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Chemical investigations of the Dufour gland contents of Attine ants
Insect Biochem., Vol. 11, No. 3, pp. 343-351, 1981. Printed in Great Britain.
0020-1790/81/030343-08502.00/0 © t981 Pergamon Press Ltd.
CHEMICAL INVESTIGATIONS OF THE DUFOUR GLAND CONTENTS OF ATTINE ANTS R. P. EVERSHEDand E. D. MORGAN Department of Chemistry, University of Keele, Staffordshire ST5 5BG, England (Received 30 September 1980)
Abstract--The Dufour glands of workers of the two subspecies Atta sexdens sexdens and Atta sexdens rubropilosa are completely filled with linear alkanes and alkenes in the C)s to C23 chain length range in microgram amounts. There are quantitative differencesin the hydrocarbon composition betweenworkers of the two subspecies.The major glandular component ofA. s. sexdens is (Z)-9-tricosene,in A. s. rubropilosa the most abundant component is (Z)-9-nonadecene.From a chemicaltaxonomic viewpointA. s. rubropilosa and A. s. sexdens could be regarded as separate species. The Dufour gland of Atta cephalotes is only partially filled with linear alkanes and alkenes in the Clz to C19 chain length range in nanogram amounts; n-heptadecane is the most abundant alkane and (Z)-9nonadecene is the most abundant alkene. Key Word Index: Ants, Dufour gland, Atta sexdens, Atta cephalotes, hydrocarbons
INTRODUCTION WE HAVE recently reported a chemical study of the Dufour gland of the two Attine ants, Atta cephalotes and Acromyrmex octospinosus (EVERSHED and MORGAN, 1980). The results showed that, in common with the other myrmicines that have been studied, these two tropical species also produce volatile hydrocarbons in their Dufour glands. In contrast to the temperate myrmicines (CAMMAERTSet al., 1978), the volatile material of these two Attines constituted only a very small fraction of the total gland volume. It has been proposed that the primary evolutionary function of the Dufour gland secretion is one of sting or egg lubrication (WHEELER, 1910; ROnERTSON,1968). The sting of these Attines is rudimentary, its principal function being to deposit the trail pheromone on the substratum. On this basis we suggested that the absence of lubricating chemicals in their Dufour glands is the result of there being little necessity for them. A. cephalotes workers produce mainly n-alkanes in their Dufour gland with n-heptadecane as the major constituent. The hydrocarbons produced by A. octospinosus are of a rather different type; the major component being a terpenoid hydrocarbon, homofarnesene. With the exception of Pogonomyrmex rugosus and P. barbatus, which produce a number of methyl-branched alkanes (REGNIER et al., 1973), the tendency of myrmicines to produce either predominantly terpenoid hydrocarbons or predominantly linear hydrocarbons is one which has been shown to be common to all those myrmicines examined to date (BLUM and HERMANN, 1978; MORGAN et al., 1979; CAMMAERTSet al., 1980). To act as a basis for future biosynthetic and ethological studies we have extended our investigations to include Atta sexdens sexdens and A. sexdens rubropilosa and this paper reports the identity of the volatile chemicals of their Dufour glands. Greater experience with the techniques has enabled us 343
to present the results of some further work on the Dufour gland volatiles of A. cephalotes. The Dufour glands of the two subspecies of A. sexdens are almost completely filled with hydrocarbons which are for the most part monosaturated alkenes. The compositions of the gland contents in A. s. sexdens and A. s. rubropilosa are sufficiently different to enable them to be considered separate species, from a chemical taxonomic viewpoint.
MATERIALS AND METHODS Sources of insect material Colonies of A. cephalotes from Trinidad were maintained in the laboratory at 26--28°C and at 85-95% r.h. (relative humidity) and supplied with a variety of plant materials for their fungus cultivation. Additional A. cephalotes soldiers were obtained from other colonies from various parts of South America maintained at the University College of North Wales, Bangor. Workers of A. s. sexdens and A. s. rubropilosa were also supplied from colonies maintained at Bangor originating from Guyana and Paraguay respectively. The worker ants were killed by momentary immersion in liquid nitrogen or by exposure to solid carbon dioxide. The ants wereeither used immediatelyor stored in a deep freeze at 20°C. -
Gas chromatography ( GC) Pye 104 series gas chromatographs fitted with flame ionisation detectors (FID) were used throughout these investigations. The three types of GC phase used were: A. 5~o (w/w) OV 101 on Chromosorb W, acid washed and hexamethyldisilazane treated (AW-HMDS) 100-120 mesh packed in a 1.5 m x 4 mm i.d. glass column. B. 5% (w/w) diethyleneglycol succinate (DEGS) on Supersorb (AW-HMDS) 100-120 mesh packed in a 3 m x 4 mm i.d. glass column. C. 10~/o (w/w) polyethyleneglycol (PEG 20M) on Chromosorb W (AW-H MDS) 100-120mesh packed in a 1.5 m x 4 mm i.d. glass column.
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R.P. EVERSHEDAND E. D. MORGAN
Single dissected glands, capillary extracts (MORGAN and TYLER, 1977) and whole gasters were analysed by GC in conjunction with a solid sampling technique (MORGAN and WADHAMS, 19723. Tissue extracts were prepared by macerating whole gasters with redistilled hexane (at approx. 10 kd/mg tissue) in a small tissue grinder. The decanted extracts were concentrated to a volume of 4-5 #1 before analysis by exposure to a stream of dry nitrogen at room temperature. Gas chromatography-mass spectrometry (GC-MS) Initial GC-MS studies were perfomed on a Pye 104 series GC with FID linked to a Hitachi-Perkin Elmer RMU 6E mass spectrometer through a Watson-Bieman separator maintained at 190°C. Helium was the carrier gas at a flow rate of 15 ml/min. Further investigations were carried out on a Pye 204 series GC linked to a VG Micromass 7070F mass spectrometer through a glass jet separator. This system incorporated a VG 2000 data system. In both cases analyses were performed on tissue extracts. Identifications were made by interpretation of fragmentation patterns and by comparison with reference spectra. Reference compounds The GC retention times and mass spectra of the glandular components were compared with those of commercially available n-alkanes. Alkenes were synthesised via a Wittig route; the appropriate phosphonium salt was prepared by refluxing the alkyl bromide with triphenylphosphine for 5 hr in dry dimethylformamide (DMF) (dried by refluxing for 5 hr over P2Os, followed by distillation from P2Os). The Wittig reaction was carried out in dry tetrahydrofuran (THF) (distilled from sodium) under a dry nitrogen atmosphere. The ylid was formed by adding an equimolar solution of n-butyl lithium in hexane to the salt solution at 0°C. This was followed by dropwise addition of the appropriate aldehyde to give the alkene. The reaction was over in a few seconds at 0°C. The mixture of E and Z isomers produced was separated by medium pressure column chromatography (MPCC) on 20% (w/w) AgNO 3 on Kielselgel (EVERSHED, MORGAN and THOMPSON, in preparation). GC analysis showed the relative proportion of Z to E isomers to be 4:1 under the experimental conditions described. The purity of all the reference compounds was checked by GC; and by infrared spectrometry (IR) in the case of the alkenes. Bromination Micro-scale bromination of the glandular components was performed by adding 0.5 #l of a 1:1 solution of bromine in carbon