The Composition of Beeswax and Other Waxes Secreted by

A. P. TULLOCH.
National Research Council of Canad
Prairie Regional Laboratory,
Saskatoon, Saskatchewan
ABSTRACT
This n:vicw deals with waxes of me mners of two quite different groups of'
insects the bees and the scale insects,
which ~eerete large amounts of wax, The
former use wax as a structural material
and the latter as a protective material.
The compositions of waxes from sorne of
these insects are described and particular
attention is paid to the compositions of
the unhydrolyzed
waxes and to the
presence of hydroxy acids. New analyses
of beeswax and of wax of a species of
.humble bee are reported. The structures
of the diesters, hydroxyesters and diols of
neeswax are elucidated. The bumble bee
wax con tains major proportions of saturated and unsaturated hydrocarbons, and
of long chain saturated, mono- and diunsaturated esters. The relationship between
stru<.:ture and function of the waxes is discussed.
INTRODUCTION
1nsects bclong to the class of animais with
Ihe fQllowing characteristics:
the body is
divided into head, thorax and abdomen; the
head carries a single pair of antennae; and the
thorax carries three pairs of legs and usually
one or two pairs of wings. These features dislinguish them from crabs, lobsters, spiders,
mites. millepedes etc.
Ncarly a million species of insects have been
dl'scribed and several times this number may
actually exist. 1nsects are divided into about 28
orders; severa] include a very large number of
specics but most have only a few thousand. A
simple introduction to insect biology has been
wrillen by Wigglesworth (1).
Thl' orders which con tain sorne of the best
known insects arc listed in Table 1 together
with the approximate number of species in the
onkr: for convenience the other, less weil
known, orders have been omitted. The first 13
orders con tain those insects whose young are
1Issued
as National
Research
Council
of Canada
o different from the adults and the last
seven con tain those whose young are radically
different from the adults.
Most or ail insects are protected from water
loss by a thin film of wax in the cuticle; this
type of wax is discussed by Jackson and Baker
(this symposium). Clearly the study of the composition of insect waxes is an enormous field
which has barely been scratched. Jackson and
Baker review two different species of cricket,
four different species of cockroach, one species
of moth and one species of scale insect. The
huge orders of flies and beetles do not seem to
have been examined at ail.
ln this review 1 shall deal with a few members of two orders of insects which secrete
much larger amounts of wax than those which
produce only a thin waxy cuticle. The most
important one to be discussed is the honey bee
(genus Apis, family Apidae). 1 shall also deal
with the wax of bumble bees (genus Bombus,
family Apidae). Bees are considered to be
among the most highly developed insects.
The other, more primitive, group of insects
which secretes large amounts of wax is that
comprising the scale insects. They are given this
name because, in many species, the female is
protected by a scale or shield consisting of a
mixture of wax and cast skins. These insects are
members of several families of the sub order
Homoptera 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 commercial
value.
The appearance and function of the waxes is
discussed later.
The chemistry of waxes secreted by insects
has been studied over the last 150 years. Literature prior to 1954 was reviewed by Warth (2),
but more prominence was given to early
theories of composition than to later, more
reliable, results. The present review will de al
only with what seem to have been the most
important advances, particularly those obtained
by modern chromatographic methods, and will
report new analyses of beeswax and wax of a
species of bumble bee.
No. 1 1260.
20nc of six papers to be published
from the
Symposium on Natural Waxes, presented at the AOCS
Mcl'linlt. San Francisco, April 1969.
Beeswax
At one time the word wax meant only bees[ 1)
A. P. TULLOCH
TABLE
1
Sorne Orders of Insects (J)
Order
Approx.
5000
1500
6000
1500
1700
1100
2000
10000
1100
2600
230
3000
55000
5000
5000
200000
85000
1100
100000
275000
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)
wax and as the most important insect wax it
has attracted the most attention, in fact, Ikuta
(3) has remarked that there were about 140
publications dealing with beeswax chemistry
between 1848 and 1930. Beeswax generally
refers to wax of the European bee, Apis
mel/i/era,
but Asiatic species A. dorsata, A.
Jlona and A. indica are sometimes also commercial sources of wax. This wax is known as
East Indian beeswax or Ghedda wax. Functional group analysis of Ghedda wax (3-5) indica t cs only minor qualitative
differences
betwcen its composition and that of common
bceswax. Results of investigations of Ghedda
wax will, therefore, be included with those of
bceswax. Waxes of wild bees of the genera
Trigona and Me/ipona (also in family Apidae)
have been examined (6), but not by modern
methods. Wax of a few species of Bombus has
also been investigated (7).
