A. P. TULLOCH. National Research Council of CanadPrairie Regional Laboratory, Saskatoon, Saskatchewan

ABSTRACT

This n:vicw deals with waxes of mem­ners of two quite different groups of'insects the bees and the scale insects,which ~eerete large amounts of wax, Theformer use wax as a structural materialand the latter as a protective material.The compositions of waxes from sorne ofthese insects are described and particularattention is paid to the compositions ofthe unhydrolyzed waxes and to thepresence of hydroxy acids. New analysesof beeswax and of wax of a species of.humble bee are reported. The structuresof the diesters, hydroxyesters and diols ofneeswax are elucidated. The bumble beewax con tains major proportions of satu­rated and unsaturated hydrocarbons, andof long chain saturated, mono- and diun­saturated esters. The relationship betweenstru<.:tureand function of the waxes is dis­cussed.

INTRODUCTION

1nsects bclong to the class of animais withIhe fQllowing characteristics: the body isdivided into head, thorax and abdomen; thehead carries a single pair of antennae; and thethorax carries three pairs of legs and usuallyone or two pairs of wings. These features dis­linguish them from crabs, lobsters, spiders,mites. millepedes etc.

Ncarly a million species of insects have beendl'scribed and several times this number mayactually exist. 1nsects are divided into about 28orders; severa] include a very large number ofspecics but most have only a few thousand. Asimple introduction to insect biology has beenwrillen by Wigglesworth (1).

Thl' orders which con tain sorne of the bestknown insects arc listed in Table 1 togetherwith the approximate number of species in theonkr: for convenience the other, less weilknown, orders have been omitted. The first 13orders con tain those insects whose young are

1Issued as National Research Council of CanadaNo. 1 1260.

20nc of six papers to be published from theSymposium on Natural Waxes, presented at the AOCSMcl'linlt. San Francisco, April 1969.

o different from the adults and the lastseven con tain those whose young are radicallydifferent from the adults.

Most or ail insects are protected from waterloss by a thin film of wax in the cuticle; thistype of wax is discussed by Jackson and Baker(this symposium). Clearly the study of the com­position of insect waxes is an enormous fieldwhich has barely been scratched. Jackson andBaker review two different species of cricket,four different species of cockroach, one speciesof moth and one species of scale insect. Thehuge orders of flies and beetles do not seem tohave been examined at ail.

ln this review 1 shall deal with a few mem­bers of two orders of insects which secretemuch larger amounts of wax than those whichproduce only a thin waxy cuticle. The mostimportant one to be discussed is the honey bee(genus Apis, family Apidae). 1 shall also dealwith the wax of bumble bees (genus Bombus,family Apidae). Bees are considered to beamong the most highly developed insects.

The other, more primitive, group of insectswhich secretes large amounts of wax is thatcomprising the scale insects. They are given thisname because, in many species, the female isprotected by a scale or shield consisting of amixture of wax and cast skins. These insects aremembers of several families of the sub orderHomoptera of the order Hemiptera (Bugs,Table 1). The scale insects have been investi­gated because many of them are serious agri­cultural pests though a few are of commercialvalue.

The appearance and function of the waxes isdiscussed later.

The chemistry of waxes secreted by insectshas been studied over the last 150 years. Litera­ture prior to 1954 was reviewed by Warth (2),but more prominence was given to earlytheories of composition than to later, morereliable, results. The present review will dealonly with what seem to have been the mostimportant advances, particularly those obtainedby modern chromatographic methods, and willreport new analyses of beeswax and wax of aspecies of bumble bee.

Beeswax

At one time the word wax meant only bees-

[ 1)

A. P. TULLOCH

TABLE 1

Sorne Orders of Insects (J)

Order

Ilragonflies (Odonata)May·flic~ (Ephemeroptera)Cockroaches and Mantids (Dictyoptera)Stone-flies (Plecoptera)Termites (Isoplera)Earwigs (Dermaptera)Stick-;n,ects (Phasmida)Grasshoppers, Locusts, Crickets (Orthoptera)Book-lice (Psocoptera)Bird-Iice (Mallophaga)Sucking·lice (Anoplura)Thrip~ (Thysanoptera)Bugs, Aphids, Scale Insects etc. (Hemiptera)Lacewings etc. (Neuroptera)Caddis flies (Trichoptera)Uutlcrflies and Moths (Lepidoptera)Flics and Mosquitoes (Diptera)Fleas (Siphonaptera)AnIs, Bees, Wasps etc. (Hymenoptera)Beellcs (Coleoptera)

Approx. No. of Species

50001500600015001700110020001000011002600230

30005500050005000

200000850001100

100000275000

wax and as the most important insect wax ithas attracted the most attention, in fact, Ikuta(3) has remarked that there were about 140publications dealing with beeswax chemistrybetween 1848 and 1930. Beeswax generallyrefers to wax of the European bee, Apismel/i/era, but Asiatic species A. dorsata, A.Jlona and A. indica are sometimes also com­mercial sources of wax. This wax is known asEast Indian beeswax or Ghedda wax. Func­tional group analysis of Ghedda wax (3-5) indi­ca t cs only minor qualitative differencesbetwcen its composition and that of commonbceswax. Results of investigations of Gheddawax will, therefore, be included with those ofbceswax. Waxes of wild bees of the generaTrigona and Me/ipona (also in family Apidae)have been examined (6), but not by modernmethods. Wax of a few species of Bombus hasalso been investigated (7).