disulphide to a single Dufour gland in a solid sample glass capillary tube (MORGAN and WAOHAMS, 1972). The sealed capillary was then placed in the solid sampler with the injection port heater set at 200°C. Five minutes were allowed to elapse before crushing to ensure complete bromination of any unsaturated components. Analysis after bromination was performed using column A, in the cases of the two A. sexdens subspecies, a nitrogen carrier gas flow rate of 60 ml/min was employed and the GC oven temperature programmed from 180-210°C at 2°C/min. In the investigation of A. cephalotes the GC oven temperature was held isothermally at 165°C. Ozonolysis The positions of the double bonds in the alkenes were determined by ozonolysis of fractions trapped from the GC using a micropreparative apparatus (BAKERet al., 1976). The trapping was performed on extracts obtained through the maceration of twenty-five gasters with 200 #1 of redistilled hexane. This volume was reduced to 2 #1 under a stream of nitrogen at room temperature before fractionation by GC. A 1:100 (FID:trap) split ratio was employed to avoid unnecessary loss of sample to the detector. The samples were
trapped in 20 cm ~U' tubes of 0.5 mm i.d. cooled in liquid nitrogen. The trapped material was washed from the 'U' tube with 100 ,ul of dichloromethane. Ozonolysis was then performed by exposing the alkene solution, cooled in an ice bath, to a slow stream of ozone from a micro-ozone generator (BEROZA and BIERL, 1969) for a few minutes. The ozonides were reduced by addition of a small crystal of triphenylphosphine in order to yield the carbonyl compounds. The micropreparative work was carried out on column A. Analysis of the ozonolysis products was performed on column B. Thin layer chromatography Thin layer chromatography was employed to determine the configurations of the double bonds in the unsaturated glandular components. Silica gel plates (0.3 mm thickness) impregnated with 10% (w/w) AgNO 3 were used in conjunction with an eluent of 1~,~, diethyl ether in light petroleum (b.p. 40-60°C). In the analysis of the alkenes in the two subspecies of A. sexdens a tissue extract of poison gland complexes from five workers was chromatographed together with synthetic samples of (E) and (Z)-9-nonadecene and (Z)9-tricosene. An extract from sixty workers gasters of A. cephalotes was also analysed under similar conditions. The plates were developed by spraying with 20~o (w/w) concentrated sulphuric acid followed by heating in an oven at 120°C for 15 min. Determination of glandular volume The dimensions of the glands were determined using an eye graticule. The glands appear as gently tapering sacs, being hemispherical at their posterior end (Fig. 1). On this basis the glandular volume (V) was calculated from the following formula where r is the radius at greatest cross section and h is the length of the gland: V= z3ztr2h+ ~3Tcr3 RESULTS A. s. sexdens and A. s. rubropilosa The D u f o u r glands of A. s. rubropilosa a n d A. s. sexdens are almost completely filled with mixtures of the same alkanes a n d alkenes (Tables 1 and 3). In b o t h subspecies the q u a n t i t y of h y d r o c a r b o n in the gland is directly p r o p o r t i o n a l to the body weight of the ant (Fig. 2). The relative p r o p o r t i o n s of the individual c o m p o n e n t s differ significantly in the two subspecies (Table 1, Fig. 3). In A. s. rubropilosa the most a b u n d a n t c o m p o n e n t is (Z)-9-nonadecene, while in A. s. sexdens, (Z)-9-tricosene predominates. G a s c h r o m a t o g r a p h y of a single gland from each subspecies revealed twenty-one volatile c o m p o n e n t s (Fig. 3). C o m p o n e n t s 2, 4, 6, 9, 12 a n d 15 were found to have retention times corresponding to n-alkanes in the C l s to C2o chain length range, on b o t h polar a n d n o n - p o l a r G C phases (columns A and C, see Materials a n d Methods). Plotting the logarithm o f retention time (log tr) against the n u m b e r of c a r b o n a t o m s in the h y d r o c a r b o n chain suggested t h a t c o m p o n e n t 17 is p r o b a b l y n-heneicosane. C o m p o n e n t s 5 a n d 11 h a d retention times o n columns A a n d C c o r r e s p o n d i n g to heptadecene and nonadecene. A further plot of log t r against the n u m b e r of c a r b o n atoms in the chain indicated that c o m p o n e n t s 1, 8, 14, 16, 19 a n d ' 2 1 were p r o b a b l y m o n o - u n s a t u r a t e d alkenes, namely pentadecene, octadecene, eiconsene, heneicosene, docosene a n d tricosene respectively.
345
Fig. 1. Sketches of the Dufour gland, poison gland complexes o f Atta cephalotes and of A. sexdens rubropilosa, showing D F = Dufour gland, PG = poison glands, PV = poison vesicle and S = sting lance.
Dufour glands of Attine ants
347
Table 1. Dufour gland compositions of A. s. rubropilosa and A. s. sexdens together with the analytical evidence for the assignments A. s. rubropilosa
Mean % composition by weight (_+ S.D. n = 10)
Compound
1 Pentadecene 2 Pentadecane 3 Homofarnesene 4 Hexadecane 5 Heptadecene 6 Heptadecane 7 8 9-Octadecene 90ctadecane 10 8,11-Nonadecadiene I1 (Z)-9-Nonadecene 12 Nonadecane 13 14 Eicosene 15 Eicosane 16 9-Heneicosene 17 Heneicosane 18 19 Docosene 20 21 (Z)-9-Tricosene
0.06 0.32 0.32 0.39 0.88 8.35 0.24 3.27 1.15 5.81 38.9 7.17 0.01 0.84 0.38 4.03 0.92 0.01 1.72 0.01 25.24
+ 0.04 + 0.16 + 0.08 + 0.16 + 0.21 ___ 2.80 + 0.10 + 1.03 ___ 0.36 + 0.52 ___ 3.50 __+ 1.70 +__ 0.02 +__ 0.45 _ 0.20 +_ 0.93 ___ 0.37 + 0.02 __+ 0.55 + 0.02 _ 6.50
A. s. sexdens
Range of values found (ng)
Mean % composition by weight (+__ S.D. n = 10)
0.05.5 1.0 23.6 2.8 23.6 4.641.3 6 . 4 - 41.3 65.0- 374.0 1.6- 88.6 9.8 - 205.0 9.8 68.9 56.8- 403.0 380 -2697 65.0- 532.0 0.02.8 5.5 - 138.8 2.839.4 15.8 - 354.3 7.970.9 0.03.9 9.8 - 205.0 0.07.9 106 - 2598
0.13 + 0.06 0.09 __+ 0.05 0.10 + 0.06 0.21 + 0.11 1.80 + 0.40 0.55 _ 0.27 1.22 + 0.26 1.21 + 0.28 3.91 _ 0.70 17.7 ___ 3.10 16.0 + 2.10 0.14 + 0.17 0.61 ___ 0.26 0.97 +0.20 5.57 ___ 0.89 3.44 _ 0 . 7 1 0.01 + 0.15 2.55 + 0.43 0.01 + 0.01 43.8 + 4.70
Range of values found (rig)
1.60.8 1.41.827.63.913.8 15.8 46.1308 145 1.66.3 11.061.4 35.80.027.6 0.0485 -
15.8 11.8 8.9 25.6 142,0 73,8 83.7 94,5 216,0 984 999 22.1 63,0 63.0 425,0 271,0 21.9 193.0 11.8 2944
Evidence
GC, GC, GC, GC, GC, GC,
Br+ BrBr+, MS BrBr+ B r - , MS
GC, GC, GC, GC, GC,
Br+, MS, OZ BrBr+, OZ B r + , M S , OZ, TLC B r - , MS
GC, GC, GC, GC,
Br+ BrB R + , OZ Br-
GC, Br+ GC, Br+, MS, OZ, TLC
GC: indicates that the substance has similar retention times on both polar and non-polar GC phase to the assigned compound; B r - : component unaffected on treatment with bromine; Br +: component removed from GC profile on treatment with bromine; MS: identity confirmed from mass spectrum; OZ: the position of the double bond(s) has been determined by ozonolysis and subsequent GC analysis; TLC: conformation of the double bond has been determined by thin layer chromatography on silver nitrate-impregnated plates. These definitions also apply to Table 4.
Linked G C - M S confirmed the identities o f c o m p o n e n t s 6, 8, 11, 12 and 21. C o m p o n e n t s 6 and 12 had mass spectra identical with those o f n - h e p t a d e c a n e (M ÷ 240, C17Ha6) and n - n o n a d e c a n e (M ÷ 268, C19H4o ) respectively. C o m p o n e n t s 8, 11 and 21 had mass spectra c o r r e s p o n d i n g to those o f the m o n o unsaturated alkenes, octadecene ( M ÷ 252, ClsH3~), n o n a d e c e n e (M ÷ 266, C19H38) and tricosene (M ÷ 322, C23H46 ) respectively.
