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Identification of the components of Dufour gland secretion of the ant Myrmica rubra and responses to them
d. Insect Physiol.. 1977. Vol. 23. pp. 511 to 515. Per,qamon Pre.ss. Printed in Great Britain.
I D E N T I F I C A T I O N OF THE C O M P O N E N T S OF D U F O U R G L A N D SECRETION OF THE ANT M Y R M I C A R U B R A AND RESPONSES TO THEM E. D. MORGAN1. R. C. TYLER1. and M. C. CAMMAERTS2 1Department of Chemistry, Keele University, Keele, Staffs. ST5 5BG, England. -'Laboratoire de Biologie Animale et Cellulaire, Universite fibre de Bruxelles, 50 av. F. D. Roosevelt. 1050 Brussels, Belgium (Received 3 November 1976)
Akstraet--The Dufour gland secretion consists of a solution of volatile oxygenated compounds in a mixture of higher hydrocarbons and sesquiterpenoid alkenes. Methanol, butenone, 2-methylpropanaL and 1-butanol have now been identified as the remaining major volatile components. These substances have no strong behavioural effect upon ants. nor do they induce trail following behaviour. The principal activit3 of the Dufour gland can be traced to the three more abundant volatile components, ethanal. acetone, and butanone, together with the minor component ethanol.
INTRODUCTION
techniques especially to handle these volatile compounds and determine their chemical structure (MoRGAN and WADnA~tS. 1972b; MORGAN and TYLER. 1977). Some of these volatile components have been identified and their ethological effects reported recently (CA'~taERTS-TRlcOr et al., 1976). We now report the identification of the remainder of the volatile components with their ethological tests and draw some conclusions upon the total function of the Dufour gland secretion.
TIlE DUFOUR gland and poison gland of ants of the genus M y r m i c a are exocrine secretory organs having a common duct to the lancet of the sting. The poison gland has a muscular sphincter, but the Dufour gland apparently has not, and its contents can presumably be discharged alone or with the poison. In an attempt to understand the behavioural r61e of these secretions and in particular to locate and identify the trail pheromone of M . rubra we have carried out a systematic examination of the volatile contents of these glands. The major components of the Dufour gland were MATERIALS AND METHODS found to be straight chain hydrocarbons in the range C33 to C19. with or without double bonds, and three The colonies of M . rubra were maintained in the sesquiterpenoid compounds, farnesene, homofarnelaboratory as described in earlier papers (TRICOT et sene, and bishomofarnesene (MORGAN and WADnAMS, al., 1972: CAMMAERTS-TPdcOTet at., t976). 1972a). In ethological tests of the kind described by Material from the gland for gas chromatography CAMMAERTs-TPacox (1973). none of these compounds, was collected by first plunging the ant into liquid however, produced any recognizable behavioural renitrogen for one second which caused the lancet of sponse (unpublished results from our laboratoriesl. the sting to be everted. This was grasped with forceps The contents of the poison gland are quite different, and the whole sting and Dufour gland were removed being essentially an aqueous solution of a non-volatile intact from the gaster. The gland apparatus was then proteinaceous venom (J~'YTscn. 1969). Nevertheless, placed on a microscope stage and the Dufour gland both these secretions induce specific behavioural re- pierced with a hard glass capillary of 50 to 70/an sponses. The poison gland is not attractive to workers diameter, which drew up the liquid. The progress of at a distance (CAMMAERTS-TRICOT,1973), but it is the removal of the liquid could easily be seen through source of the trail pheromone (CAMMaERTS-TRIcoT, the microscope. The capillary containing the liquid 1974: CAMMAERTS-TRlcOTet al., 1977). The Dufour was sealed in an ampoule and heated to 220°C in gland is attractive and induces a rapid, wandering, the injection heater of the gas chromatograph and sinuous, or exploratory movement in recruited then crushed into the carrier gas stream. Chromatography was performed on a Pye Series 104 instruworkers (CAM~aAERTS-TmcoT. 1973). ment with flame ionization or thermal conductivity We had recognized in our earlier work on the detectors, using 5 ft by ¼ o.d. glass columns filled with Dufour gland (MORGAN and WADHAMS, 1972a) that there was a very volatile component to this secretion. Porapak Q, 120 to 150 mesh (Waters Associates) with either nitrogen or helium as carrier gasl The oven Lack of pheromone activity in the hydrocarbons made it necessary for us to develop microchemical temperature was in most cases at 167°C isothermal. 511
