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Hydrocarbon and other lipid constituents of the bull ant, Myrmecia gulosa
J. Insect Physiol., 1970,Vol. 16,pp. 1721to 1728.PergamonPress. Printed in Great Britain
HYDROCARBON AND OTHER LIPID CONSTITUENTS THE BULL ANT, MYRMECIA GULOSA G. W. K. CAVILL,
D. V. CLARK, M. E. H. HOWDEN, S. G. WYLLIE
OF
and
SchooI of Chemistry, The University of New South Wales, Kensington, N.S.W. 2033, Australia (Receiwed 11 March 1970) Abstract-The lipid constituents of Myrmecia gulosa comprise hydrocarbon, glyceride, and fatty acid fractions. The major hydrocarbons which are constituents of the cuticle wax are an homologous series of monomethylalkanes, C26 to C34. Each homologue is a mixture of isomers, for example, the C30 alkane includes 9-, ll-, 13-, and 15methylnonacosane. The minor hydrocarbons of the cuticle wax include the normal paraffins, Cl9 to C29. Of the free fatty acids the major components are oleic with lesser amounts of palmitic, stearic, and minor acids. 1,2- and 1,3-diglycerides and I-monoglycerides of normal type are the major lipid constituents. INTRODUCTION RECENT studies on the chemical composition of exocrine gland secretions of the primitive ant, Mymeciu gulosa (F.) (CAVILL and ROBERTSON, 1965), have established that, in addition to the characteristic proteinaceous venom of the venom gland (CAVILL et al., 1964; EWAN, 1967), the Dufour’s gland contains a range of aliphatic hydrocarbons (CAVILL and WILLIAMS, 1967). A preliminary study of the metastemal glands of M. gulosa also suggested the presence of aliphatic hydrocarbons. As such aliphatic hydrocarbons are constituents of insect cuticle waxes (GILMOUR, 1961), an examination of the hydrocarbon and other lipid constituents of M. gulosa has now been undertaken. So that sufficient material would be available for chemical characterization, the lipid constituents were obtained by total extraction of M. gulosa, and then compared, by gas chromatography, with the material obtained by extraction of the detached cuticle. Characterization of the major lipid constituents of the cuticle wax provides basic data in relation to proposed studies on ‘surface’ pheromones. MATERIALS
AND METHODS
Isolation of lipid constituents A typical extraction procedure was as follows. The ants (ZOOg) were trisected into heads, thoraxes, and gasters, and these were extracted (Soxhlet) with right petroleum, b.p. 40 to 6O”C, or methylene chloride (ZOOml) for 48 hr (cf. CAVILL and WILLIAMS, 1967). Each extract, after treatment with sodium carbonate 54
1721
1722
G. W. K. CAVILL, D. V. CLARK, M. E. H. HOWDEN, AND S. G. WYLLIE
solution, was washed (water) and dried (anhyd. sodium sulphate). The product, after removal of the solvent, was subjected to chromatography on alumina (Peter Spence, Grade H), yielding the hydrocarbon and glyceride fractions. The hydrocarbons were eluted with light petroleum and the glyceride fractions with solvents of increasing polarity. The alkaline aqueous solution, after acidification, was extracted with ether. The free fatty acids so obtained were converted into their methyl esters by treatment with an ethereal solution of diazomethane.
Gas chromatography Analytical gas chromatography was carried out on a Perk&Elmer 800 unit with nitrogen as carrier gas; the 6 ft by + in. stainless steel columns were packed with the listed stationary phases on Chromosorb G (SO-100 mesh): (a) 5% silicone oil, SE30, (b) 5% Ap iezon L, (c) 25% diethyleneglycol succinate. Preparative gas chromatography was undertaken on a Varian 705 unit with nitrogen as carrier gas; the 20 ft by # in. column was packed with 30% SE30 on Chromosorb W (SO-100 mesh).
Spectrometry Nuclear magnetic resonance spectra were recorded on a Varian A-60 spectrometer with carbon tetrachloride as solvent and tetramethylsilane as internal standard. Mass spectra were recorded on an A.E.I. MS9 instrument. 1.r. spectra were recorded as liquid capillaries on a Hilger-Watts Infrascan spectrophotometer with a grating monochromator.