M i c r o b r o m i n a t i o n o f the glandular c o m p o n e n t s resulted in the elimination o f peaks 1,3, 5, 8, 10, 11, 14, 19 and 21 from the G C profile, showing that they are unsaturated c o m p o u n d s . The log plots and mass spectrometry had previously s h o w n that with the exception o f c o m p o n e n t s 3 and 10 all these substances c o r r e s p o n d to m o n o - u n s a t u r a t e d alkenes. C o m p o n e n t 3 did not fit the log tr series o f either nalkane or alkene h o m o l o g o u s series, its reaction with
Table 2. Compounds arising through the ozonolysis of A. sexdens Dufour gland alkenes Component number (in Table 1) 8 10 11 16 21
Ozonolysis products
Assignment
Nonanal Octanal (malondialdehyde, obscured by solvent peak) Nonanal + decanal Nonanal + dodecanal Nonanal + tetradecanal
9-Octadecene 8,11-Nonadecadiene 9-Nonadecene 9-Heneicosene 9-Tricosene
Table 3. Comparison of glandular volume (calculated from eye graticule measurements) and hydrocarbon content (determined from GC analysis)
Species A. s. sexdens A. s. rubropilosa
Glandular volume (nl)
Volume of hydrocarbon (nl)
Hydrocarbon as % of total gland volume
7.95 9.72
7.86 9.65
98.9 99.3
348
R.P. EVERSHEDANDE. D. MORGAN A. s. se xdens
A . s. rubropiloso
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Fig. 2. Comparison of the variation in hydrocarbon content in individual workers ofAtta sexdens sexdens and A. sexdens rubropilosa with live body weight. bromine confirms that it is unsaturated. It has been found to have the same values of tr on both polar and non-polar GC phases as the homofarnesene (7-ethyl3, 11-dimethyldodeca-1, 3, 6, 10-tetraene) observed in A. octospinosus (EVERSHED and MORGAN, 1980), Myrmica scabrinodis and M. sabuleti (CAMMAERTSe t al., 1980). There was too little of this substance present to obtain its complete mass spectrum. However in spite of the absence of an M* peak and several of the higher mass fragments, the remainder of the spectrum
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~8 16
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12
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Fig. 3. Gas chromatographic profiles obtained from single Dufour glands from Atta sexdens rubropilosa (A) and A. s. sexdens (B) on a 5% OV-101 column programmed from 180 to 210°Cat 2°C/min, nitrogen carrier gas flow rate 60 ml/min.
is very similar to that obtained from homofarnesene. The reaction of component 10 with bromine combined with its behaviour on non-polar and polar GC phases relative to the nonadecane and nonadecene suggested that it may be a doubly unsaturated C~9 alkene, i.e. nonadecadiene. This component was trapped together with the nonadecene and ozonised. Gas chromatography of the ozonolysis products revealed two major peaks of equivalent area, subsequently identified from their retention times as nonanal and decanal and one minor component corresponding to octanal. The two major components have undoubtedly arisen from component 11, which can be more fully assigned as 9-nonadecene. The minor product of the ozonolysis, octanal, is believed to represent two C a fragments from the symetrical doubly unsaturated hydrocarbon, which is therefore 8, 11-nonadecadiene. The C a fragment resulting from the ozonolysis of this compound would be malondialdehyde, and owing to its low molecular weight would be obscured by the solvent peak in the subsequent GC analysis. Ozonolysis of the octadecene component gave only one major product, corresponding to nonanal, indicating therefore that this alkene has the unsaturation in the 9-position. Ozonolysis of the heneicosene gave two products in equivalent proportions corresponding to nonanal and dodecanal indicating unsaturation at the 9-position. Furthermore ozonolysis of the tricosene component gave two components in equivalent amounts corresponding to nonanal and tetradecanal. This compound is therefore more completely identified as 9-tricosene. The ozonolysis results are summarised in Table 2. Thin layer chromatography of a hexane extract of five workers gasters on silver nitrate-impregnated silica plates together with (Z)- and (E)-9-nonadecene and (Z)-9-tricosene standards revealed that the only detectable alkenes in the A. sexdens Dufour gland have the (Z) configuration, no component was observed corresponding to the (E) isomer. As the most abundant alkenes in the two subspecies are 9nonadecene and 9-tricosene, these have both been assigned the (Z) configuration. The remaining alkenes are present at too low a concentration to allow a confident assignment to be made.
Dufour glands of Attine ants
349
(A)
2 13
2 9
(B)
r4
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j
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20
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Min Fig. 4. Gas chromatographic traces of single Dufour glands from workers ofA. cephalotes on a 59/00V-101 column at 165°C isothermal, nitrogen carrier gas flow rate 60 ml/min, before (A) and after (B) microbromination. The identities of the various components in the glands of the two subspecies together with the analytical evidence and percentage composition of the secretion in each subspecies are shown in Table 1. The values given in the Table were obtained from ten replicates in each subspecies. The absolute amount of each substance was calculated by comparing the area of the G C peak from the analysis of a single gland with a known amount of hydrocarbon standard. The results show clearly that although the two subspecies produce similar substances in their Dufour glands, there are significant differences in the relative amounts of the individual components. The composition of the secretion is, however, constant in workers from each subspecies, especially with respect to the major components.
In the D u f o u r gland of A. s. sexdens workers (Z)-9tricosene is the most abundant component at 43.79/0 while (Z)-9-nonadecene is the second most abundant component, constituting a mean value of 17.7% of the total. In A. s. rubropilosa the reverse is the case: (Z)-9nonadecene is the major component and (Z)-9tricosene the second most abundant component.
A. cephalotes As seen in Fig. 4, the chromatograph trace A shows a typical G C profile obtained through the analysis of a single D u f o u r gland from a worker of A. cephalotes. This volatile material is composed of straight chain alkanes and alkenes. Components 1, 2, 3, 5, 7, 9, 11 and 14 have been previously designated as n-alkanes in
Table 4. Dufour gland composition of A. cephalotes together with the analytical evidence for the assignments
Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Dodecane Tridecane Tetradecane Pentadecene Pentadecane Homofarnesene Hexadecane Heptadecene Heptadecane Octadecene Octadecane Nonadecadiene* )? (Z)-9-Nonadecene j Nonadecane
Mean ~ composition by weight (+ S.D. n = 10)
Range of values found (ng)
0.38 + 0.25 3.23 + 1.20 1.23 + 1.20 1.11 _ 0.37 4.86 _ 1.60 0.77 + 0.38 2.64 -{- 0.75 1.82 + 1.20 46.7 + 4.60 1.77 _+_ 0.61 2.61 + 0.85
0.1 1.1 0 . 8 - 6.0 0.21.0 0 . 2 - 4.6 0 . 8 - 14.2 0 . 1 - 2.2 0 . 5 - 5.4 0 . 2 - 4.0 12.1 - 104 0 . 3 - 3.6 0 . 6 - 4.2
21.3 + 7.50 11.1 + 1.30
1.8- 78.2 2 . 0 - 25.2
Evidence GC, B r GC, B r GC, B r GC, Br+ GC, B r GC, Br+ GC, B r GC, Br+ GC, B r - , MS GC, Br+ GC, B r GC, B r GC, Br+,MS, OZ, TLC GC, B r - , MS
(Z)-9-nonadecene and nonadecadiene are in a ratio of approximately 9:1, peaks are not well resolved. See subscript to Table 1 for the definitions of the abbreviations in the Evidence column. *
350
R.P. EVERSHEDAND E. D. MORGAN