To compare properties of waxes and to consider their biosynthesis it is clearly important to
know the composition of the natural unhydrolyzed wax. Sorne early investigators did try
to determine this, but most investigations have
been carried out using saponification products.
Since wax components are complex mixtures of
homologs, it was difficulf to make an accurate
analysis prior to the application of gas liquid
chromatography (GLC). The early investigators,
however, were able to distinguish between components of medium chain length, with about 16
carbons, and very long chain components with
about 30 carbons. A critical review of investigations of beeswax up to 1962 was made by
Callow (8).
No. of Species
ln 1848, Brodie (9) reported that the free
acids of unhydrolyzed beeswax, obtained by
extraction with ethanol, were long chain compounds (C2 7) and also that part of the remaining wax was a palmitate of a long chain
alcohol (10). Later it was gradually established
that beeswax was a mixture of hydrocarbons,
esters and acids (2).
Further advances were made by Gascard
(11) and Damoy (12), who, however, studied
only hydrolysis products. They concluded that
the hydrocarbons, alcohols and long chain acids
were ail odd-numbered with 25-31 carbons.
Chibnall et al. (13) reinvestigated their results,
using x-ray crystallography, and showed that
though the hydrocarbons were odd-numbered
C2S-C31
compounds the alcohols and long
chain acids were in fact even-numbered with
24-34 carbons.
ln 1961, Downing et al. (14) separated the
components of hydrolyzed beeswax into hydrocarbons, alcohols, acids, diols and hydroxy
acids and reduced themall to hydrocarbons.
These were then analyzed by GLC with the
results in Table II. The hydrocarbons, 16% of
the wax, were mainly C2 5 -C3 3, the principal
alcohols were C24 -C34, palmitic acid was the
major acid and the long chain acids were
C24-C34.
These figures not only confirmed the
qualitative conclusions of Gascard and Damoy
concerning the hydrocarbons and of Chibnall et
al. concerning the alcohols and long chain acids,
but also Brodie's early isolation of palmitic
acid. As the free acids of unhydrolyzed wax
were not examined separately, the composition
of the acids is that of the total wax acids.
[ 2)
--_._--J(,
hydrolyzcd
(,;ornpOlll'nt
Trace
3.726.8
1.0
4.7
4.9
17.5
8.2
15.2
1.5
2.7
2.0COMPOSITION
5.4
0.8
0.3
0.2
0.3
0.8
8.8
7.5
31
Trace
0.6c
511.5
0.9
11.9
30.1
19.0
1.5
23.5
10.1
0.5
Trace
31.6
J4.3
Trace
3.0
Trace
19.6
14.8
13.8
30.6
6.5
1.2
1.6
2.6
31 A
Trace
16
Trace
0.4
3.7
1.6
0.4
11.9
3.7
0.3
16.5
1.3
14.8
39.2
19.3
Wax
Acids
alcohols
"Diois"
BWax
1.5e
15.5
20.8
0.9
acids
0.32.2
2.0
2.2
OF yINSECT Tracec
WAXES
hydrocarhons
Monohydric
4.1
9.8
0.9d
I!ydros
0.5
0.3
0.8
50.5
8.5b
wax
in
Olerived Nalurlllly
From Ikcswax
Fraclions
occurring
TABLE"
(wl. ,;:.)a
aDowning el al. (14), with permission.
and 0.7% saturated
bConsisls of 7.11%monounsaturated
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 the
hydrocarbons
were entirely straight chain and
satllratcd, unsatllrated
hydrocarbons
have been
frcqucnt1y 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 alkanes
arc e2 5 -C2 9 compounds; very small amounts of
branched
chain hydrocarbons
(16) and trans
nlcfins (17) were also isolated and identified.
Table
Il contains
two other
interesting
ilcllls. First diols (3% of the total), which were
isolalcd for the first time, though without elucidating their structure
apart from chain length,
and second hydroxy acids (13% of total), which
havc a longer history.
Bccswax hydroxy acids were first mentioned
in 1919 when Lipp and Kovacs (18) reported
that thc acids of saponificd Ghedda wax were
mainly CI 7 and hydroxy CI 7 acids. Free acids
of this wax were very long chain compounds
and differcnt
from combined
acids (19). ln
19J3 Ikuta (20), working with Japanese bees-
wax, which cornes from a variety of A. indica
and is similar to Ghedda wax, showed that the
hydroxy
acid is a hydroxypalmitic
acid and
that
the major acid is palmitic
acid (21).
Toyama and Hirai (22), in 1951, reported that
Japanese and European
beeswaxes contain the
same hydroxy
acid.