Model compounds The reference compounds, 1 l-, 14-, and Kmethylnonacosane were synthesized for purposes of gas chromatographic and mass spectrometric comparison with the natural constituents. The following synthesis of 15-methylnonacosane from tetradecanol is typical. The alcohol was converted into 1-iodotetradecane which, via the Grignard reaction with solid carbon dioxide, gave pentadecanoic acid. The acid chloride on treatment with dimethylcadmium gave hexadecan-Zone (cf. CASON, 1947). This ketone, separated and purified using chromatography on alumina, was then subjected to a Wittig reaction with tetradecyltriphenylphosphonium iodide, also prepared from 1-iodotetradecane (cf. GREEN~ALDet al., 1963). The product, a mixture of cis- and trans-15-methylnonacos-14-ene was purified by chromatography on silica gel impregnated with silver nitrate. The olefins were hydrogenated under pressure, in the presence of Adam’s catalyst, to yield the required lSmethylnonacosane. Similarly, 11-methylnonacosane was prepared from dodecan-Zone and octadecyltriphenylphosphonium iodide, and 14-methylnonacosane from pentadecan-a-one and pentadecyltriphenylphosphonium iodide.
HYDROCARBON AMI OTHER LIPID CONSTITUENTS
OF THE BULL ANT
1723
RESULTS
Lipid constituents
of M.
gulosa
M. g&osa workers collected in the RoyaI National Park, south of Sydney, yielded lipid constituents which, in total, comprised from 2 per cent in summer up to 5 per cent in winter of the body weight of the insect. Of this lipids, approximately 14 per cent were isolated from the heads, 14 per cent from the thoraxes, and 72 per cent from the gasters, The constituents were readily separated into a neutral fraction consisting essentially of hydrocarbons and glycerides, and a fatty acid fraction. The latter comprised 20 to 30 per cent of the total extract. The neutral fraction, after chromatography on alumina (or silica), was further separated into hydrocarbons and the glyceride-ester constituents, in the approximate ratio 1 : 2.
The hydrocarbons Gas chromatography of the hydrocarbons showed that they could be divided into two main groups. The first and minor group consists of a mixture of normal and branched chain hydrocarbons, C9 to C14. Fig. 1 shows a typical gas chroSOIV
5% s-30.
I
35
1t
1100
I
30
I
25
I
20 Time,
FIG.
I
I5
1
IO
/
5
min
1. GLC of low molecular weight hydrocarbons.
matographic trace of these hydrocarbons, n-octane being added as a standard. A plot of log retention time vs. carbon number showed the major peaks, that is 1 to 5, to correspond to the normal alkanes, Cl0 to C14. Other hydrocarbons, whose peaks are also unchanged on hydrogenation, comprise several series of branched-chain alkanes. Peaks 6 to 12 may belong to the iso- or ante&-series.
1724
G. W. K. CAVILL, D. V. CLARK, M. E. H. HOWDEN, AND S. G. WYLLIE
The hydrocarbons previously characterized from Dufour’s gland in M. gulosa, including pentadecane, heptadecane, and heptadec-cis-8-ene (CAVILL and WILLIAMS, 1967), are present in the total extract, and in range span the above and the following groups. The second and major group of hydrocarbons comprises some 95 per cent of the total. On gas chromatography these hydrocarbons gave a complex series of peaks (Fig. 2). By the use of reference hydrocarbons, including a mixture of
5%SE-30,250’ &ml C,, &, Cz3,C,, and C,, dkanes
added
Time,
min
FE. 2. GLC of high molecular
weight hydrocarbons.
hydrocarbons from wool wax known to contain normal, iso and anteiso constituents (DOWNING et al., 1961) and by plots of log retention time vs. chain length, peaks 1 to 11 were identified as normal alkanes of chain length Cl9 to C29, with the odd numbered chains predominating (Figs. 2, 3). Similarly peaks 12 to 17, present only in small amounts, were shown to correspond to members of either the iso or anteiso series. The peaks, 18 to 24, correspond to members of an homologous series from C28 to C34, the even-numbered alkanes predominating. These hydrocarbons were unaffected by treatment with molecular sieve under conditions which separated the normal alkanes. Further, NMR and mass spectral data for these homologues suggested they were monomethylalkanes, the branch occurring towards the centre of the chain. Recently, comparable monomethylalkanes have been reported as minor constituents from beeswax (STRANSKY et al., 1966) and wool wax (MOLD et al., 1966). In the present work the more abundant methylalkanes, represented by peaks 18, 20, and 22 (Fig. 2), were separated by preparative gas chromatography, and their mass spectra examined. These data confirmed the branched nature of the alkanes and also showed that each of the purified homologues was a mixture of isomeric monomethylalkanes, with the methyl branch at various positions along the chain. To assist in the interpretation of the mass spectra, the reference compounds ll-, 14-, and 15-methylnonacosane were synthesized. These synthetic compounds could not be separated from each other under our gas chromatographic conditions and had a retention time identical with that of the C30 member of the
HYDROCARBON
AND
OTHER
LIPID CONSTITUENTS
172.5
OF THE BULL ANT
unknown series. Furthermore, comparison of the mass spectra of the synthetic compounds with those of the purified homologues enabled the major components of each homologue to be characterized.
e g g
I I%I.0o-so.e0.7 -
E 0.6 2 0,5-
o n-Alkanes l ;SOOr &7/&O A
20
21
22
23
24
25
26
27 Carbon
28
2!3
30
olkanes Centrally bmnched monamethyl alkanes
31
32
33
34
number
FIG. 3. Log retention time vs. carbon number plot for high molecular weight
hydrocarbons.