To compare properties of waxes and to con­sider their biosynthesis it is clearly important toknow the composition of the natural unhy­drolyzed wax. Sorne early investigators did tryto determine this, but most investigations havebeen carried out using saponification products.Since wax components are complex mixtures ofhomologs, it was difficulf to make an accurateanalysis prior to the application of gas liquidchromatography (GLC). The early investigators,however, were able to distinguish between com­ponents of medium chain length, with about 16carbons, and very long chain components withabout 30 carbons. A critical review of investi­gations of beeswax up to 1962 was made byCallow (8).

ln 1848, Brodie (9) reported that the freeacids of unhydrolyzed beeswax, obtained byextraction with ethanol, were long chain com­pounds (C2 7) and also that part of the re­maining wax was a palmitate of a long chainalcohol (10). Later it was gradually establishedthat beeswax was a mixture of hydrocarbons,esters and acids (2).

Further advances were made by Gascard(11) and Damoy (12), who, however, studiedonly hydrolysis products. They concluded thatthe hydrocarbons, alcohols and long chain acidswere ail odd-numbered with 25-31 carbons.Chibnall et al. (13) reinvestigated their results,using x-ray crystallography, and showed thatthough the hydrocarbons were odd-numberedC2S-C31 compounds the alcohols and longchain acids were in fact even-numbered with24-34 carbons.

ln 1961, Downing et al. (14) separated thecomponents of hydrolyzed beeswax into hydro­carbons, alcohols, acids, diols and hydroxyacids and reduced themall to hydrocarbons.These were then analyzed by GLC with theresults in Table II. The hydrocarbons, 16% ofthe wax, were mainly C2 5 -C3 3, the principalalcohols were C24 -C34, palmitic acid was themajor acid and the long chain acids wereC24-C34. These figures not only confirmed thequalitative conclusions of Gascard and Damoyconcerning the hydrocarbons and of Chibnall etal. concerning the alcohols and long chain acids,but also Brodie's early isolation of palmiticacid. As the free acids of unhydrolyzed waxwere not examined separately, the compositionof the acids is that of the total wax acids.

[ 2)

COMPOSITION OF INSECT WAXES

TABLE"Hydrocllrhons Olerived From Ikcswax Fraclions (wl. ,;:.)aNalurlllly occurring

1I·!'araftïnhydrocarhons

hydrcll"arhonMonohydricI!ydros y

~arhon No.Wax AWax Balcohols"Diois"Acidsacids--_._---12

0.30.414

0.81.51(,

50.5511.517

0.30.4III

8.5b9.8

19}0.50.3 Trace

20Tracec0.9d4.1

210.80.8

220.30.2 Trace2.01.6

233.73.7 0.30.4

240.60.411.915.217.58.2

2S7.58.8 Trace0.5

261.21.010.119.64.93.7

2726.830.1TraceTraceTrace0.3

282.21.314.839.24.31.9

2919.316.5Trace2.6Trace0.5

301.60.931.614.83.00.6c

3120.819.0Trace2.2Trace1.5e

320.91.523.56.54.70.4

3.113.815.5

.145.42.0

3S J(,2.7

Pcrccntagc of (,;ornpOlll'nt inhydrolyzcd wax16313311J

aDowning el al. (14), with permission.bConsisls of 7.11%monounsaturated and 0.7% saturated by examination of Ihe methyl esters.

eTraee indicales present but in too small amount (ca. 0.1%) 10 be estimated satisfaclorily.

dlndudes saluraled and unsaturated acids in 8pproximately equal proportions.

e A hrnad peak of the range shown; 3.4% is not absorbed by the Linde Molecular Sieve column.

Althollgh Downing et al. concluded that thehydrocarbons were entirely straight chain andsatllratcd, unsatllrated hydrocarbons have beenfrcqucnt1y reported (2). ln 1966, Streibl et al.(15) showcd that about 31% of beeswax hydro­carbons l:onsist of ds olefins which were mainly<:31 and C33 compounds, whereas the alkanesarc e2 5 -C2 9 compounds; very small amounts ofbranched chain hydrocarbons (16) and transnlcfins (17) were also isolated and identified.

Table Il contains two other interestingilcllls. First diols (3% of the total), which wereisolalcd for the first time, though without eluci­dating their structure apart from chain length,and second hydroxy acids (13% of total), whichhavc a longer history.

Bccswax hydroxy acids were first mentionedin 1919 when Lipp and Kovacs (18) reportedthat thc acids of saponificd Ghedda wax weremainly CI 7 and hydroxy CI 7 acids. Free acidsof this wax were very long chain compoundsand differcnt from combined acids (19). ln19J3 Ikuta (20), working with Japanese bees-

wax, which cornes from a variety of A. indicaand is similar to Ghedda wax, showed that thehydroxy acid is a hydroxypalmitic acid andthat the major acid is palmitic acid (21).Toyama and Hirai (22), in 1951, reported thatJapanese and European beeswaxes contain thesame hydroxy acid. After extensive frac­tionation a portion of the hydroxy acids (rep­resenting only about 10% of the original crudehydroxy acids) appeared to be 14-hydroxy­palmitic acid. The isolation of tetradecanedioicacid from the products of permanganate oxi­dation of the mother liquors seemed to supporttheir structure. This is more likely, however, tobe evidence for the presence of a 15-hydroxy­palmitic acid since nitric acid oxidation ofhydroxy acids with penultimate hydroxylgroups resuIts most1y in the loss of 2 carbonatoms (23).

The nuclear magnetic resonance (NMR)spectrum of beeswax hydroxy acids, examinedby Horn et al. (24) in 1964, showed con­clusively that the principal component is a

[ 3 1

A. P. TULLOCH

3.21

EFG

B

A

FIG. 1. Thin layer chromatograph of beeswax andbumble bee wax. 1, USP beeswax; 2, beeswax fromhoneycomb cappings; 3, nùxture of triacontane,octadecyl stearate, octacosanoland octacosanoic acid;4, bumble bee wax; 5, local, unrefmed beeswax. Theletters A-G refer to ester fractions of beeswax. Platewas Silica Gel G, development solvent was benzene at32 C, spots were detected by spraying with 50% sul­furie acid and heating with an infrared lamp.Standards were synthesized as previously described(40).