512
E . D . MORGAN, R. C. TYLER AND M. C. CAMMAERTS
h
la e
k
,
,
,
,
,
,
rain
Fig. 1. Gas chromatograph trace of contents of two Dufour glands from workers of M. rubra on Porapak Q at 187°C with N 2 carrier gas at 50ml rain -1. Peaks are: a, base line disturbance due to pressure change on crushing sample; b, water peak; c, methanol; d. ethanal: e. ethanol; f, propanal; g, acetone; h, 2-methylpropanal: i, butenone; j, butanone; k. l-butanol. The ethanal and acetone peaks have been attenuated to bring them on scale. Relative peak areas were obtained by photocopying the chromatogram, cutting peaks from the photocopy and weighing them. Solutions of pure compounds in water were prepared and quantities comparable to those found in the gland were chromatographed in order to relate peak area (detector response) to weight of compound chromatographed. To study the action of the substances identified in the Dufour gland secretion on the movement of ants, the pure substances were diluted 10'* or 106 times with pure liquid paraffin. Small portions t0.01 cm 3) of these solutions were deposited on pieces of filter paper iWhatman No. 1, l cm21, and the paper placed on the foraging area of the colony. The orientation of workers towards the object, their linear speed, and the sinuosity of their movement were quantified in the manner described previously (CAMMAERTS-TRICOT et al.. 1976). In order to reveal any incidental trail pheromone activity among the substances found in the Dufour gland, solutions of the pure substances (0.1 cm 3) in a mixture of hexane and acetone (5cm s, 85:15 V/V) were prepared, and 25 #l of the solution i.e. 0.5 ~tl of the substance placed in a normograph pen and used to trace a circle (radius 5 cmt on millimetre graph paper. The circumference of the circle was divided into arcs of 10 degrees. The paper was placed in the foraging area of the colony and for 30 min any ants crossing the circle were observed. The number of arcs of 10: which each ant walked on the circumference
without deviating from it were counted. In this way the distribution of these distances travelled on the circle were collected for each substance. This procedure was developed by PAS'rEELSand VEmiAEGaE (1974) and used in an earlier paper (CAMMAERTS'-TRICOT and VERHAEGHE. 1974). RESLrLTS By removing the Dufour gland contents with a fine capillary and chromatographing the liquid on Porapak Q, 9 major components were eventually recognized in the volatile faction (i.e. C1 to C5). Because compounds elute from Porapak in order of molecular size, some guess could be made about the nature of the compound. This was checked by comparing retention times with those of pure standards until a compound was found that coincided in retention. To avoid accidental coincidence, retentions were compared also on columns of Chromosorb 102 and the functional group was checked by reaction gas chromatography (e.g. reduction of aldehydes and ketones to alcohols, or removal of alcohols with boric acid) by methods described by MORGAN and TYLER (1977). The compounds are listed in Table 1. together with their relative peak areas on the chromatogram. Because with low mol. wt compounds the flame ionization detector response is dependant upon a number of factors, the absolute amount of material represented by each peak was calculated from known
Dufour gland secretion of M. ruhra
513
Table 1. Composition of the volatile part of the secretion of the Dufour gland of M. rubra
Compound methanol ethanal ethanol propanal acetone 2-methylpropanal butenone butanone 1-butanol
Relative peak area ('~o)
Average quantity per gland (ng)
1.3 15.3 5.1 1.6 30.6
2.8 48.3 4.3 1.3 27.4
11.2
11.8
15.7 13.7 5.5 100.0
amounts of pure compound. The absolute quantity and wt ~o of total volatiles in the gland are also given in Table 1. with the range found for a number of nests from different areas and workers of differing size and age. The presence of water in the gland was sought using a thermal conductivity detector with the gas chromatograph. The water peak obtained was no bigger than that found when an empty capillary was sealed and submitted to the same chromatographic conditions. The water content must therefore be less than 1 ng per gland. The total volume of liquid removable from a single gland was found to be 3.0hi to 16nl (average 7.5 nlt by measuring the volume of liquid drawn into a capillary, or an average of 5.8 #g (assuming an average density for C16 hydrocarbons of 0.77g ml-1). The volatile portion therefore represents 2°~ by weight of the total, and a concentration of 16 mg m l - 1 or 0.4 M l -~ for ethanal, the most abundant volatile compound.