Thus the position of the methyl branch in the synthetic 11-methylnonacosane is clearly shown by the increased intensity of the charactersitic doublet of peaks at m/e 168, 169; 252, 253; and 280, 281 (Fig. 4), reflecting the tendency of the chain to fragment preferentially at the tertiary-branched carbon atom on electron impact. The intensity of the peaks at m/e 168, 169; 196, 197; 224, 225; 252, 253; 280, 281; and 308, 309 in the mass spectrum of the unknown C30 compound (Fig. 4) corresponds to methyl branches at carbon atoms 9, 11, 13, and 15. Comparable peaks were noted by STRANSKY et al. (1966) for their C28 hydrocarbon from beeswax. Thus the C30 fraction consists mainly of a mixture of 9-, ll-, 13-, and 15-methylnonacosane. It seems likely that smaller amounts of other isomers are also present. The C28 and C32 homologue consists of a similar mixture with methyl branches at positions 9, 11, 13, and 15 predominating. Finally, pieces of cuticle excised from the heads, thoraxes and gasters of M. gulosa workers (we are indebted to Dr. PHYLLIS L. ROBERTSON for these dissections) were extracted with light petroleum, and the extracts examined by gas chromatography. The above hydrocarbons were confirmed as constituents of the cuticle wax, with minor variations in their relative proportions from head, thorax, and gaster. The fatty
acids
The acidic constituents of M. gdosa which were separated from the total extracts of heads, thoraxes, and gasters, have been analysed by gas chromatography
1726
G. W. K. CAVILL, D. V. CLARK, M. E. H. HOWDEN, AND S. G. WYLLIE
The major constituent is oleic acid (approx. 70 per cent), after methylation. plus palmitic and stearic acid. The above compounds were characterized by a comparison of their methyl esters with standard specimens on three gas chromatographic columns. II-Methylnonacosane
422
14 - Methylnonacosane
60 u” 40 : -$
20
.z 0 0, .? %
0 100
z K
80
15- Methylnonacosane
60
40 20 0 00
r
Branched
2:”
C,,+omers
150 180 7.00 220 240 260 280 ZOO 320 340 360 380 400 420 m/e FIG. 4.
Mass
spectra of branched C30 alkanes.
The identification of minor constituents is based on plots of log retention times vs. carbon number, before and after hydrogenation. The minor acids, tentatively characterized, include tridecanoic, tetradecanoic, and heptadecanoic acids, branched C13, C16, C17, C19, and C20 acids, plus a normal Cl6 and a
IWDROCARBON AND OTHER LIPID CONSTITUENTS
OF THE BULL
ANT
1727
branched C20 unsaturated acid. The composition of the acids from heads, thoraxes, and gasters does not appear to vary to any marked extent. A total extract from heads and thoraxes from a summer collection of M. gulosa, on treatment with sodium hydrogen carbonate solution, gave an acid fraction which, after methylation and chromatography on alumina, yielded an ester having a terpene-like odour. Comparative gas chromatography, using methyl octanoate, nonanoate, and decanoate as standards, suggested the product is both branched and unsaturated. The ester showed retention times on two columns, identical with those of methyl citronellate.