After
extensive
fractionation
a portion of the hydroxy acids (representing only about 10% of the original crude
hydroxy
acids) appeared
to be 14-hydroxypalmitic acid. The isolation of tetradecanedioic
acid from the products
of permanganate
oxidation of the mother liquors seemed to support
their structure. This is more likely, however, to
be evidence for the presence of a 15-hydroxypalmitic
acid since nitric acid oxidation
of
hydroxy
acids
with
penultimate
hydroxyl
groups resuIts most1y in the loss of 2 carbon
atoms (23).
The
nuclear
magnetic
resonance
(NMR)
spectrum
of beeswax hydroxy acids, examined
by Horn et al. (24) in 1964, showed conclusively
that the principal
component
is a
[ 3 1
A. P. TULLOCH
IS-hydroxypalmiti,
a'ld. ln connedion
with
Ihis finding it is a r,markable
coincidcnœ
that
an osmoi)hilic
yeast of thl' genllS Tom/opsis.
whit'h was isolated
from flowers and from
bumhle
hl'l' nests.
produces
glycosides
of
several hydroxy
acids including
15-hydroxypalmili,
acid and also hydroxylates
palmitk
a,id giving a mixture of glycosides of 15- and
16-hydroxy palmitic
acids (25). Aiso 16-hydroxypalmitic
acid, as the ma crocy clic lactone,
is the major constituent
of the scent of two
species of solitary bec (genus Halictus) (26).
Presumably
this acid is produced
by the bee
concerned,
but 1 thought
that
the yeast
Toru/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
by
Tom/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
acid
with [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 usually
optically
active, but racemic
hydroxy
fatty
acids have sometimes been isolated (27).
Beeswax has been fractionated
by column
chromatography
(28) and by thin layer chromatography
(TLC) (29) though the fractions
were not clearly identified.
Since different
optical
isorners
of 15-hydroxypalmitic
acid
might be present in different
wax fractions,
1
have investigated
the chromatographie
separation of the whole wax. Honeycomb
cappings
were used since commercial
wax might have
been altered by blcaching
and refining.
ln a
TLC chromatogram
of beeswax
samples,
ail
show thcsame
components;
in particular
there
arc several components
with Rf's smaller than
that of long chain monoester (Fig. 1). Most of
the fractions observed by TLC were isolated by
silicic 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 to
that reported bcfore (14). Chromatography
on
silver nitrate silicic acid (15) gave alkanes and
l'is olefins (26%), and the compositions
of these
two fractions,
determined
by GLC, were very
similar to those reported by Streibl et al. (15).
. Monoesters A (35%) contained 40-50 carbon
aloms with C46 and C48 as major components.
[4
A
B
C
D
E
F
G
1
.2
3
FIG. 1. Thin layer chromatograph of beeswax and
bumble bee wax. 1, USP beeswax; 2, beeswax from
honeycomb cappings; 3, nùxture of triacontane,
octadecyl stearate, octacosanoland octacosanoic acid;
4, bumble bee wax; 5, local, unrefmed beeswax. The
letters A-G refer to ester fractions of beeswax. Plate
was Silica Gel G, development solvent was benzene at
32 C, spots were detected by spraying with 50% sulfurie acid and heating with an infrared lamp.
Standards were synthesized as previously described
(40).
J
COMPOSITION
Of INSECT WAXES
TABLE
Composition
III
of Unhydrolyzed
Beeswax
Compositiolla
Si02 ('OIUIIlIl chromatography
!Iydrocarhons
Esters A (monoesters)
Es"'rs Il (diesters)
Eskrs C (hydroxy esters)
Estersi) (hydroxy esters)
Es"'rs E (hydroxy esters)
Esters F (hydroxy esters)
Esters G (hydroxy esters)
Free acids
Not identified
aCalculated
hy Findley
23
45
15
35
12
4
4
4
8
4
8
6
(,
Acid esters
Free alcohols
9
5
1
12
and Brown (30).
Hydrolysis yielded palmitic acid and only traces
of longer çhain acids, and C24-C34 alcohols,
the original esters are thus palmitates of these
alwhols. Very recently Holloway (31) has report cd similar results for the composition
of
beeswax monoesters.
The presence of 15-hydroxypalmitate
in esters B-G was shown by
NMR spectroscopy.
NMR can also give an estimate of the extent
to which the hydroxyl
group
is acylated,
since studies
of methyl
hydroxystearates
(32) and their acetates (A.P.
Tulloch, unpublished
work) show that the signai duc to the terminal CH3 of a hydroxy acid
with the hydroxyl
group on the penultimate
carbon atom undergoes
a downfield
displacement of about 0.05 ppm on acylation.