CD

IS-hydroxypalmiti, a'ld. ln connedion withIhis finding it is a r,markable coincidcnœ thatan osmoi)hilic yeast of thl' genllS Tom/opsis.whit'h was isolated from flowers and frombumhle hl'l' nests. produces glycosides ofseveral hydroxy acids including 15-hydroxy­palmili, acid and also hydroxylates palmitka,id giving a mixture of glycosides of 15- and16-hydroxy palmitic acids (25). Aiso 16-hy­droxypalmitic acid, as the ma crocy clic lactone,is the major constituent of the scent of twospecies of solitary bec (genus Halictus) (26).Presumably this acid is produced by the beeconcerned, but 1 thought that the yeastToru/op.l"/s might perhaps be involved in for­ma t ion of beeswax hydroxy acids.

If thcse acids had the same optical config­ur a t ion as hydroxy acid produced byTom/opsis, a common origin could be indi­caled. 1 have isolated hydroxy acids from bees­wax and measured their specifie rotation.Tom/op.üs produces 15-L-hydroxypalmitic acidwith [a] D + 4.5, but hydroxy acids from com­mercial (USP) beeswax had [a] D + 1.5, sug­gesling a mixture of racemate and L-isomer.lIydroxy acids from natural sources are usuallyoptically active, but racemic hydroxy fattyacids have sometimes been isolated (27).

Beeswax has been fractionated by columnchromatography (28) and by thin layer chro­matography (TLC) (29) though the fractionswere not clearly identified. Since differentoptical isorners of 15-hydroxypalmitic acidmight be present in different wax fractions, 1have investigated the chromatographie sepa­ration of the whole wax. Honeycomb cappingswere used since commercial wax might havebeen altered by blcaching and refining. ln aTLC chromatogram of beeswax samples, ailshow thcsame components; in particular therearc several components with Rf's smaller thanthat of long chain monoester (Fig. 1). Most ofthe fractions observed by TLC were isolated bysilicic acid column chromatography and identi­fied by NMR spectroscopy, GLC and exami­nation of their hydrolysis products (A.P.Tulloch, to be published).

Table 111 lists the fractions obtained in this

way and compares them with a beeswax com­position cakulated by Findley and Brown (30)from the results of functional group analysis.The percentage of hydrocarbons is similar tothat reported bcfore (14). Chromatography onsilver nitrate silicic acid (15) gave alkanes andl'is olefins (26%), and the compositions of thesetwo fractions, determined by GLC, were verysimilar to those reported by Streibl et al. (15).

. Monoesters A (35%) contained 40-50 carbonaloms with C46 and C48 as major components.

[4 J

COMPOSITION Of INSECT WAXES

TABLE III

Composition of Unhydrolyzed Beeswax

Si02 ('OIUIIlIl chromatography

!Iydrocarhons 15Esters A (monoesters) 35Es"'rs Il (diesters) 12Eskrs C (hydroxy esters) 4Estersi) (hydroxy esters) 4Es"'rs E (hydroxy esters) 4Esters F (hydroxy esters) 8Esters G (hydroxy esters) 4Free acids 8Not identified 6

aCalculated hy Findley and Brown (30).

Hydrolysis yielded palmitic acid and only tracesof longer çhain acids, and C24-C34 alcohols,the original esters are thus palmitates of thesealwhols. Very recently Holloway (31) has re­port cd similar results for the composition ofbeeswax monoesters. The presence of 15-hy­droxypalmitate in esters B-G was shown byNMR spectroscopy. NMR can also give an esti­mate of the extent to which the hydroxylgroup is acylated, since studies of methylhydroxystearates (32) and their acetates (A.P.Tulloch, unpublished work) show that the sig­nai duc to the terminal CH3 of a hydroxy acidwith the hydroxyl group on the penultimatecarbon atom undergoes a downfield displace­ment of about 0.05 ppm on acylation.

Esters Barc C56-C64 diesters, mainly withthe structure:

On hydrolysis they give three groups of com­ponents: aciels (almost entirely palmitic acid),hydroxy aciels together with a minor amount ofdiols, and C24-C34 alcohols (approximatelyom' molar proportion of each group). Diestersof 2-hydroxy acids and of l ,2-diols with chro­matographie properties similar to esters B havercccntly bcen isolated from the skin surfacelipids of rat (33) and other animais (N.Nicolaides, H. C. Fu and M. N. A. Ansari, thissymposium).

Esl\:rs (' and D consist partly, and esters Ealmosl entirc!y, of C40-C50 esters with a free011 ~roup. These hydroxy esters are mainlycomposeel of C24-C34 alcohols esterified with15-hydroxypalmitic acid but monoesters(Illost Iy pal mitates and lignocerates) of diols areprohahly also present. Hydrolysis of esters Fand G gavc higher proportions of hydroxy acidsamI diols than the other ester fractions indi-

Compositiolla

2345

(,9

Acid esters 5Free alcohols 1

12

cating the presence of hydroxy diesters and tri­esters.

Palmitic acid was almost the only non­hydroxy acid obtained from esters A, B, C andF, but D and E gave lignoceric acid as weil, andG gave C24-C34 acids only; the free acids wereC24-C34 and contained no palmitic acid, inagreement with Brodie's conclusions (9).Hydroxypalmitic acid formed at least 80% ofthe hydroxy acids from B-G and the remainderwas an assortment of longer chain hydroxyacids. GLC examination of the acetylatedmethyl hydroxypalmitates (34) showed thatthey consisted of mixtures of about 85% 15­acetoxypalmitate and 15% 14-acetoxypalmitateexcept for those from esters D, which hadabout 50% of each. None of the hydroxy acidsamples were optically pure, most having[al D - + 2.00• Thus there seems to be no evi­dence so far for the involvement of the yeastTorulopsis in the formation of the hydroxyacids.