The glyceri& The more polar compounds eluted from alumina after the hydrocarbons proved to be mainly glycerides, and the acid fraction obtained by hydrolysis of this material closely corresponded in composition to that of the free fatty acids reported above. By the use of ir. and, in particular, NMR spectroscopy (HOPKINS, 1965) the main constituents of the various glyceride fractions were identified. Two minor components, an ester fraction (vmax 1735 cm-l) and a triglyceride 1747 cm-l) were eluted with benzene-light petroleum, the former fraction (v,, tailing the hydrocarbons. The major component, a 1 : 2 mixture of 1,2- and 1,3-diglycerides was eluted with ether-benzene (1 : l), then a I-monoglyceride fraction was eluted with methanol-chloroform (1 : 9). The NMR spectra of these glyceride fractions were consistent with oleic acid being the predominant esterified acid (60 to 65 per cent), and with an average fatty acid chain length in the glyceride of Cl4 to Cl8 (c$ HOPKINS, 1965). This detailed spectroscopic study was carried out on the glyceride fraction from ants collected in the winter period. DISCUSSION The cuticular hydrocarbons comprise up to one-third of the neutral fraction extracted from M. gulosa workers, and, strikingly, the homologous series of centrally branched monomethylalkanes constitutes some 90 per cent of these hydrocarbons. Comparably monomethylalkanes have been detected recently as minor components of wool wax (MOLD et al.,1966) and of beeswax (STRANSKY et al., 1966). They comprise O-1 per cent of the latter. Beeswax has long been known to consist of normal long-chain esters, together with paraffin hydrocarbons and free fatty acids. More recent studies (DOWNING et al., 1961) h ave confirmed this pattern and, further, shown that the major hydrocarbons (approx. 16 per cent) are normal alkanes, Cl9 to C33, with the oddnumbered homologues, C27 to C33, predominating. In contrast, the major cuticular hydrocarbons of M. gulosa are the homologous series of monomethylalkanes, C26 to C34, with the even-numbered members predominating. However, the minor cuticular hydrocarbons include the normal alkanes, Cl9 to C29, the odd-numbered homologues again being present in relaSeasonal variation in the relative proportions of the tively greater amounts.
1728
G. W. K. CAVILL, D. V. CLARK, M. E. H. HOWDEN, ANDS. G. WYLLIE
hydrocarbon components was noted. In general, the winter extracts showed a higher proportion of normal to branched hydrocarbons; however, there was a lower proportion of the higher chain length homologues of both series of alkanes. The free fatty acids of M. gulosa comprise oleic (approx. 70 per cent) with smaller proportions of palmitic, stearic, and lesser acids. This composition corresponds to that normally found in animal fats (cf. GILBY, 1965). Moreover, the glyceride fraction
would
appear
On the assumption
to contain
the above
that the glycerides
acids as the major
are representative
then the hydrocarbons are the major components biogenesis of these centrally branched monomethylalkanes
gutosa,
fatty
acid units.
of the fat body of M.
of the cuticle wax. The has yet to be considered.
Acknozcledgements-We thank Dr. PHYLLIS L. ROBERTSON of this university for helpful discussion, and Dr. D. DOWNING,C.S.I.R.O., Division of Applied Chemistry, Melbourne, for specimens of wool wax hydrocarbons. Messrs. R. GREENWOOD and J. RIDINGS are thanked for technical assistance. This investigation has been supported by grants from the U.S. Public Health Service and the Australian Research Grants Committee. REFERENCES CASONJ. (1947) The use of organ0 cadmium reagents for the preparation of ketones. Chern. Rev. 40, 15-32. CAVILL G. W. K. and ROBERTSONP. L. (1965) Ant venoms, attractants and repellents. Science, Wash. 149, 1337-1345. CAVILL G. W. K., ROBERTSONP. L., and WHITFIELD F. B. (1964) Venom and venom apparatus of the bull ant, Myrmecia gulosa (Fabr.). Science, Wash. 146, 79-80. CAVILL G. W. K. and WILLIAMSP. J. (1967) C onstituents of Dufour’s gland in Myrmecia gulosa. J. Insect Physiol. 13, 1097-1103. DOWNINGD. T., FRANZ Z. H., LAMBERTONJ. A., MURRAYK. E., and REDCLIFFEA. H. (1961) Studies in waxes-XVIII. Beeswax: a spectroscopic and gas chromatographic examination. Aust. J. Chem. 14, 253-263. SWAN L. M. (1967) Ph.D. Thesis, The University of New South Wales. GILBY A. R. (1965) Lipids and their metabolism in insects. A. Rew. Ent. 10, 141-160. GILMOURD. (1961) Biochemistry of Insects. Academic Press, New York. GREENWALD R., CHAYKOVSKY M., and COREYE. J. (1963) The Wittig reaction using methylsuliinyl carbanion-dimethyl sulphoxide. J. org. Chem. 28, 1128-1129. HOPKINSC. Y. (1965) Nuclear magnetic resonance in fatty acids and glycerides. In Progress in the Chemistry of Fats and Lipids (Ed. by HOLMANT.) 8, 213-250. Pergamon Press, Oxford. ~IOLD J. D., MEANS R. E., STEVENSR. K., and RUTH J. M. (1966) The par&in hydrocarbon of wool wax. Homologous series of methylalkanes. Biochemistry 5, 455-461. STRANSKY K., STREIBL M., and SORM F. (1966) Naturally occurring waxes-IV. A new type of branched paraffins from the honey bee wax. Colin Czech. them. Commun. 31, 4694-4702.