Esters Barc C56-C64 diesters, mainly with
the structure:
On hydrolysis they give three groups of components: aciels (almost entirely palmitic acid),
hydroxy aciels together with a minor amount of
diols, and C24-C34
alcohols
(approximately
om' molar proportion
of each group). Diesters
of 2-hydroxy acids and of l ,2-diols with chromatographie properties similar to esters B have
rcccntly bcen isolated from the skin surface
lipids of rat (33) and other
animais
(N.
Nicolaides, H. C. Fu and M. N. A. Ansari, this
symposium).
Esl\:rs (' and D consist partly, and esters E
almosl entirc!y, of C40-C50 esters with a free
011 ~roup. These hydroxy
esters are mainly
composeel of C24-C34 alcohols esterified with
15-hydroxypalmitic
acid
but
monoesters
(Illost Iy pal mitates and lignocerates) of diols are
prohahly also present. Hydrolysis
of esters F
and G gavc higher proportions
of hydroxy acids
amI diols than the other ester fractions indi-
cating the presence of hydroxy diesters and triesters.
Palmitic
acid was almost
the only nonhydroxy acid obtained from esters A, B, C and
F, but D and E gave lignoceric acid as weil, and
acids only; the free acids were
G gave C24-C34
C24-C34
and contained
no palmitic acid, in
agreement
with
Brodie's
conclusions
(9).
Hydroxypalmitic
acid formed at least 80% of
the hydroxy acids from B-G and the remainder
was an assortment
of longer chain hydroxy
acids. GLC examination
of the acetylated
methyl
hydroxypalmitates
(34) showed that
they consisted
of mixtures of about 85% 15acetoxypalmitate
and 15% 14-acetoxypalmitate
except
for those from esters D, which had
about 50% of each. None of the hydroxy acid
samples
were
optically
pure,
most having
[al D - + 2.00• Thus there seems to be no evidence so far for the involvement
of the yeast
Torulopsis in the formation
of the hydroxy
acids.
Alcohols
(C24-C34)
were obtained
from
each ester fraction with only minor variations
in the relative amounts
of each alcohol. Diois
from B to E were C24-C28 with C24 the major
component,
but F and G gave C24-C30 diols
with C28 the major component.
Esters with
free carboxyl
groups and free alcohols, suggested by Findley and Brown (30) were not
detected in this investigation.
Free alcohols are
very min or components
of unhydrolyzed
beeswax (8).
The diols were shown to have the structure:
CH3ÇH(CH2)n
OH
CH2CH20H
(n = 20-26)
by examination
of, their NMR spectrum
and
that of their acetates
and by comparison
of
their GLC retention
times with those of synthetic model compounds.
With one primary
[ 5]
647
111
912
217
9120
25
217
152119
59
9411 28
30
638
18
894
\2Dienoicf
Saturatede
Monoenoice
Saturatedf
Monoenoicf
total
wax
numner
Composition
ofTABLE
BumbleIVBee Waxa
Hydrocarbonsc
31
12
1\
18
2861.5
A. P. TULLOCH
0.5
Estersc,d40
a"rood cells and honeypots
from nests of Bombus 11Ifocinctus supplied by G. A. Hobbs, Canada
Department
of Agriculture.
Lethbridge, Alberta. were extracted with chloroform.
The reddish orange
wax formed 30% of the original weight; the residue consisted of insect de bris and the paperlike walls
of 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 20-30
mesh glass neads coated with 0.3% silicone SE 30. He 45 ml/min, temperature
programmed
at 3°/min
from temperatures
between 100-200 C to 325 C depending on sample. Other columns were used as
ncfore (40).
cWax (2.17 g) on Si02 column (100 g Biosil A, Bio-Rad. Richmond, Calif.). Elution with hexane
gavc hydrocarbons
(0.82 g) and with hexane containing
10-25% CHCI3 gave esters (0.63 g). Polàr
fraction (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 with
hexanc 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 saturated
l'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) and
alwhols (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) gave
ethyl esters (0.11 g). akohols (0.05 g) and unidentified
gum (0.27 g) on ethanolysis.
hydroxyl group and one at the penultimate
position they wuld arisc by reduction of the
hydroxy al.:idsthough they wntain at least 8-12
more I.:arbon atoms (A.P. Tulloch to bé pubIished).