Alcohols (C24-C34) were obtained fromeach ester fraction with only minor variationsin the relative amounts of each alcohol. Diois

from B to E were C24-C28 with C24 the majorcomponent, but F and G gave C24-C30 diolswith C28 the major component. Esters withfree carboxyl groups and free alcohols, sug­gested by Findley and Brown (30) were notdetected in this investigation. Free alcohols arevery min or components of unhydrolyzed bees­wax (8).

The diols were shown to have the structure:

CH3ÇH(CH2)n CH2CH20H (n = 20-26)OH

by examination of, their NMR spectrum andthat of their acetates and by comparison oftheir GLC retention times with those of syn­thetic model compounds. With one primary

[ 5]

A. P. TULLOCH

TABLE IVComposition of Bumble Bee WaxaHydrocarbonsc

Estersc,dCarnonn numner

SaturatedeMonoenoiceSaturatedfMonoenoicfDienoicf

23

9224

1125

594726

1127

1225211

1129

121730

1131

4534

0.5136

0.5338

1.51240

122642

18171944

1\383046

69848

40201850

8111\52

22254

0.556

0.5

l'cr ccnt ofl! total wax2896194

a"rood cells and honeypots from nests of Bombus 11Ifocinctus supplied by G. A. Hobbs, CanadaDepartment of Agriculture. Lethbridge, Alberta. were extracted with chloroform. The reddish orangewax formed 30% of the original weight; the residue consisted of insect de bris and the paperlike wallsof the cells. The wax has mp 35-45 C.

bCarbon numbers measured as before (40). GLC performed with an F & M model 402 gas chro·matograph with Oame ionization de tee tors. Column was V. in. x 3 ft glass column packed with 20-30mesh glass neads coated with 0.3% silicone SE 30. He 45 ml/min, temperature programmed at 3°/minfrom temperatures between 100-200 C to 325 C depending on sample. Other columns were used asncfore (40).

cWax (2.17 g) on Si02 column (100 g Biosil A, Bio-Rad. Richmond, Calif.). Elution with hexanegavc hydrocarbons (0.82 g) and with hexane containing 10-25% CHCI3 gave esters (0.63 g). Polàrfraction (0.73 g) obtained by elution with CHCI3'

dCarhnn numbcrs of esters are only tentative as hydrolysis products not fully characterized.

CHydrocarbons (0.76 g) chromatographed on an AgN03-Si02 column (80 g. 17. 41). Elution withhexanc gave alkancs (0.57 g) and with hexane containing 10% benzene gave alkenes (0.185 g). Alkenes(0.05 g) wcrc oxidized with KMn04-NaI04 (42) and products analyzed by GLC (43).

fEstcrs (0.63 g) chromatographed on AgN03·Si02 column. Hexane-benzene (9: 1) gave saturatedl'stcrs (O.llg). hexane·henzene (3:2) gave monounsaturated esters (0.36). hexane-benzene (2:3) gave<Iiunsaturatcd esters (0.075 g). Ethanolysis of esters and separation of resulting ethyl esters and aleo·hols on 8i02 column was as previously described (40). Saturated esters (O.llg) gave ethyl esters(0.055 g) and "lcohols (0.078 g). monounsaturated esters (0.36 g) gave ethyl esters (0.13 g) andalwhols (0.26 g). diunsaturated esters (0.084 g) gave ethyl esters (0.029 g) and akohols (0.064 g).

gl{cmaindcr of wax (34%) was relatively polar, nonvolatile fraction. This fraction (0.45 g) gaveethyl esters (0.11 g). akohols (0.05 g) and unidentified gum (0.27 g) on ethanolysis.

hydroxyl group and one at the penultimateposition they wuld arisc by reduction of thehydroxy al.:idsthough they wntain at least 8-12more I.:arbon atoms (A.P. Tulloch to bé pub­Iished).

The variety of compounds obtained by sap­onification of bceswax. their pecu1iar chainlength range, the difference in composition ofthe frec and combined al.:ids and the differentproportions in which the components are com-

bined to give esters A to G, ail suggest comp1exbiosynthetic pathways. Not surprising1y, therehave been only a few reports dealing with thebiosynthesis of beeswax.

When bees were fed 1·14C-acetate thehydrocarbons and free acids of the wax werestrongly labelled but the esters (and the acidsarid alcohols of which they were composed)were not appreciably labelled (35). lt appearedthat different wax components were synthe-

[6 ]

COMPOSITION OF INSECT WAXES

TABLE V

Yields Per Cent of Hydrolysis Produets of Seale Inseet Waxesa

HydroxyInseet

HydroearbonsAlcoholsn-Aeidsacids

Gascardiamadagascariensis

0.628.038.033.4C<)ccus ceri[erus

2.647.450.0Tachardia lacca

1.877.221.0lcerya purchasi

26.932.331.49.4Ceroplastes rusci

11.823.664.60Pulvinaria floci[era

8.339.232.220.3Quadraspidiotus perniciosus

9.814.775.50

UFaurot-Bouehet and Michel (52,53), with permission.

sized in different tissues. However, when2-14C-aœtatc was injected into the body cavityof honey bees, esters and free acids bothbc,amc labelled in a few ho urs though the non­saponifiable portion of the wax was moreheavily labelled than the acids (36).

Bumble Bee Wax

Wax produœd by several species of bumblebec was examined by Sundwik. Wax from B.mU.\curUIIl had mp 35-40 C (7) and this waxand wax from B. terres tris (37) gave long chainakohols on hydrolysis. The alcohols 'werereporled to give a neutral compourid on treat­menl wilh strong alkali (38) in contrast to theakohols of a plant. louse wax which yieldeda,ids. This wuld mean that the bumble beewax alcohol was a secondary alcohol which wasdehyùrogcnatcd to a ketone, or the neutralmalerial wuld have been hydrocarbon impuri­lies in the alcohols.

