Insect Biochem., VoL 9 pp. 117 to 121. 0020--1700/79/0201-0117 $02.00/0 © Pergamon Press Ltd. 1979. Printed in Great Britain

THE CHEMICAL COMPOSITION OF THE DUFOUR GLAND SECRETION OF THE ANT MYRMICA SCRABRINODIS

E. D. MORGAN,* K. PARRY and R. C. TYLER

Department of Chemistry, University of Keele, Staffordshire. ST5 5BG, England

(Received 24 April 1978)

Abstract--The Dufour gland secretion of M. scabrinodis is largely composed of the same farnesene homofarnesene and bis-homofarnesene isomers found earlier in M. rubra, a Cla Tris-homofarnesene has also been identified. The hydrocarbons found in M. rubra are absent from M. scabrinodis except for small amounts of pentadecane, pentadecene, heptadecane and nonadecane. Methanol, ethanal, ethanol, acetone, butanone, butenone and l-butanol are also present in small quantities. Further evidence has been obtained from the dehydration of pure nerolidol isomers by a new method, to show that the Myrmica ants produce (Z,E)-ct-farnesene.

Key Words Index: Ant. Dufour gland. Myrmica scahrinodis, :t-farnesene, pheromones

I N T R O D U C T I O N

THE DUFOUR gland secretion of the common red ant Myrmica rubra was shown to consist of straight chain hydrocarbons, saturated and unsaturated and three terpenoid compounds; farnesene, homofarnesene and bishomofarnesene (MORGAN and WADHAMS, 1972a). Fur ther work has shown that low molecular weight volatile components also have a pheromone effect in this species (CAMMAERTS-TRICOT e t al., 1976; MORGAN et al., 1977).

A preliminary examinat ion by gas chromatography o f the contents o f the Dufour gland o f M. scabrinodis showed that it had a completely different profile from that o f M. rubra, that essentially there were only four components rather than more than twenty as in M. rubra. Since these two species are often found in colonies only a few feet apart, it was o f interest to study the composi t ion o f the chemicals in the Dufour gland of M. scabrinodis and to carry out behavioural studies. Closer examination showed the four main components were terpenoid hydrocarbons, and the absence o f straight chain hydrocarbons o f similar molecular weight made M. scabrinodis a better species in which to study these terpenoids.

M A T E R I A L S AND M E T I t O D S

Colonies of M. seabrinodis were collected in Staffordshire and Shropshire and maintained in artificial nests and fed a diet of an aqueous solution of sucrose and chopped desert locusts and other insects. Age of workers was determined from degree of pigmentation according to the scheme of CAMMAERTS-TRICOT (1974).

The methods used for determining the nature and percentage composition of the volatile components of the gland, the amount of water, and the volume of the gland were as described in MORGAN et al. (1977).

For the study of the less volatile hydrocarbons, seven

* Author for correspondence.

I.B. 9 . I --H

analytical gas chromatography columns were used, with either OV-101 silicone, Apiezon L, PEGA or PEG 20 M as stationary phase, and two capillary columns (30 x 0.3 mm i.d.), one coated with OV-101, the other with PEG 20M. Samples were applied to the packed analytical columns by sealing the sting apparatus (including the poison and Dufour glands) or whole gasters in glass ampoules and crushing them in the heated carrier gas stream as described by MORGAN and WADHAMS (1972b). For capillary columns, a solution in diethyl ether was prepared by grinding a number ofgasters in a small tissue grinder, centrifuging, decanting the supernatant, reducing its volume by evaporation at 25°C with a stream of nitrogen and storing for use at -20°C under nitrogen.

Mass spectra were obtained with a Pye gas chromatograph linked through a Biemann separator to an Hitachi-Perkin RMU-6 mass spectrometer, excitation energy 70 eV, accelerator 1.8 kV, and source temperature 200°C.