The variety of compounds obtained by saponification of bceswax. their pecu1iar chain
length range, the difference in composition of
the frec and combined al.:ids and the different
proportions in which the components are com-
bined to give esters A to G, ail suggest comp1ex
biosynthetic pathways. Not surprising1y, there
have been only a few reports dealing with the
biosynthesis of beeswax.
the
When bees were fed 1·14C-acetate
hydrocarbons and free acids of the wax were
strongly labelled but the esters (and the acids
arid alcohols of which they were composed)
were not appreciably labelled (35). lt appeared
that different wax components were synthe-
[6 ]
COMPOSITION
OF INSECT WAXES
TABLE
Yields Per Cent of Hydrolysis
V
Produets
of Seale Inseet Waxesa
21.0
23.6
50.0
47.4
n-Aeids
acids
Alcohols 0.6
2.6
75.5
20.3
33.4
64.6
31.4
32.2
38.0
09.4
77.2
28.0
14.7
32.3
39.2
26.9
9.8
11.8
8.3
1.8
Hydroearbons
Hydroxy
madagascariensis
perniciosus
UFaurot-Bouehet
and Michel
(52,53), with
sized in different
tissues. However, when
2-14C-aœtatc was injected into the body cavity
of honey bees, esters and free acids both
bc,amc labelled in a few ho urs though the nonsaponifiable portion of the wax was more
heavily labelled than the acids (36).
Bumble
Bee Wax
Wax produœd by several species of bumble
bec was examined by Sundwik. Wax from B.
mU.\curUIIl
had mp 35-40 C (7) and this wax
and wax from B. terres tris (37) gave long chain
akohols on hydrolysis. The alcohols 'were
reporled to give a neutral compourid on treatmenl wilh strong alkali (38) in contrast to the
akohols of a plant. louse wax which yielded
a,ids. This wuld mean that the bumble bee
wax alcohol was a secondary alcohol which was
dehyùrogcnatcd to a ketone, or the neutral
malerial wuld have been hydrocarbon impurilies in the alcohols.
1 have invcstigaled wax extracted from
hol1t~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 Band
l'stns C and D of beeswax are absent. The TLC
pattern was hardly changed by diazomethane
trcatlllcnt of the wax showing that free acids
arc not present to any extent (methyl esters
have an Rf similar to esters B). Fractionation
on a silil:ic acid (;olumn gave hydrocarbons
(37%), Illonoesters (29%) and a more polar fraction (34'}!,). The procedures used are shown as
foatnotcs ta Table IV.
NMR spedros(;opy showed the presence of
unsatllrated wmpounds with isolated double
bonds (44) in the hydrocarbons but appreciable
alllollnts of branched chain hydrocarbons were
ahsenl. The hydrocarbons were separated into
alkanes and alkenes (AgN03-Si02) and anaIyzcù by GLC with the results in Table IV. Un( 7
permission.
like beeswax hydrocarbons the two fractions
had similarchain lengths with the C25 hydrocarbon the principal component.
Infrared
spectroscopy showed that the alkenes were cis
olefins and oxidative cleavage (KMn04-NaI04)
gave heptanoic acid and CI 6-C2 2 fatty acids
showing that the double bond is at the 7,8position. Beeswax olefins con tain 10, II-unsaturation (15), but olefins with 7,8-unsaturation
have been isolated from rose waxes (45). The
composition of the hydrocarbons of bumble
bee wax is of interest since Calam (46) has
obtained saturated and unsaturated C2 1-C25
hydrocarbons
from the heads of males of
several species of bumble bee.
The esters are also part1y unsaturated and
were separated into saturated, monoenoic and
dienoic fractions by AgNOrSi02 chromatography. GLC analysis gave the results in Table
IV.
Ethanolysis of the saturated esters gave
mainly palmitate with a little stearate and a
complex misture of saturated primary alcohols.
NMR spectroscopy of these alcohols showed
them to be branched chain compounds (44)
with probably
as many as four methyl
branches. They may be related to derivatives of
the dihydrofarnesols
recent1y isolated from
bumble bees (47).
Ethanolysis of the monounsaturated esters
gave mainly oleate and saturated primary alcohols which were largely straight chain. The
principal alcohols were tentatively identified as
tetracosanol and hexacosanol and the minor
alcohols as odd-numbered CI 9-C2 3 alcohols.
The components of the diunsaturated esters
were not identified.
Ethanolysis of the most polar wax fraction
gave a complex misture of esters and alcohols
(- 30% of weight). The other products were not
identified but GLC analysis and NMR spectroscopy showed that 15-hydroxypalmitic acid
was absent. There is thus no evidence that yeast
l
A. P. TlJLLOCH
Iws bl'l'n involvl'd in hydroxy acid formation in
this wax l'itlll'r.
