinceitlikelyhasamodeofactionvery
differentfrommostchemicalinsecticidesinuse. However,resistancetomethoprenewasdocumented
inLasiodermaserricorne(F.)(Coleoptera: Anobiidae)asearlyas1994[31],andmethopreneresistancein
otherspeciesofstoredgraininsectswasreportedinrecentyears(e.g.,[32]). Nevertheless,aswithany
insecticide,judicioususeofmethopreneinstoredgrainintegratedpestmanagementcanbeeffective
andcouldservetocombatseriouspestswhilealsoreducingoverallchemicalinputs[4,11].
5. Conclusions
This research clearly shows that application of methoprene to stored wheat at the time
of bin-loading can protect that grain from insect infestation throughout 40 weeks of storage.
Theexperimentconfirmsthatthermostatically-controlledaerationtostoredwheat,suchthataeration
fansturnononlywhenoutsidetemperaturesarecoolerthanthosewithinthegrainmass,canresultin
substantialgraincoolingthatcandeterdevelopmentofinfestinginsects. Thehighlevelofinsecticidal
activityofthemethoprene-treatedwheatinourexperiments,whetherfromaeratedornon-aerated
bins, was essentially unchanged throughout 40 weeks of storage in these field bins. Methoprene
applied to just the top 50 cm of the grain elicited significant insect control compared to untreated
wheat,butadmixtureofmethoprenetotheentiregrainmasshadthebestresultsoverall. Thisresearch
providesoneofthefewcontrolledfieldvalidationsofpreviouslaboratorystudiesthatsuggestthe
applicationofmethopreneontocommercially-storedgraincanbeaneffectivealternativetotheuseof
othertraditionalinsecticidesandfumigants.
Acknowledgments:TheauthorsthankEdmondBonjourandRandyBeebefortechnicalassistance.Thisstudy
waspartiallyfundedbytheUnitedStatesDepartmentofAgriculture,AgriculturalResearchService;theUSDA
Cooperative State Research, Education, and Extension Service Pest Management Alternatives Program and
theRiskAvoidanceandMitigationProgram;theOklahomaAgriculturalExperimentStationandtheKansas
AgriculturalExperimentStation. Thisarticlereportstheresultsofresearchonly. Mentionoftradenamesor
commercialproductsinthispublicationdoesnotforthepurposeofprovidingspecificinformationanddoesnot
implyanendorsementorarecommendationbytheauthors’affiliationsforthemuse.
AuthorContributions: SamuelS.Liu,FrankH.ArthurandThomasW.Phillipsconceivedanddesignedthe
experiments;SamuelS.Liuperformedtheexperiments;SamuelS.Liu,FrankH.ArthurandThomasW.Phillips
analyzedthedata;DouglasVanGundycontributedtheDiaconII® usedinthestudyandprovidedanalytical
serviceforresidueanalyses;SamuelS.Liu,FrankH.ArthurandThomasW.Phillipswrotethepaper.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.Thefundingsponsorshadnoroleinthedesign
ofthestudy;inthecollection,analyses,orinterpretationofdata;inthewritingofthemanuscript,andinthe
decisiontopublishtheresults.