A mixture of (E)- and (Z)-nerolidol (5.0 g) was dehydrated by heating with p-toluene sulphonic acid (20 mg) in toluene (50 ml) which was refluxed in a Dean-Stark apparatus until no more water was collected. The mixture was cooled, neutralized with sodium carbonate and toluene removed under reduced pressure. The residue was chromatographed on a column of Florisil (2.5 cm i.d. x 25 cm), eluting with light petroleum (b.p. 40-60°C, 50 ml). Yield of mixed farnesenes was 819/o. Dehydrations of pure (E)- and (Z)-nerolidol were carried out on a 0.5 g scale by the same method.

Samples of pure (E)- and (Z)-nerolidol were obtained by preparative gas chromatography on a Pye 105 gas chromatograph, using a 10~o PEG 20 M on Diatomite (100-120 mesh) column (3 m x 4.5 mm i.d.) with nitrogen flow of 40 mil min -1 at 180°C. The purified isomers were trapped in glass U-tubes cooled in liquid nitrogen, and separately dehydrated, as above.

R E S U L T S

The analysis o f the very volatile port ion o f the Dufour gland contents of M. scabrinodis showed that this port ion was qualitatively identical to t h a t o f M. rubra. Quantitatively, M. scabrinodis contains an . average 114 ng of volatiles (Table I) whereas M. rubra

117

118 E.D. MORGAN, K. PARRY AND R. C. TYLER

Table !. Composition of the volatile part of the contents of the Dufour gland of M. scabrinodis

Composition Peak No, Quantity per gland in Fig. 1 Compound ng* Average Min. Max.

1 Methanol 2.9 2.5 2.0 2.8 2 Ethanal 38.9 34.1 22.2 48.5 3 Ethanol 4.2 3.7 2.6 5.0 4 Propanal 0.9 0.8 0.6 1.1 5 Acetone 22.7 19.9 18.3 21.5 6 Methylpropanal 9.9 8.7 7.8 • 9.8 7 Butenone 11.9 10.4 9.0 13.6 8 Butanone 17.3 15.1 11.8 18.5 9 l-Butanol 5.5 4.8 3.8 5.6

114.2

* Average of 10 determinations.

contains an average 126 ng (MoRGAr~ et al., 1977). Ethanal, acetone, butenone and butanone are the major constituents (Fig. 1). The gland contains no detectable amount of water (i.e. less than 1 ng). The mean total volume of the gland found from 8 determinations on darkly pigmented, foraging workers is 3.8 nl with a S.E. of the mean of 0.7 nl. In younger, lightly pigmentated and very old, deeply pigmented workers, the volume is less. The gland is therefore smaller than in M. rubra, as can be readily confirmed by comparing the glands of the two species under the microscope. The percentage composition of the volatiles differs only in that the amount of butanone is higher in M. scabrinodis, the minimum percentage of this component being higher than the maximum value found in M. rubra.

When either a whole gaster or material removed with a fine capillary from the Dufour gland of M. scabrinodis was gas chromatographed, after the very volatile compounds were eluted, a simple pattern of four major peaks was eluted (Fig. 2). The first three of

8

I 5

5 2

3

,=.-4-

. . , . ~ mln

Fig. 1. Gas chromatographic trace of the very volatile portion of the Dufour gland secretion of M. scabrinodis. Conditions: 5 ft Porpak Q column at 167°C with helium carrier gas at 50 ml min-L The first two peaks are due to pressure disturbance on injection and water. Peaks are 1 =- methanol, 2 = ethanal, 3 = ethanol, 4 = propanal, 5 = acetone, 6 = 2-methylpropanal, 7 = butenone, 8 =

butanone. 9 = 1-butanol.

these corresponded in retention times to the farnesene, homofarnesene and bishomofarnesene found in M. rubra. The identification was confirmed by mass spectrometry. The mass spectrum of farnesene was identical to that published by MORGAN and WADHAMS (1972a). The spectra of the homofarnesene and bishomofarnesene were rather more intense with slight differences in peak intensity, but showed they were from the same compounds. For bishomofarnesene the base peak was at m/e 93, and the ion at M + (232), m/e 203, 133, 105 and 83 slightly more intense. A detailed analysis of the mass spectra will be given elsewhere. The analysis showed from the relative intensities of certain ions, that of the six possible faresene isomers, that present in both M. rubra and M. scabrinodis is (Z,E)-ct-farnesene. Similar arguments applie d to the higher homologues indicates that they also possess the (Z,E)-~-arrangement of double bonds. The extra methylene is on C 7 or C 3 (probably the former) in homofarnesene and on C~t and C 7 or C 3 in bishomofarnesene. The trishomofarnesene, described here for the first time appears to belong to the same series in respect to the geometry of the double bonds at C3 and C6. A further isomeric possibility is introduced at the C10-Cll double bond in bis- and tris- homofarnesene and the geometry of this bond is not known.