Thollgh wax of only this one specics of
blllllbk bec has becn investigated in any detail,
thl' availablc evidence, as mentioned later, at
kast shows that the physical properties of the
waxes of a number of spccics are similar so that
a provisional comparison of bumble bee wax
and honey bec W<lXcan be made. My investigation shows that bumble bel' wax has a compie x composition but one that is considerably
diffcrent from that of beeswax. The principal
differences <Ireas follows:
1. Beeswax con tains appreciable proportions
of difunctional components, the hydr()xy acids
and diols, so that about half of the beeswax
esters are diestcrs (or higher esters, or hydroxy
esters). Difunctional components are apparenUy <lbsent from bumble bee wax.
2. Beeswax components are largely straight
chain and saturated, the alcohols having mainly
30-32 carbons. Bumble bee wax components
arc more unsaturated, sorne are branched chain
compounds and the alcohols and hydrocarbons
gcncr<llly con tain 4-6 carbons less than the corresponding beeswax components. The physical
propcrtics of the waxes are naturally different,
particularly the melting point, that of bumble
bec wax being about 25 C lower than that of
beeswax.
Waxes of Seale 1nseets
Sorne scale insccts produce enough wax to
be commcn.:ially important;
these are the
Chincse wax insect (Coccus ceriferus) and the
laI.:insed (Tac!lardia lacca). C. cenjerus (in the
family COl.:l.:idae)is (or was) cultivated in China
on branl.:hes of the Chinese ash; the insects
infest the twigs so closcly that they are covered
with a thick l<lyer of wax which can be scraped
off (2). T. lacca {family Lacciferidae) is cultivatcd on trces in India and is important as the
sourœ of lac from which shellac is derived.
Crude laI.: is I.:omposed mainly of a resin of
I.:ross-linked hydroxy acids, but 5-10% of wax is
also present.
Chinese insect wax, was first investigated by
Brodie (9) who concluded that it consisted
almost entircly of a long chain ester of a long
chain aleohol. Lac wax, as a by-product of the
shellal.: industry, con tains varying amounts of
free akohols depending on the method used to
separ<Jte wax from shellac (13). Gascard (II)
showcd that lac wax and Chinese wax gave long
I.:hain adds and long chain alcohols on hydrolysis and these were later found to be C2 6 -e30 in
thl' case of Chinese wax and C3o-C34 in the
case 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 Mexico
and covers itself with a thick layer of hard wax.
The wax gives 15-oxotetratriacontan-!-o1
and
13-oxo C30 and C32 acids on hydrolysis (50).
A number of other scalc insect waxes have
been investigated, particularly in Japan (2), and
long-chain monoesters seemed to be the major
components
of most of them. Wax of
Tachardina theae (family Lacciferidae) was
unusual in yielding 9-dodecenoic and 9-tetradecenoic acids on hydrolysis (51), though these
acids may have been derived from glycerides of
the body lipids rather than from the waxy shell.
The hydrolysis products of waxes of seven
species of scale insect have been separated and
analyzed by GLC by Faurot-Bouchet
and
Michel (52,53) with the results in Tables V to
VII. Appreciable amounts of hydroxy acids
were obtained from the waxes of Gascardia
madagascariensis (family Lacciferidae), Icerya
purchasi (the cottony cushion scale, family
Margaroididae or ground pearl) and Pulvinaria
floeifera (family Coccidae). These three and
that of Coccus ceriferus also gave approximately l'quai amounts of acids and alcohols but
the
waxes of Ceroplastes rusei (family
Coccidae) and Quadraspidiotus pemiciosus (the
San José scale which attacks deciduous fruit
trees, family Diaspididae) gave a large excess of
acids and Tachardia lacca a large excess of alcohols (as reported earlier by Chibnall (13») .
Hydroxy acids from G. madagascariensis
were a mixture of C3o-C34 acids with the
hydroxyl group somewhere near the middle of
the chain. The other hydroxy acids were not
investigated.
Hydrocarbons
of the waxes were oddnumbered with 25-35 carbon s, the principal
components were either C27, C29, C31 or C3 3.
ln agreement with earlier conclusions of
Chibnall et al. (J 3,49), the acids and alcohols of
Chinese insect wax were C2 6 -e2 8 compounds
and of lac wax were C28-C34. The original
esters of the former wax wolild then be mainly
CS2 and of the latter CS6-e62 esters. Acids and
alcohols of the other waxes (Table VII) were
similar, being mainly C26-C30 compounds.
Waxes, which gave hydroxy acids on hydrolysis,
were probably more complex, perhaps more
like beeswax.