1 have invcstigaled wax extracted fromhol1t~ypots and brood cells of B. rufocinctus,whi,h is a native of western North America,and a rclatively good wax producer (39). TLC(hg. 1) shows that hydrocarbons and mono­esters arc major eomponents, diesters Bandl'stns C and D of beeswax are absent. The TLCpattern was hardly changed by diazomethanetrcatlllcnt of the wax showing that free acidsarc not present to any extent (methyl estershave an Rf similar to esters B). Fractionationon a silil:ic acid (;olumn gave hydrocarbons(37%), Illonoesters (29%) and a more polar frac­tion (34'}!,). The procedures used are shown asfoatnotcs ta Table IV.

NMR spedros(;opy showed the presence ofunsatllrated wmpounds with isolated doublebonds (44) in the hydrocarbons but appreciablealllollnts of branched chain hydrocarbons wereahsenl. The hydrocarbons were separated intoalkanes and alkenes (AgN03-Si02) and ana­Iyzcù by GLC with the results in Table IV. Un-

like beeswax hydrocarbons the two fractionshad similarchain lengths with the C25 hydro­carbon the principal component. Infraredspectroscopy showed that the alkenes were cisolefins and oxidative cleavage (KMn04-NaI04)gave heptanoic acid and CI 6-C22 fatty acidsshowing that the double bond is at the 7,8­position. Beeswax olefins con tain 10, II-unsatu­ration (15), but olefins with 7,8-unsaturationhave been isolated from rose waxes (45). Thecomposition of the hydrocarbons of bumblebee wax is of interest since Calam (46) hasobtained saturated and unsaturated C2 1-C25hydrocarbons from the heads of males ofseveral species of bumble bee.

The esters are also part1y unsaturated andwere separated into saturated, monoenoic anddienoic fractions by AgNOrSi02 chromato­graphy. GLC analysis gave the results in TableIV.

Ethanolysis of the saturated esters gavemainly palmitate with a little stearate and acomplex misture of saturated primary alcohols.NMR spectroscopy of these alcohols showedthem to be branched chain compounds (44)with probably as many as four methylbranches. They may be related to derivatives ofthe dihydrofarnesols recent1y isolated frombumble bees (47).

Ethanolysis of the monounsaturated estersgave mainly oleate and saturated primary alco­hols which were largely straight chain. Theprincipal alcohols were tentatively identified astetracosanol and hexacosanol and the minoralcohols as odd-numbered CI 9-C2 3 alcohols.The components of the diunsaturated esterswere not identified.

Ethanolysis of the most polar wax fractiongave a complex misture of esters and alcohols(- 30% of weight). The other products were notidentified but GLC analysis and NMR spectro­scopy showed that 15-hydroxypalmitic acidwas absent. There is thus no evidence that yeast

( 7 l

A. P. TlJLLOCH

Iws bl'l'n involvl'd in hydroxy acid formation inthis wax l'itlll'r.

Thollgh wax of only this one specics ofblllllbk bec has becn investigated in any detail,thl' availablc evidence, as mentioned later, atkast shows that the physical properties of thewaxes of a number of spccics are similar so thata provisional comparison of bumble bee waxand honey bec W<lXcan be made. My investi­gation shows that bumble bel' wax has a com­piex composition but one that is considerablydiffcrent from that of beeswax. The principaldifferences <Ireas follows:

1. Beeswax con tains appreciable proportionsof difunctional components, the hydr()xy acidsand diols, so that about half of the beeswaxesters are diestcrs (or higher esters, or hydroxyesters). Difunctional components are appar­enUy <lbsent from bumble bee wax.

2. Beeswax components are largely straightchain and saturated, the alcohols having mainly30-32 carbons. Bumble bee wax componentsarc more unsaturated, sorne are branched chaincompounds and the alcohols and hydrocarbonsgcncr<llly con tain 4-6 carbons less than the cor­responding beeswax components. The physicalpropcrtics of the waxes are naturally different,particularly the melting point, that of bumblebec wax being about 25 C lower than that ofbeeswax.

Waxes of Seale 1nseets

Sorne scale insccts produce enough wax tobe commcn.:ially important; these are theChincse wax insect (Coccus ceriferus) and thelaI.:insed (Tac!lardia lacca). C. cenjerus (in thefamily COl.:l.:idae)is (or was) cultivated in Chinaon branl.:hes of the Chinese ash; the insectsinfest the twigs so closcly that they are coveredwith a thick l<lyerof wax which can be scrapedoff (2). T. lacca {family Lacciferidae) is culti­vatcd on trces in India and is important as thesourœ of lac from which shellac is derived.Crude laI.: is I.:omposed mainly of a resin ofI.:ross-linked hydroxy acids, but 5-10% of wax isalso present.

Chinese insect wax, was first investigated byBrodie (9) who concluded that it consistedalmost entircly of a long chain ester of a longchain aleohol. Lac wax, as a by-product of theshellal.: industry, con tains varying amounts offree akohols depending on the method used tosepar<Jte wax from shellac (13). Gascard (II)showcd that lac wax and Chinese wax gave longI.:hain adds and long chain alcohols on hydroly­sis and these were later found to be C2 6 -e30 inthl' case of Chinese wax and C3o-C34 in thecase of lac wax (J 3,48,49).

A nother commercially interesting scale

insect is Coccus cacti, the cochineal insed.which !ives on a species of Cadus in Mexicoand covers itself with a thick layer of hard wax.The wax gives 15-oxotetratriacontan-!-o1 and13-oxo C30 and C32 acids on hydrolysis (50).