HYDROCARBON AND OTHER LIPID CONSTITUENTS THE BULL ANT, MYRMECIA GULOSA G. W. K. CAVILL,
D. V. CLARK, M. E. H. HOWDEN, S. G. WYLLIE
OF
and
SchooI of Chemistry, The University of New South Wales, Kensington, N.S.W. 2033, Australia (Receiwed 11 March 1970) Abstract-The lipid constituents of Myrmecia gulosa comprise hydrocarbon, glyceride, and fatty acid fractions. The major hydrocarbons which are constituents of the cuticle wax are an homologous series of monomethylalkanes, C26 to C34. Each homologue is a mixture of isomers, for example, the C30 alkane includes 9-, ll-, 13-, and 15methylnonacosane. The minor hydrocarbons of the cuticle wax include the normal paraffins, Cl9 to C29. Of the free fatty acids the major components are oleic with lesser amounts of palmitic, stearic, and minor acids. 1,2- and 1,3-diglycerides and I-monoglycerides of normal type are the major lipid constituents. INTRODUCTION RECENT studies on the chemical composition of exocrine gland secretions of the primitive ant, Mymeciu gulosa (F.) (CAVILL and ROBERTSON, 1965), have established that, in addition to the characteristic proteinaceous venom of the venom gland (CAVILL et al., 1964; EWAN, 1967), the Dufour’s gland contains a range of aliphatic hydrocarbons (CAVILL and WILLIAMS, 1967). A preliminary study of the metastemal glands of M. gulosa also suggested the presence of aliphatic hydrocarbons. As such aliphatic hydrocarbons are constituents of insect cuticle waxes (GILMOUR, 1961), an examination of the hydrocarbon and other lipid constituents of M. gulosa has now been undertaken. So that sufficient material would be available for chemical characterization, the lipid constituents were obtained by total extraction of M. gulosa, and then compared, by gas chromatography, with the material obtained by extraction of the detached cuticle. Characterization of the major lipid constituents of the cuticle wax provides basic data in relation to proposed studies on ‘surface’ pheromones. MATERIALS
AND METHODS
Isolation of lipid constituents A typical extraction procedure was as follows. The ants (ZOOg) were trisected into heads, thoraxes, and gasters, and these were extracted (Soxhlet) with right petroleum, b.p. 40 to 6O”C, or methylene chloride (ZOOml) for 48 hr (cf. CAVILL and WILLIAMS, 1967). Each extract, after treatment with sodium carbonate 54
1721
1722
G. W. K. CAVILL, D. V. CLARK, M. E. H. HOWDEN, AND S. G. WYLLIE
solution, was washed (water) and dried (anhyd. sodium sulphate). The product, after removal of the solvent, was subjected to chromatography on alumina (Peter Spence, Grade H), yielding the hydrocarbon and glyceride fractions. The hydrocarbons were eluted with light petroleum and the glyceride fractions with solvents of increasing polarity. The alkaline aqueous solution, after acidification, was extracted with ether. The free fatty acids so obtained were converted into their methyl esters by treatment with an ethereal solution of diazomethane.
Gas chromatography Analytical gas chromatography was carried out on a Perk&Elmer 800 unit with nitrogen as carrier gas; the 6 ft by + in. stainless steel columns were packed with the listed stationary phases on Chromosorb G (SO-100 mesh): (a) 5% silicone oil, SE30, (b) 5% Ap iezon L, (c) 25% diethyleneglycol succinate. Preparative gas chromatography was undertaken on a Varian 705 unit with nitrogen as carrier gas; the 20 ft by # in. column was packed with 30% SE30 on Chromosorb W (SO-100 mesh).
Spectrometry Nuclear magnetic resonance spectra were recorded on a Varian A-60 spectrometer with carbon tetrachloride as solvent and tetramethylsilane as internal standard. Mass spectra were recorded on an A.E.I. MS9 instrument. 1.r. spectra were recorded as liquid capillaries on a Hilger-Watts Infrascan spectrophotometer with a grating monochromator.