the C12 to C19 chain length range (EVERSHEDand MORGAN, 1980). Microbromination of material from a single gland resulted in the elimination of peaks 4, 6, 8, 10, 12 and 13 from the GC profile (Fig. 4, B). This indicates that these substances are unsaturated hydrocarbons. Components 8 and 13 have the same retention times as heptadecene and nonadecene respectively. Plotting log t r against the number of carbon atoms in the chain suggested that components 4, 8, l0 and 13 were part of a homologous series of monounsaturated alkenes. On this basis components 4 and l0 would be pentadecene and octadecene respectively. Ozonolysis of component 13 gave equal amounts of nonanal and decanal, indicating that this substance is 9-nonadecene. Thin layer chromatography of a hexane extract from sixty A. cephalotes workers Dufour glands on silver nitrate loaded silica plates indicated that this alkene at least has the (Z) configuration. Component 6 is readily brominated and has the same retention time as component 3 in the two A. sexdens subspecies. The retention time of this substance corresponds to that of homofarnesene identified in Acromyrmex octospinosus (EVERSHEDand MORGAN, 1980) and M. sabuleti and M. scabrinodis (CAMMAERTSe t aL, 1980). Component 13 is also eliminated from the GC profile by bromination and has the same retention time as the component identified as 8, I 1-nonadecadiene in the two A. sexdens subspecies. Table 4 summarises the assignments together with the analytical evidence, and lists the relative proportions of the individual components together with standard deviations and range of amounts of each substance. All quantitative determinations were performed by comparing the areas of the GC peaks with a known amount of hydrocarbon standard. DISCUSSION Workers of A. s. rubropilosa and A. s. sexdens produce microgram amounts of saturated and unsaturated hydrocarbons in their Dufour glands. This material is composed predominantly of singly unsaturated alkenes in the C~s to C23 chain length range (Table 1). This material originates only from the Dufour gland as has been proved by analysing single dissected glands and capillary extracts (MORGAN and TYLER, 1977). From the glandular volumes and quantities calculated from GC measurements it has been shown that this volatile material almost completely fills the glands (Table 3). Furthermore the amount of volatile material in the individual glands has been found to be directly proportional to the live body weight of the insect (Fig. 2). This corresponds to the relationship found in A. cephalotes (EVERSHEDand MORGAN, 1980). The variability in hydrocarbon content at a given body weight may reflect differences in age or some other factor. This variability may explain the apparent non-linearity in the relationship between hydrocarbon content and body weight of M. scabrinodis workers (MORGANet al., 1979). The size of these Attine workers varies over a wide range, for example we examined workers of A. s. rubropilosa
having body weights between 5.5 and 51.8 rag. This wide spread reveals, for these ants, a linear relationship between body weight and hydrocarbon content, but deviation from the mean is clearly apparent. In the Myrmicine ants, this large variation in body weights is not found (values between 1.1 and 3.1 mg were recorded for seventy workers of M. scabrinodis) and the variation in hydrocarbon content is more obvious. Similar sized workers of A. s. sexdens and A. s. rubropilosa produce similar quantities of hydrocarbons in their Dufour glands. Although the two subspecies produce the same chemicals there are significant quantitative differences in the relative proportions of the individual components. The differences in composition of the Dufour gland secretions of A. s. sexdens and A. s. rubropilosa are as great as the differences between species in the Myrmica genus, so from this chemical taxonomic point of view they may be considered as separate species. Only one colony of each variety was available and so further work with colonies from other areas would be necessary to establish this possibility clearly. Since the two colonies were obtained from areas geographically far apart, it would be interesting to examine colonies from an intermediate area in Brazil. However, our work on A. cephalotes and A. octospinosus was carried out on a number of colonies of both species and at no time did we observe any differences approaching that between the two A. sexdens subspecies. Relatively little study of the Dufour gland contents of myrmicine ants has been made, other than our own work on Myrmica species (cf. CAMMAERTS et al., 1981, and earlier references therein). Other studies are confined to Aphaenogaster longiceps, Novomessor cockerelli, Pogonornyrmex rugosus, P. barbatus, Solenopsis invicta, S. richteri and S. geminata, (see Table p. 809 in BLUM and HERMANN, 1978). These species have been shown to contain straight chain alkanes or methyl-branched alkanes from dodecane to nonadecane and (in Pogonomyrmex) two dimethylalkanes. No study has been made of the quantitative composition or the functions of the hydrocarbons. We report here the identification of six further, higher molecular weight hydrocarbons in the C2o to C23 range for myrmicine Dufour glands. Tricosene, the major component of the Dufour glands of A. s. sexdens and the second most abundant component of A. s. rubropilosa has been observed previously in ants of the Formicinae (BERGSTR6M and I_6FQVIST, 1973). A much larger number of species of formicines has been examined and a wider variety of compounds found (see Table p. 812 in BLUM and HERMANN, 1978). Hydrocarbons from nonane to tricosone, methylalkanes and alkenes have been found together with alkanols, alkanones, alkyl and alkenyl acetates in the Dufour glands, but for only some have quantitative comparison been made. Generally, among formicines undecane and tridecane predominate, but the variety of other components makes it possible that each species has its characteristic blend of substances that renders it detectably distinctive. This possible species specificity has been discussed by BERGSTR~3ra and LOFQVIST (1973). Suggestions that in the formicines the hydrocarbons are alarm pheromones or act as
Dufour glands of Attine ants spreading agents for formic acid have not been rigorously tested by experiment. A t this early stage o f the study, we can only p o i n t to the discovery t h a t in Myrmica the D u f o u r gland contents a p p e a r to act as a territorial m a r k i n g p h e r o m o n e , used by pioneer workers first entering a new territory to encourage other foragers to explore that territory for prey (CAMMAERTS, et al., 1977). T h e tricosene present in the two subspecies of A. sexdens has been positively identified as (Z)-9tricosene. This c o m p o u n d also occurs as the sexual a t t r a c t a n t of the male housefly (CARLSON et al., 1971), where it is k n o w n as muscalure a n d it has also been identified in the cuticle waxes of a n u m b e r of species of Periplaneta (JACKSON, 1970) a n d in the c o m b and cuticular waxes of Apis mellifera (STREIBL et al., 1966; BLOMQUIST et al., 1980). It is interesting to note that A. cephalotes a n d A. octospinosus produce n o n e of the higher molecular weight c o m p o u n d s identified in the A. sexdens subspecies. We are u n a b l e to explain why in A. cephalotes a n d A. octospinosus the h y d r o c a r b o n s f o u n d constitutes only a very small fraction of the total gland volume, while in the workers of the two A. sexdens subspecies the D u f o u r gland is almost completely filled with volatile h y d r o c a r b o n s . The earlier suggestion that the p r o d u c t i o n o f h y d r o c a r b o n s is related purely to stinging ability seems n o t to hold true in this case.
Acknowledgements--The authors thank Dr. J. M. CHERRETT of Bangor University for supplying workers of A. s. sexdens, A. s. rubropilosa and A. cephalotes, and Dr. A. MUDD of Rothamstead Experimental Station for his assistance in obtaining the mass spectra. This work was supported by a CASE award to RPE in collaboration with Rothamstead Experimental Station whom we also thank for the provision ofA. cephalotes colonies. The work was carried out under MAFF licence No. HH12230/11 issued under the Destructive Pests and Diseases of Plants Order 1965.
REFERENCES BAKER R., BRADSHAWJ. W. S., EVANSD. A., HIGGS i . D. and WADHAMSL. J. (1976) An efficient all-glass splitter and trapping system for gas chromatography, J. Chromat. Sci., 14, 425-427. BERGSTROMG. and LOFQVlSTJ. (1973) Chemical congruence of the complex odoriferous secretions from Dufour's gland in three species of ants of the genus Formica. J. Insect Physiol. 19, 877-907.