A number of other scalc insect waxes havebeen investigated, particularly in Japan (2), andlong-chain monoesters seemed to be the majorcomponents of most of them. Wax ofTachardina theae (family Lacciferidae) wasunusual in yielding 9-dodecenoic and 9-tetra­decenoic acids on hydrolysis (51), though theseacids may have been derived from glycerides ofthe body lipids rather than from the waxy shell.

The hydrolysis products of waxes of sevenspecies of scale insect have been separated andanalyzed by GLC by Faurot-Bouchet andMichel (52,53) with the results in Tables V toVII. Appreciable amounts of hydroxy acidswere obtained from the waxes of Gascardiamadagascariensis (family Lacciferidae), Iceryapurchasi (the cottony cushion scale, familyMargaroididae or ground pearl) and Pulvinariafloeifera (family Coccidae). These three andthat of Coccus ceriferus also gave approxi­mately l'quai amounts of acids and alcohols butthe waxes of Ceroplastes rusei (familyCoccidae) and Quadraspidiotus pemiciosus (theSan José scale which attacks deciduous fruittrees, family Diaspididae) gave a large excess ofacids and Tachardia lacca a large excess of al­cohols (as reported earlier by Chibnall (13») .

Hydroxy acids from G. madagascariensiswere a mixture of C3o-C34 acids with thehydroxyl group somewhere near the middle ofthe chain. The other hydroxy acids were notinvestigated.

Hydrocarbons of the waxes were odd­numbered with 25-35 carbon s, the principalcomponents were either C27, C29, C31 or C3 3.ln agreement with earlier conclusions ofChibnall et al. (J 3,49), the acids and alcohols ofChinese insect wax were C2 6 -e2 8 compoundsand of lac wax were C28-C34. The originalesters of the former wax wolild then be mainlyCS2 and of the latter CS6-e62 esters. Acids andalcohols of the other waxes (Table VII) weresimilar, being mainly C26-C30 compounds.Waxes, which gave hydroxy acids on hydrolysis,were probably more complex, perhaps morelike beeswax.

There have been conflicting reports aboutthe wax of Ceroplastes pseudoceriferus;Hashimoto and Mukai (54) found mainly C26acid and alcohols after hydrolysis, but Tamaki(55) found most of the alcohols to be branchedor cyclic and that di- and triunsaturated CI 8acids were present in addition to saturated C26and C28 acids; resin acids were also present.

Illi

TABLE VI

Conslituents of Coccid Waxes in Per Cent of Each GroupaGascardia madagascariensis

Coccus ceriferusTachardia lacca

No. of

Hydro- NonhydroxyHydro- NonhydroxyHydro- NonhydroxyC aloms

carbonsAlcoholsacidscarbonsAlcoholsacidscarbonsAlcoholsacids

r-tO

0.10Il

0.2s:12

0.1..,013

0.3Vl

146.9-- 1.0=i

16

----0.4----2.4---- 3.4018

----0.1--..3.4_.-- 0.4Z\CI

20 ...-0.1.-.-Traces --_ .0.30..,22

-- Traces..--1.2 .-0.2Z24

--4.6 0.5..7.014.4--.- 0.2Vl25 3.4--

--3.9----..-.tT1

261.472.026.00.663.049.0--0.6 0.3(")

-l2772.0 ----5.2-- 42.0--1.2

~282.211.6 4.41.028.015.54.166.6 18.2>

2920.0 ---.7.6----35.1--1.7

><

30

--5.9 5.82.72.05.62.821.0 25.1tT1

Vl311.0--

--42.1 ..--13.4--LI32

--5.919.81.0--1.5Traces9.027.233

----0.129.2----2.6--0.2

34----39.50.5------2.8 17.6

35.-.- ..6_2

36----3.2---- ----.. 0.5

aFaurot·Bouchet and Michel (52), with permission.

TABLE VII

Constituents of Coccid Waxes in Per Cent of Each Groupa QuadraspidiotusIcerya purchasi

Ceroplastes ruseiPulvinaria flocifera'pemiciosus

No. of

Hydro- Hydro-Hydro-Hydro-C atoms

carbonsAlcoholsAcidscarbonsAlcoholsAcidscarbonsAlcoholsAcidscarbonsAlcoholsAcids

14

------------- --2.5------;10-

16---loi----1.9----11.7 -- 14.7

18--.-4.7-..4.2.---1.9-- --4.4:"

20

--_.6_1----5.7.--1.8-- --4.7..;c:c 22 --0.99.4-.----.- 1.0--0.42.9t"'

24--7.37.4.-7.01.4-- 1.7.-2.78.0t"'

250.8---1.0 ----3.3 ------0

.- (")26

--49.236.41.081.21.2--4.12.9 77.812.5:r27

7.8.---58.0 ----10.6 ----LI

28--33.822.72.711.80.9 50.348.2--19.126.4

2950.4 ----26.4 ----33.6 --5.9

30--8.810.71.7--15.2 --35.919.8-- 12.5

3132.3 ----9.2 ----17.2 -72.1

32--..1.1--.-56.3 --9.78.02.7 --12.5

338.4.-_.----_.3.9 --_.18.2

34----0.4_.

--13.2 ----0.5-- 1.4

350.3

aFaurot-Bouchet and Michel (53), with permission.

COMPOSITION OF INSECT WAXES

TABLE VIII

Melting l'oints of Sorne Waxes Secreted by Insects

But later Hashimoto et al. (56) stated that,whilc the saturated esters of this wax were true

wax esters, containing long straight-chain acids<lnu akohols, the unsaturated esters wereolcates and Iinoleates of branched (dite<rpenoidetl:.) akohols. A report that wax of theComstot:k mcaly bug Pseudococcus comstocki(f<lmily Pseudocot:cidae or mealy bugs) gives10-18% of tetradecanedioic acid (57) onhydrolysis, seems ta he the first mention ofuit:arhoxylit: at:ids in waxes secreted by insects.