Model compounds The reference compounds, 1 l-, 14-, and Kmethylnonacosane were synthesized for purposes of gas chromatographic and mass spectrometric comparison with the natural constituents. The following synthesis of 15-methylnonacosane from tetradecanol is typical. The alcohol was converted into 1-iodotetradecane which, via the Grignard reaction with solid carbon dioxide, gave pentadecanoic acid. The acid chloride on treatment with dimethylcadmium gave hexadecan-Zone (cf. CASON, 1947). This ketone, separated and purified using chromatography on alumina, was then subjected to a Wittig reaction with tetradecyltriphenylphosphonium iodide, also prepared from 1-iodotetradecane (cf. GREEN~ALDet al., 1963). The product, a mixture of cis- and trans-15-methylnonacos-14-ene was purified by chromatography on silica gel impregnated with silver nitrate. The olefins were hydrogenated under pressure, in the presence of Adam’s catalyst, to yield the required lSmethylnonacosane. Similarly, 11-methylnonacosane was prepared from dodecan-Zone and octadecyltriphenylphosphonium iodide, and 14-methylnonacosane from pentadecan-a-one and pentadecyltriphenylphosphonium iodide.
HYDROCARBON AMI OTHER LIPID CONSTITUENTS
OF THE BULL ANT
1723
RESULTS
Lipid constituents
of M.
gulosa
M. g&osa workers collected in the RoyaI National Park, south of Sydney, yielded lipid constituents which, in total, comprised from 2 per cent in summer up to 5 per cent in winter of the body weight of the insect. Of this lipids, approximately 14 per cent were isolated from the heads, 14 per cent from the thoraxes, and 72 per cent from the gasters, The constituents were readily separated into a neutral fraction consisting essentially of hydrocarbons and glycerides, and a fatty acid fraction. The latter comprised 20 to 30 per cent of the total extract. The neutral fraction, after chromatography on alumina (or silica), was further separated into hydrocarbons and the glyceride-ester constituents, in the approximate ratio 1 : 2.
The hydrocarbons Gas chromatography of the hydrocarbons showed that they could be divided into two main groups. The first and minor group consists of a mixture of normal and branched chain hydrocarbons, C9 to C14. Fig. 1 shows a typical gas chroSOIV
5% s-30.
I
35
1t
1100
I
30
I
25
I
20 Time,
FIG.
I
I5
1
IO
/
5
min
1. GLC of low molecular weight hydrocarbons.
matographic trace of these hydrocarbons, n-octane being added as a standard. A plot of log retention time vs. carbon number showed the major peaks, that is 1 to 5, to correspond to the normal alkanes, Cl0 to C14. Other hydrocarbons, whose peaks are also unchanged on hydrogenation, comprise several series of branched-chain alkanes. Peaks 6 to 12 may belong to the iso- or ante&-series.
1724
G. W. K. CAVILL, D. V. CLARK, M. E. H. HOWDEN, AND S. G. WYLLIE
The hydrocarbons previously characterized from Dufour’s gland in M. gulosa, including pentadecane, heptadecane, and heptadec-cis-8-ene (CAVILL and WILLIAMS, 1967), are present in the total extract, and in range span the above and the following groups. The second and major group of hydrocarbons comprises some 95 per cent of the total. On gas chromatography these hydrocarbons gave a complex series of peaks (Fig. 2). By the use of reference hydrocarbons, including a mixture of
5%SE-30,250’ &ml C,, &, Cz3,C,, and C,, dkanes
added
Time,
min
FE. 2. GLC of high molecular
weight hydrocarbons.
hydrocarbons from wool wax known to contain normal, iso and anteiso constituents (DOWNING et al., 1961) and by plots of log retention time vs. chain length, peaks 1 to 11 were identified as normal alkanes of chain length Cl9 to C29, with the odd numbered chains predominating (Figs. 2, 3). Similarly peaks 12 to 17, present only in small amounts, were shown to correspond to members of either the iso or anteiso series. The peaks, 18 to 24, correspond to members of an homologous series from C28 to C34, the even-numbered alkanes predominating. These hydrocarbons were unaffected by treatment with molecular sieve under conditions which separated the normal alkanes. Further, NMR and mass spectral data for these homologues suggested they were monomethylalkanes, the branch occurring towards the centre of the chain. Recently, comparable monomethylalkanes have been reported as minor constituents from beeswax (STRANSKY et al., 1966) and wool wax (MOLD et al., 1966). In the present work the more abundant methylalkanes, represented by peaks 18, 20, and 22 (Fig. 2), were separated by preparative gas chromatography, and their mass spectra examined. These data confirmed the branched nature of the alkanes and also showed that each of the purified homologues was a mixture of isomeric monomethylalkanes, with the methyl branch at various positions along the chain. To assist in the interpretation of the mass spectra, the reference compounds ll-, 14-, and 15-methylnonacosane were synthesized. These synthetic compounds could not be separated from each other under our gas chromatographic conditions and had a retention time identical with that of the C30 member of the
HYDROCARBON
AND
OTHER
LIPID CONSTITUENTS
172.5
OF THE BULL ANT
unknown series. Furthermore, comparison of the mass spectra of the synthetic compounds with those of the purified homologues enabled the major components of each homologue to be characterized.
e g g
I I%I.0o-so.e0.7 -
E 0.6 2 0,5-
o n-Alkanes l ;SOOr &7/&O A
20
21
22
23
24
25
26
27 Carbon
28
2!3
30
olkanes Centrally bmnched monamethyl alkanes
31
32
33
34
number
FIG. 3. Log retention time vs. carbon number plot for high molecular weight
hydrocarbons.
