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Offlclal [ournal of the |nstltut Natlonalde la Pecherche Agronomlque (|NPA)and Deutschen |mkerbundes e.v. (D.|.8.) |SSN 0044-8435 ApldologleDO| l0.l007/sl3592-0l2-0l62-9A scientific note on the comparison ofairborne volatiles produced by commercialbumble bee (Bombus impatiens) andhoney bee (Apis mellifeia) coloniesJason R.Graham, Mark J.Carroll, PeterE.A.Teal & James D.Ellis1 3Your article is protected by copyright andall rights are held exclusively by INRA, DIBand Springer-Verlag, France. This e-offprintis for personal use only and shall not be self-archived in electronic repositories. If youwish to self-archive your work, please use theaccepted authors version for posting to yourown website or your institutions repository.You may further deposit the accepted authorsversion on a funders repository at a fundersrequest, provided it is not made publiclyavailable until 12 months after publication.A scientific note on the comparison of airborne volatiles producedby commercial bumble bee (Bombus impatiens) and honey bee(Apis mellifera) coloniesJason R. GRAHAM1, Mark J. CARROLL2, Peter E. A. TEAL2, James D. ELLIS11Entomology Nematology Department, University of Florida, PO Box 110620, Bldg. 970 Natural Area Dr., Gainesville,FL 32611, USA2Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, Chemistry Research Unit, U.S.Department of Agriculture, 1600 Southwest 23rd Drive, Gainesville, FL 32608, USAReceived 12 March 2012 Revised 30 June 2012 Accepted 19 July 2012Investigators haveshown(Stanghellini et al. 2000;SpiewokandNewman2006; Hoffmanet al. 2008)that commercial bumble bee (Bombus impatiensCresson, Hymenoptera: Apidae) colonies canserveas potential alternative hosts for the small hive beetle(SHB, AethinatumidaMurray, Coleoptera: Nitiduli-dae), ahoneybeepest(Lundie1940;NeumannandElzen 2004; Ellis and Hepburn 2006). Using olfacto-ry choice tests, Graham et al. (2011) found that SHBattraction to bumble bee colonies is chemicallymediated. SinceSHBs are attractedtobumblebeeand honey bee colonies, we expected the volatileprofiles produced by bumble bee and honey beecolony components to be similar. To test this,airborne volatiles produced by bumble bee and honeybeeadults, brood(eggs, larvae, andpupae), honey,storedpollen(=beebread), andwaxwerecollectedand analyzed.In June 2007, two commercial bumble bee (B.impatiens)quads(=eightcolonies;KoppertBiolog-ical Systems, Inc., Romulus, MI) were established atthe University of Floridas Bee Biology Unit inGainesville, FL (37.62929 N, 21.40582 W). Bothquads consisted of four bumble bee colonies contain-ingareproductivequeen, 200250workers, brood,and nesting material. Eight queenright honey beecolonies were housed in typical Langstroth styleequipment at the University of Floridas Bee BiologyUnit in Gainesville, FL.Volatileswerecollectedfromcolonycomponentsusingmethods modifiedfromSuazoet al. (2003).Thecolonycomponents (adult bees, brood, honey,pollen, andwax) wereextractedfromeight bumblebee and eight honey bee colonies. The samples wereplacedinglassvolatilecollectionchambers(3.8L)by component and colony at the USDA-ARS CenterforMedical, Agricultural, and Veterinary Entomology(CMAVE, Gainesville, FL). All volatile collectionswere conducted in a controlled environmental chambermaintained without light and at an ambient temperatureof 33 C. Charcoal-filtered and humidified air,controlled by carboloy flowmeters, was passed throughthe volatile collection chambers and into SuperQ filtersat arateof0.5L/min. All component volatileswerecollected for 14 h with the exception of brood volatilesthat were collected for 7 h. The abbreviated time framefor volatile collection ofbroodwasintendedto avoidstressing the unattended brood, recognizing the fact thatbroodmaystill be stressedinthe absenceof adultworkers. Adult beeswereprovisionedwithacottonwick saturated with a 50 % sugar water solution and anautoclavedsteel meshplatformonwhichtheadultscould crawl to minimize stress. Volatiles trapped on theSuperQ filters were extracted by eluting the filter with500Lof methylenechloride. Thesesamples werepooledbycomponent andbee type and stored in a70 C freezer (Revco Scientific, Inc., Asheville, NC)at CMAVE until use.volatile / Bombus impatiens / bumble bee / Apis mellifera / honey bee / olfactionCorresponding author: J.R. Graham,jgraham@ufl.eduManuscript editor: Monique GauthierApidologieScientific note* INRA, DIB and Springer-Verlag, France, 2012DOI: 10.1007/s13592-012-0162-9Author's personal copyThe pooled volatile samples were analyzed forboth bee types by component