Ethological activity of newly identified substances The activity of ethanal (acetaldehyde), ethanol, acetone, and butanone (methyl ethyl ketone) have been described earlier (CAMMAERTS-TRICOTet al., 1976). The results of the ethological test on the remaining substances are summarized in Table 2. None of the substances tested attracted the workers. The movement of the ants was hardly affected by butenone (methyl vinyl ketone). Butanol and 2-methylpropanal (isobutyraldehyde) only affected their movement when presented in rather high concentration. This effect disappeared when the substance was diluted 106 times. The responses to the Dufour gland cannot be explained then by the presence of methanol, butenone, 2-methylpropanal and butanol in the secretion. The effect recognized earlier as due to the Dufour gland secretion can be attributed to four of the nine substances identified. These are ethanal (acet~.l'
23 4
I
',o of total
Range ot values rain '~o max '~o
13.3 11.5 5.5
2.2 38.3 3.4 1.0 21.7 9.4 10.5 9.1 4.4
1.7-3.2 30-49 2.9~,. 1 0.4-2.2 20-24 8.1-11 9.3-12 7.7-11 3.8-5.6
126.2
100.0
aldehyde) which, synergized by ethanol, produces an attractive effect on foragers; acetone, which increases their linear speed: and ethanol, synergized by butanone, which causes a more sinuous exploratory motion. The poison gland and Dufour gland possess a common excretory duct, and the possibility always exists that a little of the duct is excised with the gland and that cross-contamination of the contents occurs. This is easily recognized in gas chromatography and occurs from time to time. The profile of the gas chromatograms of the votatiles from the two glands are very different (MORGAN and TYLER, 1977). But in order to check whether the Dufour gland produced any trail-following effect in workers, the less volatile compounds identified here have been tested on a circular trail. The compounds tested, together with the results, are given in Table. 3. Butenone (methyl vinyl ketone) potymerizes readily on keeping, and the polymer so formed was also tested. The results show that the substances were highly significantly different from the poison gland contents, and contamination with these substances could not account for the activity shown by poison gland secretion. The workers approaching the circle impregnated with the chemicals tested here moved slowly, touching the paper with their antennae, sometimes stopping or walking along the circumference for a few cm. They never ran towards the circle or along its circumference for long distances as they usually did when it had been impregnated with poison gland extract. A chemical analysis of the volatile fraction of the poison gland contents has already been described (CAMMAERTS-TRICOTet al., 1977). Methanol, ethanal, ethanol, acetone, and butanone are present in both secretions but in different proportions, and the total concentration is about 5 times higher in the Dufour gland. This study confirms that the secretion considered as emitted by the Dufour gland is effectively produced by that gland.
E. D. MORGAN, R. C. TYLER ANt) M. C. CAMMAERT$
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Dufour gland secretion of M. rubra CONCLUSIONS AND DISCUSSION The Dufour gland of M. rubra workers emits a mixture of straight chain hydrocarbons (C13 to C~91 and sesquiterpenoid alkenes (C15 to C~ 7i in which volatile oxygenated compounds are dissolved, and there is essentially no water present, into which the volatile compounds might partition preferentially. The volume of the gland found, by chemical methods, to be an average value of 7.5 nl, confirms accurately that found from observation under the microscope of the volume of older workers" Dufour glands (CAMMAERTSTRICOT and VERHAEGHE. 1974). A freshly removed Dufour gland is known to ato'act the workers and induces in them a rapid and rather sinuous activity (CAMMAERTS-TRICOT, 1973). These ethological activities can be attributed to the volatile substances emitted by the gland. Ethanal, the major volatile compound, is a true attractant for worker ants. Acetone, the second major volatile compound, incites the ants to move quickly. Ethanol, together with butanone, induces a side-to-side, wandering or sinuous movement. Methanol and butenone hardly affect the ants" movement: 2-methylpropanal and 1-butanol increase the wandering or sinuous motion when presented in relatively large quantities. but since many unfamiliar chemicals, if "presented in sufficient concentration, will also alarm the ants and induce them to walk sinuously this is not considered particularly significant. The ethologically active volatile compounds have been shown to act when presented in the very low concentrations that would be expected to be emitted by the ants (CAMMAERTSTRICOT et al.. 1976). The non-volatile hydrocarbons m the Dulour gland apparently have no ethological effect directly, and in the absence of more direct information are presumed to act as a non-volatile solvent for the active volatile portion, and would have the effect of lowering their vapour pressures and causing them to be released more slowly into the atmosphere. It therefore is entirely appropriate that there should be no water present as well. otherwise the small molecules such as ethanal and ethanol would partition themselves into the aqueous (more volatile solvent) portion to a large extent and the 'keeping' property of the hydrocarbons would be lost. The components of the Dufour gland secretion have therefore been analysed in a very careful man-
515
ner. and the ethological effect of a freshly removed gland explained. Whether there is a less volatile substance, still active after the very volatile materials have evaporated is still under investigation. Glands removed from the ants for a time lose some of their activity, but retain an effect. We suspect that the less volatile portion still contains an active compound or compounds. Acknowledgement--E D. M. wishes to thank the Science Research Council for their support of this work through the purchase of equipment.