In order to confirm the identification of farnesene as the(Z,E)-ct-isomer, amixtureof(E)- and(Z)- nerolidol was dehydrated by the method of At,~T (1970), to give a 30~ yield of mixed farnesene'isomers. It was evident from the large differences in retention times on gas chromatography that the natural isomer was not a fl- farnesene or(Z,Z)-~t- or (E,E)-ct-farnesene. It was less easy to distinguish between the possibilities of (Z,E)-a- and (E,Z)~-farnesene (Fig. 3) because of their similar retention times, and because the separation was not complete enough to give uncontaminated mass spectra of each isomer on our columns. Investigation of the dehydration reaction enabled us to improve the yield to give 81~ of mixed farnesenes with a higher proportion of the less stable isomers using p-toluene sulphonic acid.

The mixture of nerolidols was separated by preparative gas chromatography into pure (E)- nerolidol and (Z)-nerolidol, the purity checked by gas chromatography, and the isomeric identification

Dufour gland of M. scabrinodis 119

5

4

A Fig. 2. Gas chromatographic trace of the less volatile portion of the Dufour gland secretion ofM. scabrinodis on a 30 m SPI000 capillary column at 150°C with nitrogen flow rate of 3 ml min- i. Peaks are 1 = pentadecane, 2 = pentadecene, 3 = unknown 4 = heptadecane, 5 = farnesene, 6 = homofarnesene, 7 =

nonadecane, 8 = bishomofarnesene, 9 = Trishomofarnesene.

checked by 100 MHz NMR spectrometry (ANET, 1970). The nerolidols were then separately dehydrated with p-toluene sulphonic acid. (E)-Nerolidol gave a mixture of (E)-~-farnesene, (Z,E)-a-farnesene, (E,E)- n-farnesene and a cyclized product. Isomerization of the less stable cis double bond of (Z)-nerolidol occurred and a more. complex mixture of (Z)-/~- farnesene (Z,Z,)-a-farnesene, (E,Z)-g-farnesene and (E,E,)-~-farnesene with some cyclized product was produced. However although the mass spectra of (Z,E)-~-farnesene could not be obtained reliably free from other isomers, the retention times of all isomers could be accurately measured, and the natural isomer from M. scabrinodis was shown to be identical to that of (Z,E)-~-farnesene on the two capillary columns.

The amounts of the terpenoid hydrocarbons, present in a worker varied considerably over a range of values. The average quantity per insect was 200 ng farnesene, 500 ng homofarnesene and 500 ng

H H

I rr

rn IE

Fig. 3. Structures of (E)-nerolidol, I; (Z)-nerolidol, II; (Z,E)- ~-farnesene, III; and (E,Z)-~,-farnesene, IV.

bishomofarnesene and 250 ng trishomofarnesene. The ratio of homofarnesene to bishomofarnesene remains close to one throughout the wide range of composition encountered. The mean values obtained from seven nests collected at various times together with their standard errors are listed in Table 2 for farnesene, homofarnesene and bishomofarnesene. The values for trishomofarnesene, are less reliable because of the long retention times, possible loss through isomerization and poorer peak shape.

In addition to the terpenoid compounds n- pentadecane, heptadecane and nonadecane were also identified, from their easily recognized mass spectra, and from comparision of retention times with standard samples, a pentadecene, p r o b a b l y 7- pentadecene, was detected.

DISCUSSION

We have recently shown that the volatile compounds ethanal, ethanol, acetone and butanone together act as a short term attractant for workers of M. rubra to encourage them to forage for food on a new area (C~AERTS et al., 1977). The presence of the same compounds in M. scabrinodis in roughly the- same proportions is not surprising and presumably these compounds act in the same way in both species. It will be interesting to see if this hypothesis is borne out in further work.

