The previously suggested protection from microbial attack of ripening nectar by the addition of glucose oxidase by the common honey bee, Apis mellifera L., has been found to occur in 9 other eusocial Hymenoptera from 3 superfamilies. The antibacterial effect results from the accumulation of hydrogen peroxide produced by the enzyme during ripening of the stores

Burgett, D. Michael

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Glucose Oxidase: A Food Protective Mechanism in Social Hymenoptera'D. MICHAEL BURGETT2Department of Entomology, Cornell University, Ithaca, NY 14850ABSTRACTThe previously suggested protection from microbial from 3 superfamilies. The antibacterial effect resultsattack of ripening nectar by the addition of glucose oxi- from the accumulation of hydrogen peroxide produced bydase by the common honey bee, Apis mellifera L., has the enzyme during ripening of the stores.been found to occur in 9 other eusocial HymenopteraHoney-storing social insects are confronted withthe problem of protecting colony stores against micro-bial contamination. Ripened honey produced by thehoney bee, Apis mellif era L., is protected by its hy-perosmotic state. Only honey with less than about18.6% moisture shows such bacteriostatic activity.Dilute or unripened honey was shown by Sackett(1919) to display antibiotic activity.The antibacterial character in dilute Apis mellif erahoney, termed inhibine (Dold et al. 1937), was iden-tified as the enzymic production and accumulation ofhydrogen peroxide (White et al. 1962, 1963). Theenzyme responsible is a glucose oxidase. The pres-ence of a glucose oxidase in the hypopharyngealglands of the honey bee was reported by Gauhe(1941), who did not connect its presence in theseglands with the antibacterial nature of dilute honey.This was the first discovery of a glucose oxidasefrom an animal source.The following is a report on the taxonomic breadthof glucose oxidase in the stored carbohydrates ofsocial insects.MATERIALS AND METHODSAn assay involving the colorimetric determinationof evolved hydrogen peroxide in dilute honey was de-veloped for the analysis of glucose oxidase in Apismellif era honey (White and Subers 1963). Colorformation by an oxidized chromogen (o-dianisidine)in the presence of hydrogen peroxide and peroxidaseis the basis of this assay. With modifications forsmall sample sizes (0.2-1.0 g), this peroxidase-dyeassay was employed for the determination of glucoseoxidase activity in honeys of eusocial Hymenoptera.Ten-mi breakers were substituted for White andSubers 8-oz (225 ml) bottles in the incubation of thediluted honeys and/or honey protein extracts. Incu-bation was done in an agitating (100 shakes/mm)water bath.Enzyme activities were determined for dilute wholehoneys and crude honey protein extracts prepared bydialyzing honey with running tap-water (20°C) for24 h. Enzyme activity was expressed as the g H202/mg honey protein/h. Protein content was deter-mined by the method of Lowry (1951). The resultsof the protein determination are seen in Table 1.Honeys for investigation came from the followingi Received for publication Jan. 8, 1974.' Present address: Dept. of Entomology, Oregon State Univ.,Corvallis 97331.545species, Formicoidea: Myrmecocystus 'mexicanusWesmael (honey ant); Vespoidea: Protonectarinasylveirae (Saussuer) (honey wasp); and Apoidea:Bombus perplezus Cresson, B. fervidus (F.) (bumblebees), Trigona (Trigona) fuscipennis Friese, T.