REFERENCES CAMMAERTS-TRICOTM. C. (1973) Pheromones agregeant les ouvrieres de Myrmica rubra. J. Insect Physiol. 19, 1299-1315. CAMMAERTSoTRICOT M. C. (1974) Recrutement d'Ouvrieres chez Myrmica ruhra par les pheromones de l'appareil /t venin. Behaviour 50, 111-122. CAMMAERTS-TRICOT M. C.. MORGAN E. D.. TYLER R. C..
and BRAEKMANJ.-C. (1976) Dufour's gland secretion of Myrmica rubra: chemical, electrophysiological and ethological studies. J. Insect Physiol. 22, 927-932. CAMMAERTS-TRICOTM. C.. MORGANE. D., and TYLER R. C. (]977) Isolation of the Trail pheromone of M. rubra. d. Insect Physiol. 23, In press. CAMMAER'FS-TmcoTM. C. and VERHAEGHE J. C. (19741 Ontogenesis of trail pheromone production and trail following behaviour in the workers of Myrmica ruhra L. (Formicidae). Insectes Soc. 21, 275-282. JENTSCHJ. (19691 A procedure for purification of Myrmica venom: the isolation of the convulsive components, l'Ith Congr. int. Union Study Soc. Insects 6, 69-75. MORGAN E. D. and TYLER R. C. (1977) Microchemical methods for the identification of volatile pheromones. d. Chromalog. 134, 174-177. MORGANE. D. and WADHAMSL. J. (1972a) Chemical constituents of Dufours gland in the ant M)~'mica rubra. J. Insect. Physiol. 18. 1125-1135. MORGAN E. D. and WADHAMSL. J. (1972b1 Gas chromatography of volatile compounds in small samples of biological materials. J. Chromato#. Sci. 10, 528-529. PASTEELSJ-M. and VERHAEGHEJ-C. (1974) Dosage biologique de la pheromone de piste chez les fourrageuses et les reines de Myrmica rubra. Insectes Soc. 21, 167-180. TRICOT M. C.. PASTEELS J. M., and TURSCH B. 11972) Pheromones stimulant et inhibant l'argressivite chez Myrmica rubra, d. Insect Physiol. 18, 499-509.
I D E N T I F I C A T I O N OF THE C O M P O N E N T S OF D U F O U R G L A N D SECRETION OF THE ANT M Y R M I C A R U B R A AND RESPONSES TO THEM E. D. MORGAN1. R. C. TYLER1. and M. C. CAMMAERTS2 1Department of Chemistry, Keele University, Keele, Staffs. ST5 5BG, England. -'Laboratoire de Biologie Animale et Cellulaire, Universite fibre de Bruxelles, 50 av. F. D. Roosevelt. 1050 Brussels, Belgium (Received 3 November 1976)
Akstraet--The Dufour gland secretion consists of a solution of volatile oxygenated compounds in a mixture of higher hydrocarbons and sesquiterpenoid alkenes. Methanol, butenone, 2-methylpropanaL and 1-butanol have now been identified as the remaining major volatile components. These substances have no strong behavioural effect upon ants. nor do they induce trail following behaviour. The principal activit3 of the Dufour gland can be traced to the three more abundant volatile components, ethanal. acetone, and butanone, together with the minor component ethanol.