The difference between the two species in the less volatile portion of the secretion is very striking. Farnesene, homofarnesene and bishomofarnesene are practically the only components in the C 1 , -C 18 range. Though the same terpenoid compounds were present

120 E.D. MORGAN, K. PARRY AND R. C. TYLER

Table 2. The mean amounts of farnesene, homofarnesene and bishomofarnesene found for a total of seventy-two determinations on seven nests of Myrmica rubra, together with the standard errors of the means,

(S.E.M.)

Farnesene Homolhrnesene Bishomofarnescne No. of Mean value Mean value Mean value

Nest samples #g S.E.M. /~g S.E.M. /~g S.E.M.

1 12 0.25 0.03 0.56 0.06 0.47 0.09 2 9 0.31 0.08 0.65 0.16 0.81 0.13 3 18 0.13 0.02 0.30 0.05 0.30 0.05 4 5 0.16 0.04 0.18 0.08 0.21 0.06 5 10 0.32 0.04 0.90 0.13 0.92 0.13 6 7 0.14 0.05 0.37 0.14 0.41 0.17 7 11 0.19 0.04 0.31 0.05 0.27 0.06

Mean 0.21 0.47 0.48

in M. rubra, there they were mixed with C 13 -C~ 9 linear hydrocarbons, saturated and unsaturated. No correlation could be found between the amount of terpenes present in an individual or the relative proportion of them and any factor such as the colony, size of the worker, area of collection or food. Age of workers was not noted. It is possible a correlation might be found with degree of pigmentation.

The occurrence of farnesenes in insects has been recorded a number of times, in particular (E)-fl- farnesene has been identified as an alarm pheromone for several species of aphids (BowERs et al., 1972; EDWARDS et al., 1973; WIENTJENS et al., 1973). A fl- farnesene of undetermined stereochemistry has been shown to be the sole constituent of the Dufour gland of the ant Aphaenogaster longiceps (CAVlLL et al., 1967). BER(;STR(iM and Lr~OWST (1968) found an ~- farnesene isomer in the Dufour glands of three formicine ants. From their published mass spectrum it was clearly the same isomer as is found in M. rubra. This, it is concluded could not be a fl-farnesene or the most stable all-trans-ct-farnesene or (E,E)-~t-farnesene. The most probable structure was either (Z,E)-ct- farnesene or (E,Z)-~-farnesene (Fig. 3, III and IV respectively), and it can now be identified as (Z,E)-~- farnesene.

It was clear that in the present work, the same isomer was present and further efforts were made to identify it with certainty. Until such time as a pure farnesene of the correct stereochemistry can be synthesized by a multi-stage operation, the most useful stratagem to identify the isomer present would be a sample of material from ants sufficiently large (1 mg) for a proton magnetic resonance spectrum. Assuming 50~o efficiency of trapping by preparative gas chromatography the latter would require ten thousand insects. Another alternative is a mixture of synthetic farnesene from which pure isomers could be obtained by preparative gas chromatography. ANET (1970) has described a method of dehydrating a mixture of(E)- and (Z)-nerolidol (Fig. 3, I and II) to a mixture of farnesenes using phosphoryl chloride in pyridine. This gives appreciable quantities of cyclized derivatives, such as ~- and/3-bisabolene and not very much of the two required (and less stable) ~t- farnesenes. We therefore investigated a number of mild dehydrating conditions for converting nerolidol to farnesenes. The best of these, giving much higher yields of a clean mixture of farnesenes was using p-

toluene sulphonic acid, and by the dehydration of pure (E)-nerolidol by this method, retention time evidence was obtained to show that the Myrmica substance is (Z,E)-ct-farnesene.

An analysis of the relative intensities of certain important ions in the mass spectrum confirms the identification as (Z,E)-ct-farnesene, and by extension of the mass spectral arguments, it is possible to identify tentatively, the isomer of the homofarnesene, and the partial structure of bishomofarnesene and trishomofarnesene (Fig. 4), except for the isomerism introduced at the C 10 double bond in the latter two by the additional methylene group. All these structures require confirmation by independent synthesis.