Thus the position of the methyl branch in the synthetic 11-methylnonacosane is clearly shown by the increased intensity of the charactersitic doublet of peaks at m/e 168, 169; 252, 253; and 280, 281 (Fig. 4), reflecting the tendency of the chain to fragment preferentially at the tertiary-branched carbon atom on electron impact. The intensity of the peaks at m/e 168, 169; 196, 197; 224, 225; 252, 253; 280, 281; and 308, 309 in the mass spectrum of the unknown C30 compound (Fig. 4) corresponds to methyl branches at carbon atoms 9, 11, 13, and 15. Comparable peaks were noted by STRANSKY et al. (1966) for their C28 hydrocarbon from beeswax. Thus the C30 fraction consists mainly of a mixture of 9-, ll-, 13-, and 15-methylnonacosane. It seems likely that smaller amounts of other isomers are also present. The C28 and C32 homologue consists of a similar mixture with methyl branches at positions 9, 11, 13, and 15 predominating. Finally, pieces of cuticle excised from the heads, thoraxes and gasters of M. gulosa workers (we are indebted to Dr. PHYLLIS L. ROBERTSON for these dissections) were extracted with light petroleum, and the extracts examined by gas chromatography. The above hydrocarbons were confirmed as constituents of the cuticle wax, with minor variations in their relative proportions from head, thorax, and gaster. The fatty
acids
The acidic constituents of M. gdosa which were separated from the total extracts of heads, thoraxes, and gasters, have been analysed by gas chromatography
1726
G. W. K. CAVILL, D. V. CLARK, M. E. H. HOWDEN, AND S. G. WYLLIE
The major constituent is oleic acid (approx. 70 per cent), after methylation. plus palmitic and stearic acid. The above compounds were characterized by a comparison of their methyl esters with standard specimens on three gas chromatographic columns. II-Methylnonacosane
422
14 - Methylnonacosane
60 u” 40 : -$
20
.z 0 0, .? %
0 100
z K
80
15- Methylnonacosane
60
40 20 0 00
r
Branched
2:”
C,,+omers
150 180 7.00 220 240 260 280 ZOO 320 340 360 380 400 420 m/e FIG. 4.
Mass
spectra of branched C30 alkanes.
The identification of minor constituents is based on plots of log retention times vs. carbon number, before and after hydrogenation. The minor acids, tentatively characterized, include tridecanoic, tetradecanoic, and heptadecanoic acids, branched C13, C16, C17, C19, and C20 acids, plus a normal Cl6 and a
IWDROCARBON AND OTHER LIPID CONSTITUENTS
OF THE BULL
ANT
1727
branched C20 unsaturated acid. The composition of the acids from heads, thoraxes, and gasters does not appear to vary to any marked extent. A total extract from heads and thoraxes from a summer collection of M. gulosa, on treatment with sodium hydrogen carbonate solution, gave an acid fraction which, after methylation and chromatography on alumina, yielded an ester having a terpene-like odour. Comparative gas chromatography, using methyl octanoate, nonanoate, and decanoate as standards, suggested the product is both branched and unsaturated. The ester showed retention times on two columns, identical with those of methyl citronellate.