INTRODUCTION
techniques especially to handle these volatile compounds and determine their chemical structure (MoRGAN and WADnA~tS. 1972b; MORGAN and TYLER. 1977). Some of these volatile components have been identified and their ethological effects reported recently (CA'~taERTS-TRlcOr et al., 1976). We now report the identification of the remainder of the volatile components with their ethological tests and draw some conclusions upon the total function of the Dufour gland secretion.
TIlE DUFOUR gland and poison gland of ants of the genus M y r m i c a are exocrine secretory organs having a common duct to the lancet of the sting. The poison gland has a muscular sphincter, but the Dufour gland apparently has not, and its contents can presumably be discharged alone or with the poison. In an attempt to understand the behavioural r61e of these secretions and in particular to locate and identify the trail pheromone of M . rubra we have carried out a systematic examination of the volatile contents of these glands. The major components of the Dufour gland were MATERIALS AND METHODS found to be straight chain hydrocarbons in the range C33 to C19. with or without double bonds, and three The colonies of M . rubra were maintained in the sesquiterpenoid compounds, farnesene, homofarnelaboratory as described in earlier papers (TRICOT et sene, and bishomofarnesene (MORGAN and WADnAMS, al., 1972: CAMMAERTS-TPdcOTet at., t976). 1972a). In ethological tests of the kind described by Material from the gland for gas chromatography CAMMAERTs-TPacox (1973). none of these compounds, was collected by first plunging the ant into liquid however, produced any recognizable behavioural renitrogen for one second which caused the lancet of sponse (unpublished results from our laboratoriesl. the sting to be everted. This was grasped with forceps The contents of the poison gland are quite different, and the whole sting and Dufour gland were removed being essentially an aqueous solution of a non-volatile intact from the gaster. The gland apparatus was then proteinaceous venom (J~'YTscn. 1969). Nevertheless, placed on a microscope stage and the Dufour gland both these secretions induce specific behavioural re- pierced with a hard glass capillary of 50 to 70/an sponses. The poison gland is not attractive to workers diameter, which drew up the liquid. The progress of at a distance (CAMMAERTS-TRICOT,1973), but it is the removal of the liquid could easily be seen through source of the trail pheromone (CAMMaERTS-TRIcoT, the microscope. The capillary containing the liquid 1974: CAMMAERTS-TRlcOTet al., 1977). The Dufour was sealed in an ampoule and heated to 220°C in gland is attractive and induces a rapid, wandering, the injection heater of the gas chromatograph and sinuous, or exploratory movement in recruited then crushed into the carrier gas stream. Chromatography was performed on a Pye Series 104 instruworkers (CAM~aAERTS-TmcoT. 1973). ment with flame ionization or thermal conductivity We had recognized in our earlier work on the detectors, using 5 ft by ¼ o.d. glass columns filled with Dufour gland (MORGAN and WADHAMS, 1972a) that there was a very volatile component to this secretion. Porapak Q, 120 to 150 mesh (Waters Associates) with either nitrogen or helium as carrier gasl The oven Lack of pheromone activity in the hydrocarbons made it necessary for us to develop microchemical temperature was in most cases at 167°C isothermal. 511
512
E . D . MORGAN, R. C. TYLER AND M. C. CAMMAERTS
h
la e
k
,
,
,
,
,
,
rain
Fig. 1. Gas chromatograph trace of contents of two Dufour glands from workers of M. rubra on Porapak Q at 187°C with N 2 carrier gas at 50ml rain -1. Peaks are: a, base line disturbance due to pressure change on crushing sample; b, water peak; c, methanol; d. ethanal: e. ethanol; f, propanal; g, acetone; h, 2-methylpropanal: i, butenone; j, butanone; k. l-butanol. The ethanal and acetone peaks have been attenuated to bring them on scale. Relative peak areas were obtained by photocopying the chromatogram, cutting peaks from the photocopy and weighing them. Solutions of pure compounds in water were prepared and quantities comparable to those found in the gland were chromatographed in order to relate peak area (detector response) to weight of compound chromatographed. To study the action of the substances identified in the Dufour gland secretion on the movement of ants, the pure substances were diluted 10'* or 106 times with pure liquid paraffin. Small portions t0.01 cm 3) of these solutions were deposited on pieces of filter paper iWhatman No. 1, l cm21, and the paper placed on the foraging area of the colony. The orientation of workers towards the object, their linear