Linear hydrocarbons form a minor portion of the M. scahr#mdis Dulbur gland contents, however, (Z)- 8-heptadecene, the major component of M. ruhra is absent from M. scahrinodis, but pentadecene and sometimes nonadeccne are present in the latter.

Thc lindings that M. ;'llhl'[I and M. scal~rimMis posscss almost the same composition of volatile substances but very different less volatile substances in the Dufour gland accords very well with the behavioural observations on these two species, described by CAMMAERTS et al. (1978). Older workers of both species explore new foraging territory, moving slowly and laying down droplets of their oily Dufour gland secretion with the tip of the sting lance. Following workers are stimulated to move more rapidly over the territory, moving sinuously, presumably scouring it

8 6 4 OF

9 3

Fig. 4. Structures of the terpenoids found in Dufour glands from worker M. scabrinodis: !11 farnesene. V homofarnesene (two possibilities not yet distinguishable). VI bishomofar-

nesene (two possibilities). VII trishomofarnesene.

Dufour gland of M. scabrinodis 121

for food. Both species perform this behaviour and the following workers are unable to tell if the secretion has been laid by their own, or the other species.

When the volatile alcohols and carbonyl compounds evaporate away after a few minutes, the mixture of higher molecular weight hydrocarbons is left. The foraging workers detect this pheromone, moving less quickly over the territory than they did when the secretion was freshly laid, but more quickly than on a new territory; moreover, they recognised whether the secretion was deposited by their own species.

Though our early electo-antennograph studies were unable 'to detect specific response to these hydrocarbons (MORGAN and WADHAMS, unpublished results), presumably workers are able to distinguish between the predominantly linear hydrocarbons of M. rubra and the predominantly terpenoid hydrocarbons of M. scabrinodis (they are readily distinguishable by the human nose).

We are interested in extending this study to other species of Myrmica. Preliminary results show that while there is considerable variation between individuals and colonies, other species use variations on the same theme of C13--C19 linear and terpenoid hydrocarbons for their Dufour gland secretion.

Acknowledgements--We wish to thank the SCR for a grant for the purchase of gas chromatography equipment, and also for provision of the facilities of the Physico-chemical measurements Unit, Harwell, for 100 MHz NMR spectra.

REFERENCES

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BOWF~S W. S., NAULT L. R., WEBB R. E. and DUTKY S. R. (1972) Aphid alarm pheromones; isolation, identification, synthesis. Science 177, 1121-22.

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CAMMAERTS M.-C., INWOOD M. R., MORGAN E. D., PARRY K. and TYLER R. C. (1978) Comparative study of the pheromones emitted by workers of the ants Myrmica rubra and Myrmica scabrinodis. J, Insect Physiol. 24, 207-214.

CAMMAERTS M.-C., MORGAN E. D. and TYLER R. C. (1977) Territorial marking in the ant Myrmica rubra L. (Formicidae). Biol. Behav. 2, 263-272.

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CAVILL G. W. K.. WILLIAMS P. J. and WHITEIELD F. B. (1967) ct-Farnesene, Dufour's gland secretion in the ant Apheno.~ster hmgiceps. Tet. Letters No. 23. 2201-2205.

EDWARDS L. J., SIDDALL J. B., DUNHAM L. L., UDEN P. and KISLOW C. J. (1973) Trans-[~-farnesene, alarm pheromone of the green peach aphid Myzus persicae (Sulzer). Nature 241, 126-127.

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MORGAN E. D. and WADHAMS L. J. (1972a) Chemical constituents of Dufour's gland in the ant Myrmica rubra. J. Insect Physiol. 18, 1125-1135.

MORGAN E. D. and WADHAMS L. J. (1972b) Gas chromatography of volatile compounds in small samples of biological materials. J. ChrDmatog. Sci. 10, 528-529.

WIENTJENS W. H. J. M., KAKW~JK A. C. and VAN DER MAREL T. (1973) Alarm pheromones of grain aphids. Experientia 29, 658-660.