There have been conflicting reports about
the
wax of Ceroplastes pseudoceriferus;
Hashimoto and Mukai (54) found mainly C26
acid and alcohols after hydrolysis, but Tamaki
(55) found most of the alcohols to be branched
or cyclic and that di- and triunsaturated CI 8
acids were present in addition to saturated C26
and C28 acids; resin acids were also present.
Illi
0..,
-l
Vl
..
--..
-..
..
--.
..
--.---.6_2
--tT1
7.0
0.3.4
1.2
.1
0.4
0.2
0.5
1.7
LI
Traces
(")
Z
4.6
0.6
5.9
35.1
42.1
13.4
39.5
3.9
5.2
7.6
0.4
0.1.0.1
3.2
2.4
1.5
3.4
1.2
2.8
2.6
-0.3
-_.
-..
-.--..
-----..
-27.2
25.1
4.1
4.4
20 =i
><
s:
14.4
2.0
49.0
Alcohols
acids
acids
20.0
66.6
11.6
28.015.5
2.2
1.0
0Vl..,
3.4
Traces
29.2
19.8
2.8
2.7
5.8
0.5
5-0.5
1.0
0.3
.6acids
17.6
72.026.0
63.0
1.4
0.6
72.0
Alcohols
carbons
carbons
1.0
18.2
9.0
HydroHydroVl> 21.0
~
00Z
r-
_
tT1
1.0
Traces
42.0
.Nonhydroxy
Nonhydroxy
ascariensis
s
of Coccid Waxes in Per Cent of Each Groupa
Tachardia
Coccus lacca
ceriferus
TABLE
VI
6.9
0.2
0.1
0.3
0t"'..;
--_.
._.-_.
------------------.-----------..
.----4.7
4.4
4.7
11.7
LI
19.1
4.1
0.4
7.0
18.2
72.1
26.4
58.0
33.6
10.6
17.2
5.9
15.2
13.2
2.5
0.5
1.9
1.8
49.2
35.9
3.3
33.8
9.2
3.9
1.0
6_1
1.9
1.1
0.9
9.7
7.3
8.8
56.3
:" 12.5
4.2
0.4
5.7
.--._.
-2.7
------.--.
12.5
22
81.2
1Acids
.2
8.0
Acids
Alcohols
26.4
2.9
2.9
48.2
11.8
1.4
2.7
0.9
:r carbons
2.7
10.7
8.0
19.8
9.4
7.4
36.4
1.7
1.0
32.3
carbons
carbons
Acids
50.4
22.7
7.8
0.8
8.4
0.3
t"'
Hydroloi
c:
;10-
-
14.7
1.4
12.5
1.7
1.0
77.8
50.3--
saFaurot-Bouchet
of Coccid Waxes
Per Cent
Each permission.
Groupa
andinMichel
(53),of with
--Hydro(")
Hydro'pemiciosus
Pulvinaria
Ceroplastes
flocifera
rusei
TABLE VII
Quadraspidiotus
COMPOSITION
OF INSECT WAXES
TABLE VIII
l'rom
larvae
with
(58),
a mixture
of pollen and wax (60). The
also
spin
cocoons
which are later coated
Melting l'oints of Sorne Waxes Secreted by Insects
wax and converted
ta honeypots.
Sladen
Wax
presumably
referring ta B. lapidarius and
Melting point, C
B. terres tris , remarked that the wax was much
lIulIlble hee
softer than that of the honey bee, 1 have found
35-45
(lteL 7 an<l (his work)
that waxes of B. rufoeinctus and B. flavifrons
63-65
HOlley lIee (2)
have mp 35-45 C and Sundwik
(7) gave mp
(,hinese Inseel (2)
82-84
35-40 C for wax of B. ml/searum.
Lac Wax (2)
72-82
/c,'rya pl/relias; (2)
78
Bumble bees are commonly
found only in
CO""/IS
euefi (50)
99-101
temperate
climates, the nest temperature
rarely
exceeding
35 C (61); Hobbs (personal
communication)
has suggested
that this probably
accounts for the mu ch lower melting point of
But later Hashimoto
et al. (56) stated that,
bumble bee wax compared
ta honey bee wax.
whilc the saturated esters of this wax were true
Also
the
relatively
simple
nest
does not require
wax esters, containing long straight-chain
acids
<lnu akohols,
the unsaturated
esters
were a hard strong wax.
Wax of Scale Jnseets. Ali scale insects, as
olcates and Iinoleates of branched (dite<rpenoid
etl:.) akohols.