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BEROZA M. and BIERL B. A. (1969) Ozone generator for microanalysis Mikrochem. Acta 720-3. BLOMQUIST G. J., HOWARD R. W., MCDANIEL C. A., REMALEY S., DWYER L. A. and NELSON D. R. (1980) Application of methoxymercuriation-demercuriation followed by mass spectrometry as a convenient microanalytical technique for double bond location in insect derived alkenes. J. chem. Ecol. 6, 257-269. BLUM M. S. and HERMANN H. R. (1978) Venom and the venom apparatuses of the Formicidae: Myrmecinae, Ponerinae, Dorylinae, Pseudomyrmecinae, Myrmicinae and Formicinae. In Arthropod Venoms (Ed. by BETTINIS.), Handbuch der Experimentallen Pharmacologie 48, 801-894. Springer, Berlin. CAMMAERTS M. C., EVERSHED R. P. and MORGAN E. n . (1981) Comparative study of the pheromones emitted by different species of Myrmica. Proc. Int. Symp. Biosystematics of Social Insects. Academic Press (in press). CAMMAERTSM. C,, INWOODM. R., MORGANE. D., PARRYK 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. CAMMAERTSM. C., MORGANE. D. and TYLER R. C. (1977) Territorial marking in the ant Myrmica rubra L. (Formicidae). Biol. Behav. 2, 263-272. CARLSON D. A., MAYERM. S., SILHACEKO. L., JAMESJ. D., BEROZA M. and BIERL B. A. (1971) Sex attractant pheromone of the House fly, isolation, identification and synthesis. Science 174, 76. EVERSHED R. P. and MORGANE. D. (1980) A chemical study of the Dufour glands of two Attine ants. Insect Biochem. 10, 81-86. JACKSON L. L. (1970) Hydrocarbons of the cockroaches Periplaneta australasiae, Periplaneta brumea and Periplaneta fuliginosa. Lipids 5, 38-43. MORGAN E. D., PARRY K. and TYLER R. C. (1979) The chemical composition of the Dufour gland secretion of the ant Myrmica scabrinodis. Insect Biochem. 9, 117-121. MORGAN E. D. and TYLER R. C. (1977) Microchemical method for the identification of volatile pheromones. J. Chromat. 134, (1), 174-7. MORGAN E. D. and WADHAMS L. J. (1972) Ga* chromatography of volatile compounds in small samples of biological materials. J. Chromat. Sci. 10, 528-529. REGNIER E. E., NIEM M. and H6LLDOBLER B. (1973) The volatile Dufour gland components of the harvester ants Pogonomyrmex rugosus and Pogonomyrmex barbatus. J. Insect Physiol. 19, 981-992. ROBERTSON P. L. (1968) A morphological and functional study of the venom apparatus in representatives of some major groups of Hymenoptera. Aust. J. Zool. 16, 133-166. STREIBL M., STRANSKYK. and SORM F. (1966) Uber einige neue kohlenwasserstoffe im wachs der h6nigbiene (Apis mell(/bra L.) Fette Se(fen 68, 799-805. WHEELER W. M. (1910) Ants, Their Structure, Development and Behaviour. Colombia University Press, New York.
0020-1790/81/030343-08502.00/0 © t981 Pergamon Press Ltd.
CHEMICAL INVESTIGATIONS OF THE DUFOUR GLAND CONTENTS OF ATTINE ANTS R. P. EVERSHEDand E. D. MORGAN Department of Chemistry, University of Keele, Staffordshire ST5 5BG, England (Received 30 September 1980)
Abstract--The Dufour glands of workers of the two subspecies Atta sexdens sexdens and Atta sexdens rubropilosa are completely filled with linear alkanes and alkenes in the C)s to C23 chain length range in microgram amounts. There are quantitative differencesin the hydrocarbon composition betweenworkers of the two subspecies.The major glandular component ofA. s. sexdens is (Z)-9-tricosene,in A. s. rubropilosa the most abundant component is (Z)-9-nonadecene.From a chemicaltaxonomic viewpointA. s. rubropilosa and A. s. sexdens could be regarded as separate species. The Dufour gland of Atta cephalotes is only partially filled with linear alkanes and alkenes in the Clz to C19 chain length range in nanogram amounts; n-heptadecane is the most abundant alkane and (Z)-9nonadecene is the most abundant alkene. Key Word Index: Ants, Dufour gland, Atta sexdens, Atta cephalotes, hydrocarbons
INTRODUCTION WE HAVE recently reported a chemical study of the Dufour gland of the two Attine ants, Atta cephalotes and Acromyrmex octospinosus (EVERSHED and MORGAN, 1980). The results showed that, in common with the other myrmicines that have been studied, these two tropical species also produce volatile hydrocarbons in their Dufour glands. In contrast to the temperate myrmicines (CAMMAERTSet al., 1978), the volatile material of these two Attines constituted only a very small fraction of the total gland volume. It has been proposed that the primary evolutionary function of the Dufour gland secretion is one of sting or egg lubrication (WHEELER, 1910; ROnERTSON,1968). The sting of these Attines is rudimentary, its principal function being to deposit the trail pheromone on the substratum. On this basis we suggested that the absence of lubricating chemicals in their Dufour glands is the result of there being little necessity for them. A. cephalotes workers produce mainly n-alkanes in their Dufour gland with n-heptadecane as the major constituent. The hydrocarbons produced by A. octospinosus are of a rather different type; the major component being a terpenoid hydrocarbon, homofarnesene. With the exception of Pogonomyrmex rugosus and P. barbatus, which produce a number of methyl-branched alkanes (REGNIER et al., 1973), the tendency of myrmicines to produce either predominantly terpenoid hydrocarbons or predominantly linear hydrocarbons is one which has been shown to be common to all those myrmicines examined to date (BLUM and HERMANN, 1978; MORGAN et al., 1979; CAMMAERTSet al., 1980). To act as a basis for future biosynthetic and ethological studies we have extended our investigations to include Atta sexdens sexdens and A. sexdens rubropilosa and this paper reports the identity of the volatile chemicals of their Dufour glands. Greater experience with the techniques has enabled us 343
to present the results of some further work on the Dufour gland volatiles of A. cephalotes. The Dufour glands of the two subspecies of A. sexdens are almost completely filled with hydrocarbons which are for the most part monosaturated alkenes. The compositions of the gland contents in A. s. sexdens and A. s. rubropilosa are sufficiently different to enable them to be considered separate species, from a chemical taxonomic viewpoint.
MATERIALS AND METHODS Sources of insect material Colonies of A. cephalotes from Trinidad were maintained in the laboratory at 26--28°C and at 85-95% r.h. (relative humidity) and supplied with a variety of plant materials for their fungus cultivation. Additional A. cephalotes soldiers were obtained from other colonies from various parts of South America maintained at the University College of North Wales, Bangor. Workers of A. s. sexdens and A. s. rubropilosa were also supplied from colonies maintained at Bangor originating from Guyana and Paraguay respectively. The worker ants were killed by momentary immersion in liquid nitrogen or by exposure to solid carbon dioxide. The ants wereeither used immediatelyor stored in a deep freeze at 20°C. -
Gas chromatography ( GC) Pye 104 series gas chromatographs fitted with flame ionisation detectors (FID) were used throughout these investigations. The three types of GC phase used were: A. 5~o (w/w) OV 101 on Chromosorb W, acid washed and hexamethyldisilazane treated (AW-HMDS) 100-120 mesh packed in a 1.5 m x 4 mm i.d. glass column. B. 5% (w/w) diethyleneglycol succinate (DEGS) on Supersorb (AW-HMDS) 100-120 mesh packed in a 3 m x 4 mm i.d. glass column. C. 10~/o (w/w) polyethyleneglycol (PEG 20M) on Chromosorb W (AW-H MDS) 100-120mesh packed in a 1.5 m x 4 mm i.d. glass column.