at the CMAVE using aHP-6890gaschromatograph(GC, Hewlett Packard,Palo Alto, CA) equipped with a HP-1 column(30m0.25 m,J&W Scientific,Folsom, CA). Thecolumn was linked to an HP 5973 mass spectrometerusing electron impact mode (70 eV, Agilent, PaloAlto, CA) withthegas carriedbyhelium. Volatilecompoundswereidentifiedbycomparisonofreten-tion times with available synthetic standards (Sigma-Aldrich, St. Louis, MO) and massspectra with massspectra libraries (NIST, University of GottenburgChemical Ecology, Sweden).Contrary to our hypothesis, we found that there isa high level of variability between the volatilescollected frombumble bee and honey bee colonycomponents as well as volatiles collected from wholecolonies. Only 6 of 147 detected volatiles werecommon to both bee species (Table I).2-Heptanone, present amongvolatilesemittedbyadults of bothbeetypes (TableI), is knowntobeproducedfrommandibular glandsof worker honeybees, acts as a forage marking pheromone, and canattract/repel guardhoneybees (Vallet et al. 1991).Limolene is the only volatile common to the brood ofboth species. Limonene, along with 2-heptanone,heptanal, pinene, octanal, terpenine, methyl benzoate,nonanal, anddecanal, was foundtobe honeybeehivevolatilescapableofbeingdetectedbysensillaeon the SHB antennae in Gas ChromatographyElectro-Antennographic Detection (GC-EAD) bio-assays (Torto et al. 2007a).The compounds 1-phenoxy-2-propanol and butyl-ated hydroxytoluene were detected in the honeyvolatiles of bothspeciesandthis isthefirst reportof these compounds being associated with eitherspecies. Butylated hydroxytoluene has been usedextensively in the food industry as a food antioxidantadditive (Branen 1975) and may have been present inthe nesting materials or artificial diets provided to thecolonies. Volatiles collected frombumble bee andhoneybeewaxcontainedthesharedcompounds2-nonanone and nonanal, while volatiles collected frombumble bee and honey bee pollen contained only theshared compound nonanal (Table I). Nonanal wasfound by Saleh et al. (2007) to be a chemosensory cue,left behind by Bombus terrestris at food locations, nestlocations, andneutral areas (areas neither associatedwithfoodlocationsor withnest locations). Nonanaland 2-nonanone were used in wind tunnel studieswhere SHBs werefound tobe attracted toablendofcompounds whichwere collectedfromadult honeybees; nonanal alsowasdetectedbySHBandhoneybees in GC-EAD (Torto et al. 2005, 2007a, b).In conclusion, commercial bumble bee and honeybeecolonies, whilesimilarenoughtohostthesameand similar pests and pathogens, remain differentregardingtheir volatileprofiles. It islikelythat thevolatileprofilesofhoneybeesandbumblebeesarecomplex,varyingwithtemperament,beehealth,andother circumstances. This studyprovides aninitialcomparisonof thechemical ecologyof commercialbumble bees and honey bees. With an understandingof the chemical ecology associated with both types ofTable I. Chemical compounds of volatiles collected from bumble bee and honey bee colony componentsColony componentChemicalcompoundQuality Molecularweight (amu)CAS number Adults Brood Honey Pollen Wax2-Heptanone 81 114.104 000110-43-0 xLimonene 90 136.125 000138-86-3 x2-Nonanone 90 142.128 000821-55-6 xNonanal 98 142.136 000124-19-6 x x1-Phenoxy-2-propanol 96 152.084 000770-35-4 xButylatedhydroxytoluene94 220.183 000128-37-0 xQuality refersto how closely the compound matches the resultfrom the GCMS library search (with 100 being an absolutematch and 0 being no match atall). Only like chemical compounds discovered in similar components with a quality rating80 that have been verified by the respective synthetic standard chemical compounds are reported hereinJ.R. Graham et al.Author's personal copybeecolonies, atool maybefoundtohelpconservetheseimportant pollinators andcontrol thedamagecaused by SHBs and other nest invaders.Note scientifique sur lacomparaisondes compossvolatiles librs dans lair entre les colonies dubourdoncommercial(Bombusimpatiens)etcellesdelabeille (Apis mellifera)EinewissenschaftlicheNotizberdenVergleichvonflchtigenSubstanzen, dievonkommerziellenHum-melvlkern (Bombus impatiens) und Vlkern derHonigbiene (Apis mellifera) in die Luft abgegebenwerdenREFERENCESBranen, A.L. (1975)Toxicologyandbiochemistryofbutylatedhydroxyanisoleandbutylatedhydroxytoluene. J. Am. OilChem. Soc 2(52), 5963Ellis, J.D., Hepburn, H.R. (2006) Anecological digest of thesmall hivebeetle(Aethinatumida), asymbiont inhoneybee colonies (Apis mellifera). Insectes Soc 53, 819Graham, J.R., Ellis, J.D., Carroll, M.J., Teal, P.E.A. 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