There secm ta have been no investigations of1he hiosynthesis of waxcs of scale insects.

Function of WaKes Secreted by Beesand Scala 1nsects

Waxes produt:ed by these two groups ofinsct:ls have entirely diffcrent functions but, inhol h grollps, produt:tion of large amounts ofwax is relateu ta the specialized way of lifeauopLed by the inscds. Wax is secreted in waxgl<lnus whit:h t:onsist of one or more specializedt:ells at or near the surface of the abdomen.

Wax IIf Becs. Honey bees use wax ta buildLhe f<lmiliar honey comb. Wax is chewed byworker becs until soft and molded piece bypiet:e to form the network of hexagonal cells.Larvae arc reared in ce lis of the comb, differentsized t:ells being used for workers, males andqueens. Cc lis arc also used ta store honey andpollen,

Sinrc the strut:tural basis of the cell consistsonly of wax, the wax must have suit ablephysit:al propertics. Species of Apis occur inmany tropit:al countries sa that the meltingpoint of the wax must be reasonably high; inIllost t:<lses it is 62-65 C. Presumably sornedegree of plastit:ity and kneadability are alsoucsirablc, The lInsaturated hydrocarbons ofbeeswax m<lYad as plasticizers.

The nest of the bumble bee is usually on orunder the ground and is much less elaborateth<ln that of the honcy bee. It consists of asll1<111grollp of rounded cells in which the larvae<Ire r<lised and a few honeypots ta store honey.The œlls are t:Onstructt:d of wax (58,59), or

l'rom a mixture of pollen and wax (60). Thelarvae also spin cocoons which are later coatedwith wax and converted ta honeypots. Sladen(58), presumably referring ta B. lapidarius andB. terres tris , remarked that the wax was muchsofter than that of the honey bee, 1 have foundthat waxes of B. rufoeinctus and B. flavifronshave mp 35-45 C and Sundwik (7) gave mp35-40 C for wax of B. ml/searum.

Bumble bees are commonly found only intemperate climates, the nest temperature rarelyexceeding 35 C (61); Hobbs (personal com­munication) has suggested that this probablyaccounts for the mu ch lower melting point ofbumble bee wax compared ta honey bee wax.Also the relatively simple nest does not requirea hard strong wax.

Wax of Scale Jnseets. Ali scale insects, asmembers of the order of bugs, have the mouthparts modified for piercing and suc king upfluids. The adult females are degenerate, fre­quently having lost their legs, and are attachedta the hast plant by the mouth parts. It isprobably because they are stationary that manyspecies protect themselves with a waxycovering. The wax may also protect the eggsand young insects; lac of the lac insect has asimilar funcUon. ln general scale insects requirea hard, high melting wax (particularly as manyoccur in hot climates) ta protect them l'rominsect predators and l'rom the weather.

There is considerable variation in the way inwhich the wax is attached ta the insect andsorne do not have a true scale. The San Joséscale (Q. perniciosus) has a hard scale of waxand cast skins which shelters the insect and itseggs. The female of C. eeriferus and of sornespecies of Ceroplastes is covered with thickplates of wax. Other species, such as C. cactiand Pulvinaria spp., excrete a cottony mass ofwax in which the eggs are laid. Others still havepowdery lumps of wax on the surface,examples of these are J. purehasi and theComstock mealie bug (and mealie bugs ingeneral, as their na me implies).

One interesting problem which apparentlyhas not been solved is that of how the insect

can exude a very high melting wax. Thisproblem applies ta honey bees as well as tascale insects although, as Table VIII shows, thelatter have the highest melting waxes. Wax of C.caeti has a melting point as high as 100 C.

Wax presumably exudes through pores, butthis has been disputed in the case of the honeybee (35). Sorne insects exude cuticle wax con­taining a volatile solvent (1), but there is noevidence that this method is used by bees orscale insects. It wou Id probably require taomuch solvent. Beeswax- is exuded as a liquid

35-4563-6582-8472-8278

99-101

Melting point, CWax

lIulIlble hee(lteL 7 an<l (his work)

HOlley lIee (2)(,hinese Inseel (2)Lac Wax (2)

/c,'rya pl/relias; (2)CO""/IS euefi (50)

[ Il]

A. P. TULLOCH

and hardcns to a waxy scale (2). This may betruc of ail tlH' high melting insed waxes thoughthe mannl'r in which it occurs is not under­slood. PolYlllcrizalion and cross linking ofIlnsa t Ilraled components cannot be theexplanation as such components are found toonly a slllall extent.

CONCLUSION

Though only a minute fraction of the totalnllmber of insects has been investigated, it isc1e.iJrthat thcre is considerable variation in com­plcxity of composition of waxes secreted byinsects. Ali reports indicate that Chinese insectwax has a simple composition (consistingmainly of C52 monoester), but sorne of theother waxes, particularly those of bees andbumble bees, contain a very large number ofcomponents. Generally, when they are investi­gated carefully by the most modern methods,waxcs are found to be more comp!ex than wasoriginally thought. ln addition to straight chainsaturated wmponents, severa! series of unsatu­rated and branched chain components may bepresent, thus it was not until very recently thatthe exact nature of the hydrocarbons of bees­wax was established (15-17). Before any bio­synthctic investigations l'an be carried out, itwould seem essential that the exact nature ofIhe major groups of components be established.