344
R.P. EVERSHEDAND E. D. MORGAN
Single dissected glands, capillary extracts (MORGAN and TYLER, 1977) and whole gasters were analysed by GC in conjunction with a solid sampling technique (MORGAN and WADHAMS, 19723. Tissue extracts were prepared by macerating whole gasters with redistilled hexane (at approx. 10 kd/mg tissue) in a small tissue grinder. The decanted extracts were concentrated to a volume of 4-5 #1 before analysis by exposure to a stream of dry nitrogen at room temperature. Gas chromatography-mass spectrometry (GC-MS) Initial GC-MS studies were perfomed on a Pye 104 series GC with FID linked to a Hitachi-Perkin Elmer RMU 6E mass spectrometer through a Watson-Bieman separator maintained at 190°C. Helium was the carrier gas at a flow rate of 15 ml/min. Further investigations were carried out on a Pye 204 series GC linked to a VG Micromass 7070F mass spectrometer through a glass jet separator. This system incorporated a VG 2000 data system. In both cases analyses were performed on tissue extracts. Identifications were made by interpretation of fragmentation patterns and by comparison with reference spectra. Reference compounds The GC retention times and mass spectra of the glandular components were compared with those of commercially available n-alkanes. Alkenes were synthesised via a Wittig route; the appropriate phosphonium salt was prepared by refluxing the alkyl bromide with triphenylphosphine for 5 hr in dry dimethylformamide (DMF) (dried by refluxing for 5 hr over P2Os, followed by distillation from P2Os). The Wittig reaction was carried out in dry tetrahydrofuran (THF) (distilled from sodium) under a dry nitrogen atmosphere. The ylid was formed by adding an equimolar solution of n-butyl lithium in hexane to the salt solution at 0°C. This was followed by dropwise addition of the appropriate aldehyde to give the alkene. The reaction was over in a few seconds at 0°C. The mixture of E and Z isomers produced was separated by medium pressure column chromatography (MPCC) on 20% (w/w) AgNO 3 on Kielselgel (EVERSHED, MORGAN and THOMPSON, in preparation). GC analysis showed the relative proportion of Z to E isomers to be 4:1 under the experimental conditions described. The purity of all the reference compounds was checked by GC; and by infrared spectrometry (IR) in the case of the alkenes. Bromination Micro-scale bromination of the glandular components was performed by adding 0.5 #l of a 1:1 solution of bromine in carbon disulphide to a single Dufour gland in a solid sample glass capillary tube (MORGAN and WAOHAMS, 1972). The sealed capillary was then placed in the solid sampler with the injection port heater set at 200°C. Five minutes were allowed to elapse before crushing to ensure complete bromination of any unsaturated components. Analysis after bromination was performed using column A, in the cases of the two A. sexdens subspecies, a nitrogen carrier gas flow rate of 60 ml/min was employed and the GC oven temperature programmed from 180-210°C at 2°C/min. In the investigation of A. cephalotes the GC oven temperature was held isothermally at 165°C. Ozonolysis The positions of the double bonds in the alkenes were determined by ozonolysis of fractions trapped from the GC using a micropreparative apparatus (BAKERet al., 1976). The trapping was performed on extracts obtained through the maceration of twenty-five gasters with 200 #1 of redistilled hexane. This volume was reduced to 2 #1 under a stream of nitrogen at room temperature before fractionation by GC. A 1:100 (FID:trap) split ratio was employed to avoid unnecessary loss of sample to the detector. The samples were
trapped in 20 cm ~U' tubes of 0.5 mm i.d. cooled in liquid nitrogen. The trapped material was washed from the 'U' tube with 100 ,ul of dichloromethane. Ozonolysis was then performed by exposing the alkene solution, cooled in an ice bath, to a slow stream of ozone from a micro-ozone generator (BEROZA and BIERL, 1969) for a few minutes. The ozonides were reduced by addition of a small crystal of triphenylphosphine in order to yield the carbonyl compounds. The micropreparative work was carried out on column A. Analysis of the ozonolysis products was performed on column B. Thin layer chromatography Thin layer chromatography was employed to determine the configurations of the double bonds in the unsaturated glandular components. Silica gel plates (0.3 mm thickness) impregnated with 10% (w/w) AgNO 3 were used in conjunction with an eluent of 1~,~, diethyl ether in light petroleum (b.p. 40-60°C). In the analysis of the alkenes in the two subspecies of A. sexdens a tissue extract of poison gland complexes from five workers was chromatographed together with synthetic samples of (E) and (Z)-9-nonadecene and (Z)9-tricosene. An extract from sixty workers gasters of A. cephalotes was also analysed under similar conditions. The plates were developed by spraying with 20~o (w/w) concentrated sulphuric acid followed by heating in an oven at 120°C for 15 min. Determination of glandular volume The dimensions of the glands were determined using an eye graticule. The glands appear as gently tapering sacs, being hemispherical at their posterior end (Fig. 1). On this basis the glandular volume (V) was calculated from the following formula where r is the radius at greatest cross section and h is the length of the gland: V= z3ztr2h+ ~3Tcr3 RESULTS A. s. sexdens and A. s. rubropilosa The D u f o u r glands of A. s. rubropilosa a n d A. s. sexdens are almost completely filled with mixtures of the same alkanes a n d alkenes (Tables 1 and 3). In b o t h subspecies the q u a n t i t y of h y d r o c a r b o n in the gland is directly p r o p o r t i o n a l to the body weight of the ant (Fig. 2). The relative p r o p o r t i o n s of the individual c o m p o n e n t s differ significantly in the two subspecies (Table 1, Fig. 3). In A. s. rubropilosa the most a b u n d a n t c o m p o n e n t is (Z)-9-nonadecene, while in A. s. sexdens, (Z)-9-tricosene predominates. G a s c h r o m a t o g r a p h y of a single gland from each subspecies revealed twenty-one volatile c o m p o n e n t s (Fig. 3). C o m p o n e n t s 2, 4, 6, 9, 12 a n d 15 were found to have retention times corresponding to n-alkanes in the C l s to C2o chain length range, on b o t h polar a n d n o n - p o l a r G C phases (columns A and C, see Materials a n d Methods). Plotting the logarithm o f retention time (log tr) against the n u m b e r of c a r b o n a t o m s in the h y d r o c a r b o n chain suggested t h a t c o m p o n e n t 17 is p r o b a b l y n-heneicosane. C o m p o n e n t s 5 a n d 11 h a d retention times o n columns A a n d C c o r r e s p o n d i n g to heptadecene and nonadecene. A further plot of log t r against the n u m b e r of c a r b o n atoms in the chain indicated that c o m p o n e n t s 1, 8, 14, 16, 19 a n d ' 2 1 were p r o b a b l y m o n o - u n s a t u r a t e d alkenes, namely pentadecene, octadecene, eiconsene, heneicosene, docosene a n d tricosene respectively.
345
Fig. 1. Sketches of the Dufour gland, poison gland complexes o f Atta cephalotes and of A. sexdens rubropilosa, showing D F = Dufour gland, PG = poison glands, PV = poison vesicle and S = sting lance.
Dufour glands of Attine ants
347
Table 1. Dufour gland compositions of A. s. rubropilosa and A. s. sexdens together with the analytical evidence for the assignments A. s. rubropilosa
Mean % composition by weight (_+ S.D. n = 10)
Compound
1 Pentadecene 2 Pentadecane 3 Homofarnesene 4 Hexadecane 5 Heptadecene 6 Heptadecane 7 8 9-Octadecene 90ctadecane 10 8,11-Nonadecadiene I1 (Z)-9-Nonadecene 12 Nonadecane 13 14 Eicosene 15 Eicosane 16 9-Heneicosene 17 Heneicosane 18 19 Docosene 20 21 (Z)-9-Tricosene
0.06 0.32 0.32 0.39 0.88 8.35 0.24 3.27 1.15 5.81 38.9 7.17 0.01 0.84 0.38 4.03 0.92 0.01 1.72 0.01 25.24
+ 0.04 + 0.16 + 0.08 + 0.16 + 0.21 ___ 2.80 + 0.10 + 1.03 ___ 0.36 + 0.52 ___ 3.50 __+ 1.70 +__ 0.02 +__ 0.45 _ 0.20 +_ 0.93 ___ 0.37 + 0.02 __+ 0.55 + 0.02 _ 6.50
A. s. sexdens
Range of values found (ng)
Mean % composition by weight (+__ S.D. n = 10)
0.05.5 1.0 23.6 2.8 23.6 4.641.3 6 . 4 - 41.3 65.0- 374.0 1.6- 88.6 9.8 - 205.0 9.8 68.9 56.8- 403.0 380 -2697 65.0- 532.0 0.02.8 5.5 - 138.8 2.839.4 15.8 - 354.3 7.970.9 0.03.9 9.8 - 205.0 0.07.9 106 - 2598
0.13 + 0.06 0.09 __+ 0.05 0.10 + 0.06 0.21 + 0.11 1.80 + 0.40 0.55 _ 0.27 1.22 + 0.26 1.21 + 0.28 3.91 _ 0.70 17.7 ___ 3.10 16.0 + 2.10 0.14 + 0.17 0.61 ___ 0.26 0.97 +0.20 5.57 ___ 0.89 3.44 _ 0 . 7 1 0.01 + 0.15 2.55 + 0.43 0.01 + 0.01 43.8 + 4.70
Range of values found (rig)
1.60.8 1.41.827.63.913.8 15.8 46.1308 145 1.66.3 11.061.4 35.80.027.6 0.0485 -
15.8 11.8 8.9 25.6 142,0 73,8 83.7 94,5 216,0 984 999 22.1 63,0 63.0 425,0 271,0 21.9 193.0 11.8 2944
Evidence
GC, GC, GC, GC, GC, GC,
Br+ BrBr+, MS BrBr+ B r - , MS
GC, GC, GC, GC, GC,
Br+, MS, OZ BrBr+, OZ B r + , M S , OZ, TLC B r - , MS
GC, GC, GC, GC,
Br+ BrB R + , OZ Br-
GC, Br+ GC, Br+, MS, OZ, TLC
GC: indicates that the substance has similar retention times on both polar and non-polar GC phase to the assigned compound; B r - : component unaffected on treatment with bromine; Br +: component removed from GC profile on treatment with bromine; MS: identity confirmed from mass spectrum; OZ: the position of the double bond(s) has been determined by ozonolysis and subsequent GC analysis; TLC: conformation of the double bond has been determined by thin layer chromatography on silver nitrate-impregnated plates. These definitions also apply to Table 4.