(Scaptotrigona) sp. (stingless bees), Apis me/hferaL., A. indica F., A. florea F. (honey bees).The diluted whole honeys and protein extracts (1ml of extract was added to 4 ml of a 0.4 M glucosesubstrate) were incubated in a 35°C agitating waterbath. Peroxide accumulation was determined at theend of 1 h.RESULTS AND DiSCUSSIONGlucose oxidase was a component of the honeysfrom all the species examined. No hydrogen peroxidewas found in any of the honeys prior to dilution witha soditim-potassium phosphate buffer, pH 6.4. Thebuffer served as both a diluent and to adjust the pHof the honey solutions to eliminate any photo-oxida-tion of the enzyme (White and Subers 1964). Per-oxide was liberated only upon dilution and controlledincubation of the honeys. The quantitative results forperoxide accumulation from diluted whole honeys,protein extracts, and lyophilized protein extracts aresummarized in Table 2. Peroxide accumulationvalues are valuable qualitatively but do not repre-sent strict quantitative values comparable betweenspecies.All the honey-producing species examined in thisTable 1.Protein content of eusocial Hymenopterahoney.FormicoideaMyrmecocystus mexicanusVespoideaPro tonectarina sylveiraeApoideaApis tnelhif eraApis indicaApis floreaBomubus perplexusBombus fervidusTrigona (Trigona) fuscipennisTrigona (Scaptotrigona) sp.° Mean value of actual honey examined for enzyme activity.Protein content of a given honey sample will depend largelyupon the floral source of the honey.Species mg protein/g honey°3.722.182.022.722.980.881.403.994.54546 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA [Vol. 67, no. 4Table 2.-Glucose oxidase activity in eusocial Hymen-optera honey.Whole honey not tested for enzyme activity.study are eusocial. Sociality has developed indepen-dently many times in the bees, wasps, and ants. Theability of these insects to utilize plant carbohydratesas reserve colony stores developed to a great extentbecause of the large number of workers per colonyand the advanced state of polyethism in eusocialReprinted from theANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICAHymenoptera. Selective pressure has sustained aglucose oxidase system for the protection of theseenergy stores against microbial contamination.A honey glucose oxidase represents a defense sys-tem at the colony level. This enzyme mechanism isnow seen to be present in honeys from representativemembers of 3 superfamilies of the Hymenoptera:Formicoidea, Vespoidea, and Apoidea. The probabil-ity is high that this enzyme occurs among most honeystoring social insects.REFERENCES CITEDDold, H., D. H. Du, and S. T. Dziao. 1937. Nachweisantibakterieller, hitze und lichtempfindlicher Hem-mungsstoffe (Inhibine) in Naturhonig (Bluten-honig). Z Hyg. Infektkrankh. 120: 155-67.Gauhe, A. 1941. Uber em glukoseoxydierendes Enzymin der Pharynxdruse der Honigbiene. Z. Vergl.Physiol. 28: 211-53.Lowry, 0. H., N. J. Rosebough, A. L Farr, and R. J.RandalL 1951. Protein measurement with folinphenol reagent. J. Biol. Chem. 193: 265-75.Sackett, W. G. 1919. Honey as a carrier of intestinaldiseases. Bull. Agric. Exp. Stn., Colorado Agric.College 252: 1-18.White, J. W., M. H. Subers, and A. I. Schepartz. 1962.The identification of inhibine. Am. Bee J. 102:430-31.1963. The- identification of inhibine, the antibacterialfactor in honey, as hydrogen peroxide, and itsorigins in a honey glucose oxidase system. Biochem.Biophys. Acta 73: 57-70.White, J. W., and M. H. Subers. 1963. Studies onhoney inhibine. 2. A chemical assay. J. Apic. Res.2: 93-100.1964. Studies on honey inhibine. 4. Destruction ofthe peroxide accumulation system by light. J. FoodSci. 29: 819-28.SampleActivity-beg HSO2/mghoney protein/hWholehoneydilutedDialyzed ProteinExtract-Substrate,0.4 M glucoseAqueoussolutionLyophil-izedFormicoideaMyrmecocystusme.ricanus 271.8 265.9VespoideaPro tonectarinasylveirae 493.1 459.4 320.8ApoideaApis mellif era 140.6 55.6 45.9Apis indica 20.4 22.3 15.6Apis florea 172.3 25.6 18.3Bombus perplezus 943.3 386.4Bombus fervidus 124.6 63.5Trigona (Trigona)fuscipennis 103.1 209.7 201.9Trigona (Scapto-trigona) sp. 248.6 400.0 315.3

Glucose Oxidase: A Food Protective Mechanism in Social Hymenoptera

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Glucose Oxidase: A Food Protective Mechanism in Social Hymenoptera

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