The glyceri& The more polar compounds eluted from alumina after the hydrocarbons proved to be mainly glycerides, and the acid fraction obtained by hydrolysis of this material closely corresponded in composition to that of the free fatty acids reported above. By the use of ir. and, in particular, NMR spectroscopy (HOPKINS, 1965) the main constituents of the various glyceride fractions were identified. Two minor components, an ester fraction (vmax 1735 cm-l) and a triglyceride 1747 cm-l) were eluted with benzene-light petroleum, the former fraction (v,, tailing the hydrocarbons. The major component, a 1 : 2 mixture of 1,2- and 1,3-diglycerides was eluted with ether-benzene (1 : l), then a I-monoglyceride fraction was eluted with methanol-chloroform (1 : 9). The NMR spectra of these glyceride fractions were consistent with oleic acid being the predominant esterified acid (60 to 65 per cent), and with an average fatty acid chain length in the glyceride of Cl4 to Cl8 (c$ HOPKINS, 1965). This detailed spectroscopic study was carried out on the glyceride fraction from ants collected in the winter period. DISCUSSION The cuticular hydrocarbons comprise up to one-third of the neutral fraction extracted from M. gulosa workers, and, strikingly, the homologous series of centrally branched monomethylalkanes constitutes some 90 per cent of these hydrocarbons. Comparably monomethylalkanes have been detected recently as minor components of wool wax (MOLD et al.,1966) and of beeswax (STRANSKY et al., 1966). They comprise O-1 per cent of the latter. Beeswax has long been known to consist of normal long-chain esters, together with paraffin hydrocarbons and free fatty acids. More recent studies (DOWNING et al., 1961) h ave confirmed this pattern and, further, shown that the major hydrocarbons (approx. 16 per cent) are normal alkanes, Cl9 to C33, with the oddnumbered homologues, C27 to C33, predominating. In contrast, the major cuticular hydrocarbons of M. gulosa are the homologous series of monomethylalkanes, C26 to C34, with the even-numbered members predominating. However, the minor cuticular hydrocarbons include the normal alkanes, Cl9 to C29, the odd-numbered homologues again being present in relaSeasonal variation in the relative proportions of the tively greater amounts.
1728
G. W. K. CAVILL, D. V. CLARK, M. E. H. HOWDEN, ANDS. G. WYLLIE
hydrocarbon components was noted. In general, the winter extracts showed a higher proportion of normal to branched hydrocarbons; however, there was a lower proportion of the higher chain length homologues of both series of alkanes. The free fatty acids of M. gulosa comprise oleic (approx. 70 per cent) with smaller proportions of palmitic, stearic, and lesser acids. This composition corresponds to that normally found in animal fats (cf. GILBY, 1965). Moreover, the glyceride fraction
would
appear
On the assumption
to contain
the above
that the glycerides
acids as the major
are representative
then the hydrocarbons are the major components biogenesis of these centrally branched monomethylalkanes
gutosa,
fatty
acid units.
of the fat body of M.
of the cuticle wax. The has yet to be considered.
Acknozcledgements-We thank Dr. PHYLLIS L. ROBERTSON of this university for helpful discussion, and Dr. D. DOWNING,C.S.I.R.O., Division of Applied Chemistry, Melbourne, for specimens of wool wax hydrocarbons. Messrs. R. GREENWOOD and J. RIDINGS are thanked for technical assistance. This investigation has been supported by grants from the U.S. Public Health Service and the Australian Research Grants Committee. REFERENCES CASONJ. (1947) The use of organ0 cadmium reagents for the preparation of ketones. Chern. Rev. 40, 15-32. CAVILL G. W. K. and ROBERTSONP. L. (1965) Ant venoms, attractants and repellents. Science, Wash. 149, 1337-1345. CAVILL G. W. K., ROBERTSONP. L., and WHITFIELD F. B. (1964) Venom and venom apparatus of the bull ant, Myrmecia gulosa (Fabr.). Science, Wash. 146, 79-80. CAVILL G. W. K. and WILLIAMSP. J. (1967) C onstituents of Dufour’s gland in Myrmecia gulosa. J. Insect Physiol. 13, 1097-1103. DOWNINGD. T., FRANZ Z. H., LAMBERTONJ. A., MURRAYK. E., and REDCLIFFEA. H. (1961) Studies in waxes-XVIII. Beeswax: a spectroscopic and gas chromatographic examination. Aust. J. Chem. 14, 253-263. SWAN L. M. (1967) Ph.D. Thesis, The University of New South Wales. GILBY A. R. (1965) Lipids and their metabolism in insects. A. Rew. Ent. 10, 141-160. GILMOURD. (1961) Biochemistry of Insects. Academic Press, New York. GREENWALD R., CHAYKOVSKY M., and COREYE. J. (1963) The Wittig reaction using methylsuliinyl carbanion-dimethyl sulphoxide. J. org. Chem. 28, 1128-1129. HOPKINSC. Y. (1965) Nuclear magnetic resonance in fatty acids and glycerides. In Progress in the Chemistry of Fats and Lipids (Ed. by HOLMANT.) 8, 213-250. Pergamon Press, Oxford. ~IOLD J. D., MEANS R. E., STEVENSR. K., and RUTH J. M. (1966) The par&in hydrocarbon of wool wax. Homologous series of methylalkanes. Biochemistry 5, 455-461. STRANSKY K., STREIBL M., and SORM F. (1966) Naturally occurring waxes-IV. A new type of branched paraffins from the honey bee wax. Colin Czech. them. Commun. 31, 4694-4702.