REFERENCES

1. Wigglesworlh, V. B., "The Life of Insects,"Weiuenfeld and Nicolson, London, 1964.

2. Warth, A. H" "The Chemistry and Technology ofWaxes," 2nd Edition, Reinhold Publishing Corp.,NewYork,1956,p.76-121.

3. Ikuta, H., Analyst 56:430 (1931).4. Roberts, O. D., and H. T. Islip, Ibid. 47:246

(1922).5. Phaukc, R. P., Bee World 42:149 (1961).6. Godamer, 1., Arch. Pharm. 255:425 {1917).7. Sundwik, E. E., Z. Physiol. Chem. 26: 56 (1898).8. Callow. R. K., Bee World 44:95 (1963).9. Brouie, B. C., Ann. Chem. 67: 180 (1848).

10. Brouie, B. C.,lbid. 71:144 (1849).II. Gascard, A., Ann. Chim. (Paris) 15:332 (1921).12. Damoy, G., J. Pharm. Chim. 29:148, 225 (1924).13. Chibnall, A. C., S. H. Piper, A. Pollard, E. F.

Williams anu P. N. Sahai, Biochem. 1. 28:2189(1934).

14. Downing, D. T., Z. H. Kranz, 1. A. Lamberton. K.E. Murray and A. H. Redcliffe, Australian 1.Chem. 14:253 (1961).

15. Sireibl, M., K. Sldnsky and F. Sc's'rm, Fette SeifenAnslrichmiUel 68:799 (1966).

16. Siransky, K., M. Streibl and F. ~rm, CollectionCzech. Chem. Commun. 31 :4694 (1966).

17. Streihl, M., anu K. Siransky, FeUe SeifenAnslrichmillel 70:343 (1968).

18. Lipp, A., and E. Koyacs, J. Prakl. Chem. 99:243(1919). '

19. Lipp, A., and E. Casimir, Ibid. 99:256 (1919).20. Ikuta, H., 1. Soc. Chem. Ind. Japan 36:447

( 1933).21. Ikuta, H.. Ibid. 36:373 (1933).22. Toyama, Y.. and H. Hirai. Fett<· &'ikn

Anstriehimillel 53:556 (1951).23. Tulloeh, A. P., and 1. F. T. Spencer. JAOCS

43:153 (1966).24. Horn, D. H. S., Z. H. Kranz and J. A. Lamberton,

Australian J. Chem. 17:464 (1964).25. Tulloch, A. P., J. F. T. Spencer and P. A. J. Gorin.

Cano J. Chem. 40:1326 (1962).26. Anderson, C. O., G. Bergstrclm, B. Kullenberg and

S. Stailberg-Stenhagen, ArkiY Kemi 26: 191(1966).

27. Tulloch, A. P., Cano J. Chem. 43:415 (1965).28. Fuchs, W., and A. de Jong, Felle Scifen

Anstrichmittel 56:218 (1954).29. Kaufmann, H. P., and B. Das, Ibid. 65:398

(1963).30. Findley. T. W., and 1. B. Brown, JAOCS 30:291

(1953).31. Holloway, P. J., Ibid. 46:189 (1969).32. Tulloch, A. P., Ibid. 43:670 (1966).33. Nikkari, T., and E. Haahti, Biochim. Biophys.

Acta 164:294 (1968).34. Tulloch, A. P., JAOCS 41 :833 (1964).35. Piek, T., 1. Insect Physiol. 10:563 (1964).36. Young, R. G., Life Sei. 2:676 (1963).37. Sundwik, E. E., Z. Physiol. Chem. 53:365 (1907).38. Sundwik, E. E.,lbid. 72:455 (1911).39. Hobbs, G. A., Cano Entomologist 97:1293 (1965).40. Tulloch, A. P., Cano 1. Chem. 47:3119 (1969).41. De Vries, B., JAOCS 40: 184 (1963).42. Yon Rudloff, E., Cano 1. Chem. 34:1413 (1956).43. TUlioch, A. P., and B. M. Craig, JAOCS 41 :322

(1964).44. Hopkins, C. Y., "Progress in the Chemistry of

Fats and Other Lipids," Vol. 8, Pergamon Press,Oxford, 1965, p. 213.

45. Wollrab. V., Collection Czech, Chem. Commun.33: 1584 (1968).

46. Calam, D. H., Nature 221:857 (1969). ..47. Bergstrô'm, S., B. Kullenberg, S. Stail berg­

Stenhagen and E. Stenhagen. Arkiv Kemi 28:453(1968).

48. Francis, F., S. H. Piper and T. Malkin, Proc. Roy.Soc. London Ser. A. 128:214 (1930).

49. Pollard. A., A. C. Chibnall and S. H. Piper, Bio­chem. J. 25:2111 (1931).

50. Chibnali. A. C., A. L. Latner. E. F. Williams andC. A. Ayre. Biochem. J. 28:313 (1934).

51. Kono, M., and R. Maruyama, J. Agr. Chem. Soc.Japan 15:177 (1939).

52. Faurot-Bouchet, E., and G. Michel, JAOCS41 :418 (1964).

53. Faurot-Bouchet, E., and G. Michel, Bull. Soc.Chim. Biol. 47:93 (1965).

54. Hashimoto, A., and K. Mukai, Nippon NogeiKagaku Kaishi j9:489 (1965).

55. Tamaki, Y., Lipids 1:297 (1966).56. Hashimoto, A., H. Yoshida and K. Mukai, Nippon

Nogei Kagaku Kaishi 41 :498 (1967).57. Tamaki, Y., Lipids 3: 186 (1968).58. Sladen, F. W. L., "The Humble Bee," MacMillan &

Co., Ltd., London, 1912, p. 12-58.59. Free, 1. B., and C. G. Butler, "Bumblebees,"

Collins, London, 1959, p. 5-25.60. Hobbs, G. A., Cano Entomologist 99: 127\ (1967).61. Hasselrot, T. B., Opuscula Entomol. Suppl. 17:1

( \960).

[Received April 21, 1969]

[ 12]