Linked G C - M S confirmed the identities o f c o m p o n e n t s 6, 8, 11, 12 and 21. C o m p o n e n t s 6 and 12 had mass spectra identical with those o f n - h e p t a d e c a n e (M ÷ 240, C17Ha6) and n - n o n a d e c a n e (M ÷ 268, C19H4o ) respectively. C o m p o n e n t s 8, 11 and 21 had mass spectra c o r r e s p o n d i n g to those o f the m o n o unsaturated alkenes, octadecene ( M ÷ 252, ClsH3~), n o n a d e c e n e (M ÷ 266, C19H38) and tricosene (M ÷ 322, C23H46 ) respectively.
M i c r o b r o m i n a t i o n o f the glandular c o m p o n e n t s resulted in the elimination o f peaks 1,3, 5, 8, 10, 11, 14, 19 and 21 from the G C profile, showing that they are unsaturated c o m p o u n d s . The log plots and mass spectrometry had previously s h o w n that with the exception o f c o m p o n e n t s 3 and 10 all these substances c o r r e s p o n d to m o n o - u n s a t u r a t e d alkenes. C o m p o n e n t 3 did not fit the log tr series o f either nalkane or alkene h o m o l o g o u s series, its reaction with
Table 2. Compounds arising through the ozonolysis of A. sexdens Dufour gland alkenes Component number (in Table 1) 8 10 11 16 21
Ozonolysis products
Assignment
Nonanal Octanal (malondialdehyde, obscured by solvent peak) Nonanal + decanal Nonanal + dodecanal Nonanal + tetradecanal
9-Octadecene 8,11-Nonadecadiene 9-Nonadecene 9-Heneicosene 9-Tricosene
Table 3. Comparison of glandular volume (calculated from eye graticule measurements) and hydrocarbon content (determined from GC analysis)
Species A. s. sexdens A. s. rubropilosa
Glandular volume (nl)
Volume of hydrocarbon (nl)
Hydrocarbon as % of total gland volume
7.95 9.72
7.86 9.65
98.9 99.3
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R.P. EVERSHEDANDE. D. MORGAN A. s. se xdens
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Fig. 2. Comparison of the variation in hydrocarbon content in individual workers ofAtta sexdens sexdens and A. sexdens rubropilosa with live body weight. bromine confirms that it is unsaturated. It has been found to have the same values of tr on both polar and non-polar GC phases as the homofarnesene (7-ethyl3, 11-dimethyldodeca-1, 3, 6, 10-tetraene) observed in A. octospinosus (EVERSHED and MORGAN, 1980), Myrmica scabrinodis and M. sabuleti (CAMMAERTSe t al., 1980). There was too little of this substance present to obtain its complete mass spectrum. However in spite of the absence of an M* peak and several of the higher mass fragments, the remainder of the spectrum
I
!5
(A) 21
6
~8 16
_J
k,8 All ,4 -.jv~
q(B)
12
21
0
_/ I 20
I
Io
I
o
Min
Fig. 3. Gas chromatographic profiles obtained from single Dufour glands from Atta sexdens rubropilosa (A) and A. s. sexdens (B) on a 5% OV-101 column programmed from 180 to 210°Cat 2°C/min, nitrogen carrier gas flow rate 60 ml/min.
is very similar to that obtained from homofarnesene. The reaction of component 10 with bromine combined with its behaviour on non-polar and polar GC phases relative to the nonadecane and nonadecene suggested that it may be a doubly unsaturated C~9 alkene, i.e. nonadecadiene. This component was trapped together with the nonadecene and ozonised. Gas chromatography of the ozonolysis products revealed two major peaks of equivalent area, subsequently identified from their retention times as nonanal and decanal and one minor component corresponding to octanal. The two major components have undoubtedly arisen from component 11, which can be more fully assigned as 9-nonadecene. The minor product of the ozonolysis, octanal, is believed to represent two C a fragments from the symetrical doubly unsaturated hydrocarbon, which is therefore 8, 11-nonadecadiene. The C a fragment resulting from the ozonolysis of this compound would be malondialdehyde, and owing to its low molecular weight would be obscured by the solvent peak in the subsequent GC analysis. Ozonolysis of the octadecene component gave only one major product, corresponding to nonanal, indicating therefore that this alkene has the unsaturation in the 9-position. Ozonolysis of the heneicosene gave two products in equivalent proportions corresponding to nonanal and dodecanal indicating unsaturation at the 9-position. Furthermore ozonolysis of the tricosene component gave two components in equivalent amounts corresponding to nonanal and tetradecanal. This compound is therefore more completely identified as 9-tricosene. The ozonolysis results are summarised in Table 2. Thin layer chromatography of a hexane extract of five workers gasters on silver nitrate-impregnated silica plates together with (Z)- and (E)-9-nonadecene and (Z)-9-tricosene standards revealed that the only detectable alkenes in the A. sexdens Dufour gland have the (Z) configuration, no component was observed corresponding to the (E) isomer. As the most abundant alkenes in the two subspecies are 9nonadecene and 9-tricosene, these have both been assigned the (Z) configuration. The remaining alkenes are present at too low a concentration to allow a confident assignment to be made.
Dufour glands of Attine ants
349
(A)
2 13
2 9
(B)
r4
,M
j
I
I
20
I
I0
Min Fig. 4. Gas chromatographic traces of single Dufour glands from workers ofA. cephalotes on a 59/00V-101 column at 165°C isothermal, nitrogen carrier gas flow rate 60 ml/min, before (A) and after (B) microbromination. The identities of the various components in the glands of the two subspecies together with the analytical evidence and percentage composition of the secretion in each subspecies are shown in Table 1. The values given in the Table were obtained from ten replicates in each subspecies. The absolute amount of each substance was calculated by comparing the area of the G C peak from the analysis of a single gland with a known amount of hydrocarbon standard. The results show clearly that although the two subspecies produce similar substances in their Dufour glands, there are significant differences in the relative amounts of the individual components. The composition of the secretion is, however, constant in workers from each subspecies, especially with respect to the major components.
In the D u f o u r gland of A. s. sexdens workers (Z)-9tricosene is the most abundant component at 43.79/0 while (Z)-9-nonadecene is the second most abundant component, constituting a mean value of 17.7% of the total. In A. s. rubropilosa the reverse is the case: (Z)-9nonadecene is the major component and (Z)-9tricosene the second most abundant component.