A report
that
wax of the members of the order of bugs, have the mouth
parts modified
for piercing
and suc king up
Comstot:k mcaly bug Pseudococcus comstocki
fre(f<lmily Pseudocot:cidae
or mealy bugs) gives fluids. The adult females are degenerate,
10-18% of tetradecanedioic
acid (57)
on quently having lost their legs, and are attached
hydrolysis, seems ta he the first mention
of ta the hast plant by the mouth parts. It is
probably because they are stationary
that many
uit:arhoxylit: at:ids in waxes secreted by insects.
protect
themselves
with
a waxy
There secm ta have been no investigations
of species
covering. The wax may also protect the eggs
1he hiosynthesis of waxcs of scale insects.
and young insects; lac of the lac insect has a
Function of WaKes Secreted by Bees
similar funcUon. ln general scale insects require
and Scala 1nsects
a hard, high melting wax (particularly
as many
ta protect them l'rom
Waxes produt:ed
by these two groups of occur in hot climates)
insct:ls have entirely diffcrent functions but, in insect predators and l'rom the weather.
There is considerable
variation in the way in
hol h grollps, produt:tion
of large amounts of
ta the insect and
wax is relateu ta the specialized
way of life which the wax is attached
auopLed by the inscds. Wax is secreted in wax sorne do not have a true scale. The San José
scale (Q. perniciosus) has a hard scale of wax
gl<lnus whit:h t:onsist of one or more specialized
t:ells at or near the surface of the abdomen.
and cast skins which shelters the insect and its
Wax IIf Becs. Honey bees use wax ta build
eggs. The female of C. eeriferus and of sorne
with thick
Lhe f<lmiliar honey comb. Wax is chewed by species of Ceroplastes is covered
worker becs until soft and molded piece by plates of wax. Other species, such as C. cacti
and Pulvinaria spp., excrete a cottony
mass of
piet:e to form the network of hexagonal cells.
wax in which the eggs are laid. Others still have
Larvae arc reared in ce lis of the comb, different
powdery
lumps
of wax
on the surface,
sized t:ells being used for workers, males and
of these are J. purehasi and the
queens. Cc lis arc also used ta store honey and examples
Comstock
mealie bug (and mealie bugs in
pollen,
Sinrc the strut:tural basis of the cell consists
general, as their na me implies).
One interesting
problem
which apparently
only of wax, the wax must have suit able
physit:al propertics.
Species of Apis occur in has not been solved is that of how the insect
can exude
a very high melting
wax. This
many tropit:al countries
sa that the melting
applies ta honey bees as well as ta
point of the wax must be reasonably
high; in problem
scale insects although,
as Table VIII shows, the
Illost t:<lses it is 62-65 C. Presumably
sorne
latter have the highest melting waxes. Wax of C.
degree of plastit:ity and kneadability
are also
ucsirablc,
The lInsaturated
hydrocarbons
of caeti has a melting point as high as 100 C.
beeswax m<lYad as plasticizers.
Wax presumably
exudes through pores, but
The nest of the bumble bee is usually on or
this has been disputed in the case of the honey
under the ground and is much less elaborate
bee (35). Sorne insects exude cuticle wax conth<ln that of the honcy bee. It consists of a taining a volatile solvent (1), but there is no
sll1<111
grollp of rounded cells in which the larvae
evidence that this method is used by bees or
<Ire r<lised and a few honeypots
ta store honey.
scale insects.
It wou Id probably
require tao
The œlls are t:Onstructt:d of wax (58,59), or
much solvent. Beeswax- is exuded as a liquid
[ Il]
A. P. TULLOCH
and hardcns to a waxy scale (2). This may be
truc of ail tlH' high melting insed waxes though
the mannl'r in which it occurs is not underslood. PolYlllcrizalion and cross linking of
Ilnsa t Ilraled components
cannot
be the
explanation as such components are found to
only a slllall extent.
CONCLUSION
Though only a minute fraction of the total
nllmber of insects has been investigated, it is
c1e.iJrthat thcre is considerable variation in complcxity of composition of waxes secreted by
insects. Ali reports indicate that Chinese insect
wax has a simple composition (consisting
mainly of C52 monoester), but sorne of the
other waxes, particularly those of bees and
bumble bees, contain a very large number of
components. Generally, when they are investigated carefully by the most modern methods,
waxcs are found to be more comp!ex than was
originally thought. ln addition to straight chain
saturated wmponents, severa! series of unsaturated and branched chain components may be
present, thus it was not until very recently that
the exact nature of the hydrocarbons of beeswax was established (15-17). Before any biosynthctic investigations l'an be carried out, it
would seem essential that the exact nature of
Ihe major groups of components be established.
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..
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[Received April 21, 1969]
'
[ 12]