A. cephalotes As seen in Fig. 4, the chromatograph trace A shows a typical G C profile obtained through the analysis of a single D u f o u r gland from a worker of A. cephalotes. This volatile material is composed of straight chain alkanes and alkenes. Components 1, 2, 3, 5, 7, 9, 11 and 14 have been previously designated as n-alkanes in
Table 4. Dufour gland composition of A. cephalotes together with the analytical evidence for the assignments
Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Dodecane Tridecane Tetradecane Pentadecene Pentadecane Homofarnesene Hexadecane Heptadecene Heptadecane Octadecene Octadecane Nonadecadiene* )? (Z)-9-Nonadecene j Nonadecane
Mean ~ composition by weight (+ S.D. n = 10)
Range of values found (ng)
0.38 + 0.25 3.23 + 1.20 1.23 + 1.20 1.11 _ 0.37 4.86 _ 1.60 0.77 + 0.38 2.64 -{- 0.75 1.82 + 1.20 46.7 + 4.60 1.77 _+_ 0.61 2.61 + 0.85
0.1 1.1 0 . 8 - 6.0 0.21.0 0 . 2 - 4.6 0 . 8 - 14.2 0 . 1 - 2.2 0 . 5 - 5.4 0 . 2 - 4.0 12.1 - 104 0 . 3 - 3.6 0 . 6 - 4.2
21.3 + 7.50 11.1 + 1.30
1.8- 78.2 2 . 0 - 25.2
Evidence GC, B r GC, B r GC, B r GC, Br+ GC, B r GC, Br+ GC, B r GC, Br+ GC, B r - , MS GC, Br+ GC, B r GC, B r GC, Br+,MS, OZ, TLC GC, B r - , MS
(Z)-9-nonadecene and nonadecadiene are in a ratio of approximately 9:1, peaks are not well resolved. See subscript to Table 1 for the definitions of the abbreviations in the Evidence column. *
350
R.P. EVERSHEDAND E. D. MORGAN
the C12 to C19 chain length range (EVERSHEDand MORGAN, 1980). Microbromination of material from a single gland resulted in the elimination of peaks 4, 6, 8, 10, 12 and 13 from the GC profile (Fig. 4, B). This indicates that these substances are unsaturated hydrocarbons. Components 8 and 13 have the same retention times as heptadecene and nonadecene respectively. Plotting log t r against the number of carbon atoms in the chain suggested that components 4, 8, l0 and 13 were part of a homologous series of monounsaturated alkenes. On this basis components 4 and l0 would be pentadecene and octadecene respectively. Ozonolysis of component 13 gave equal amounts of nonanal and decanal, indicating that this substance is 9-nonadecene. Thin layer chromatography of a hexane extract from sixty A. cephalotes workers Dufour glands on silver nitrate loaded silica plates indicated that this alkene at least has the (Z) configuration. Component 6 is readily brominated and has the same retention time as component 3 in the two A. sexdens subspecies. The retention time of this substance corresponds to that of homofarnesene identified in Acromyrmex octospinosus (EVERSHEDand MORGAN, 1980) and M. sabuleti and M. scabrinodis (CAMMAERTSe t aL, 1980). Component 13 is also eliminated from the GC profile by bromination and has the same retention time as the component identified as 8, I 1-nonadecadiene in the two A. sexdens subspecies. Table 4 summarises the assignments together with the analytical evidence, and lists the relative proportions of the individual components together with standard deviations and range of amounts of each substance. All quantitative determinations were performed by comparing the areas of the GC peaks with a known amount of hydrocarbon standard. DISCUSSION Workers of A. s. rubropilosa and A. s. sexdens produce microgram amounts of saturated and unsaturated hydrocarbons in their Dufour glands. This material is composed predominantly of singly unsaturated alkenes in the C~s to C23 chain length range (Table 1). This material originates only from the Dufour gland as has been proved by analysing single dissected glands and capillary extracts (MORGAN and TYLER, 1977). From the glandular volumes and quantities calculated from GC measurements it has been shown that this volatile material almost completely fills the glands (Table 3). Furthermore the amount of volatile material in the individual glands has been found to be directly proportional to the live body weight of the insect (Fig. 2). This corresponds to the relationship found in A. cephalotes (EVERSHEDand MORGAN, 1980). The variability in hydrocarbon content at a given body weight may reflect differences in age or some other factor. This variability may explain the apparent non-linearity in the relationship between hydrocarbon content and body weight of M. scabrinodis workers (MORGANet al., 1979). The size of these Attine workers varies over a wide range, for example we examined workers of A. s. rubropilosa
having body weights between 5.5 and 51.8 rag. This wide spread reveals, for these ants, a linear relationship between body weight and hydrocarbon content, but deviation from the mean is clearly apparent. In the Myrmicine ants, this large variation in body weights is not found (values between 1.1 and 3.1 mg were recorded for seventy workers of M. scabrinodis) and the variation in hydrocarbon content is more obvious. Similar sized workers of A. s. sexdens and A. s. rubropilosa produce similar quantities of hydrocarbons in their Dufour glands. Although the two subspecies produce the same chemicals there are significant quantitative differences in the relative proportions of the individual components. The differences in composition of the Dufour gland secretions of A. s. sexdens and A. s. rubropilosa are as great as the differences between species in the Myrmica genus, so from this chemical taxonomic point of view they may be considered as separate species. Only one colony of each variety was available and so further work with colonies from other areas would be necessary to establish this possibility clearly. Since the two colonies were obtained from areas geographically far apart, it would be interesting to examine colonies from an intermediate area in Brazil. However, our work on A. cephalotes and A. octospinosus was carried out on a number of colonies of both species and at no time did we observe any differences approaching that between the two A. sexdens subspecies. Relatively little study of the Dufour gland contents of myrmicine ants has been made, other than our own work on Myrmica species (cf. CAMMAERTS et al., 1981, and earlier references therein). Other studies are confined to Aphaenogaster longiceps, Novomessor cockerelli, Pogonornyrmex rugosus, P. barbatus, Solenopsis invicta, S. richteri and S. geminata, (see Table p. 809 in BLUM and HERMANN, 1978). These species have been shown to contain straight chain alkanes or methyl-branched alkanes from dodecane to nonadecane and (in Pogonomyrmex) two dimethylalkanes. No study has been made of the quantitative composition or the functions of the hydrocarbons. We report here the identification of six further, higher molecular weight hydrocarbons in the C2o to C23 range for myrmicine Dufour glands. Tricosene, the major component of the Dufour glands of A. s. sexdens and the second most abundant component of A. s. rubropilosa has been observed previously in ants of the Formicinae (BERGSTR6M and I_6FQVIST, 1973). A much larger number of species of formicines has been examined and a wider variety of compounds found (see Table p. 812 in BLUM and HERMANN, 1978). Hydrocarbons from nonane to tricosone, methylalkanes and alkenes have been found together with alkanols, alkanones, alkyl and alkenyl acetates in the Dufour glands, but for only some have quantitative comparison been made. Generally, among formicines undecane and tridecane predominate, but the variety of other components makes it possible that each species has its characteristic blend of substances that renders it detectably distinctive. This possible species specificity has been discussed by BERGSTR~3ra and LOFQVIST (1973). Suggestions that in the formicines the hydrocarbons are alarm pheromones or act as
Dufour glands of Attine ants spreading agents for formic acid have not been rigorously tested by experiment. A t this early stage o f the study, we can only p o i n t to the discovery t h a t in Myrmica the D u f o u r gland contents a p p e a r to act as a territorial m a r k i n g p h e r o m o n e , used by pioneer workers first entering a new territory to encourage other foragers to explore that territory for prey (CAMMAERTS, et al., 1977). T h e tricosene present in the two subspecies of A. sexdens has been positively identified as (Z)-9tricosene. This c o m p o u n d also occurs as the sexual a t t r a c t a n t of the male housefly (CARLSON et al., 1971), where it is k n o w n as muscalure a n d it has also been identified in the cuticle waxes of a n u m b e r of species of Periplaneta (JACKSON, 1970) a n d in the c o m b and cuticular waxes of Apis mellifera (STREIBL et al., 1966; BLOMQUIST et al., 1980). It is interesting to note that A. cephalotes a n d A. octospinosus produce n o n e of the higher molecular weight c o m p o u n d s identified in the A. sexdens subspecies. We are u n a b l e to explain why in A. cephalotes a n d A. octospinosus the h y d r o c a r b o n s f o u n d constitutes only a very small fraction of the total gland volume, while in the workers of the two A. sexdens subspecies the D u f o u r gland is almost completely filled with volatile h y d r o c a r b o n s . The earlier suggestion that the p r o d u c t i o n o f h y d r o c a r b o n s is related purely to stinging ability seems n o t to hold true in this case.
Acknowledgements--The authors thank Dr. J. M. CHERRETT of Bangor University for supplying workers of A. s. sexdens, A. s. rubropilosa and A. cephalotes, and Dr. A. MUDD of Rothamstead Experimental Station for his assistance in obtaining the mass spectra. This work was supported by a CASE award to RPE in collaboration with Rothamstead Experimental Station whom we also thank for the provision ofA. cephalotes colonies. The work was carried out under MAFF licence No. HH12230/11 issued under the Destructive Pests and Diseases of Plants Order 1965.
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