speed, and the sinuosity of their movement were quantified in the manner described previously (CAMMAERTS-TRICOT et al.. 1976). In order to reveal any incidental trail pheromone activity among the substances found in the Dufour gland, solutions of the pure substances (0.1 cm 3) in a mixture of hexane and acetone (5cm s, 85:15 V/V) were prepared, and 25 #l of the solution i.e. 0.5 ~tl of the substance placed in a normograph pen and used to trace a circle (radius 5 cmt on millimetre graph paper. The circumference of the circle was divided into arcs of 10 degrees. The paper was placed in the foraging area of the colony and for 30 min any ants crossing the circle were observed. The number of arcs of 10: which each ant walked on the circumference
without deviating from it were counted. In this way the distribution of these distances travelled on the circle were collected for each substance. This procedure was developed by PAS'rEELSand VEmiAEGaE (1974) and used in an earlier paper (CAMMAERTS'-TRICOT and VERHAEGHE. 1974). RESLrLTS By removing the Dufour gland contents with a fine capillary and chromatographing the liquid on Porapak Q, 9 major components were eventually recognized in the volatile faction (i.e. C1 to C5). Because compounds elute from Porapak in order of molecular size, some guess could be made about the nature of the compound. This was checked by comparing retention times with those of pure standards until a compound was found that coincided in retention. To avoid accidental coincidence, retentions were compared also on columns of Chromosorb 102 and the functional group was checked by reaction gas chromatography (e.g. reduction of aldehydes and ketones to alcohols, or removal of alcohols with boric acid) by methods described by MORGAN and TYLER (1977). The compounds are listed in Table 1. together with their relative peak areas on the chromatogram. Because with low mol. wt compounds the flame ionization detector response is dependant upon a number of factors, the absolute amount of material represented by each peak was calculated from known
Dufour gland secretion of M. ruhra
513
Table 1. Composition of the volatile part of the secretion of the Dufour gland of M. rubra
Compound methanol ethanal ethanol propanal acetone 2-methylpropanal butenone butanone 1-butanol
Relative peak area ('~o)
Average quantity per gland (ng)
1.3 15.3 5.1 1.6 30.6
2.8 48.3 4.3 1.3 27.4
11.2
11.8
15.7 13.7 5.5 100.0
amounts of pure compound. The absolute quantity and wt ~o of total volatiles in the gland are also given in Table 1. with the range found for a number of nests from different areas and workers of differing size and age. The presence of water in the gland was sought using a thermal conductivity detector with the gas chromatograph. The water peak obtained was no bigger than that found when an empty capillary was sealed and submitted to the same chromatographic conditions. The water content must therefore be less than 1 ng per gland. The total volume of liquid removable from a single gland was found to be 3.0hi to 16nl (average 7.5 nlt by measuring the volume of liquid drawn into a capillary, or an average of 5.8 #g (assuming an average density for C16 hydrocarbons of 0.77g ml-1). The volatile portion therefore represents 2°~ by weight of the total, and a concentration of 16 mg m l - 1 or 0.4 M l -~ for ethanal, the most abundant volatile compound.
Ethological activity of newly identified substances The activity of ethanal (acetaldehyde), ethanol, acetone, and butanone (methyl ethyl ketone) have been described earlier (CAMMAERTS-TRICOTet al., 1976). The results of the ethological test on the remaining substances are summarized in Table 2. None of the substances tested attracted the workers. The movement of the ants was hardly affected by butenone (methyl vinyl ketone). Butanol and 2-methylpropanal (isobutyraldehyde) only affected their movement when presented in rather high concentration. This effect disappeared when the substance was diluted 106 times. The responses to the Dufour gland cannot be explained then by the presence of methanol, butenone, 2-methylpropanal and butanol in the secretion. The effect recognized earlier as due to the Dufour gland secretion can be attributed to four of the nine substances identified. These are ethanal (acet~.l'
23 4
I
',o of total
Range ot values rain '~o max '~o
13.3 11.5 5.5
2.2 38.3 3.4 1.0 21.7 9.4 10.5 9.1 4.4
1.7-3.2 30-49 2.9~,. 1 0.4-2.2 20-24 8.1-11 9.3-12 7.7-11 3.8-5.6
126.2
100.0
aldehyde) which, synergized by ethanol, produces an attractive effect on foragers; acetone, which increases their linear speed: and ethanol, synergized by butanone, which causes a more sinuous exploratory motion. The poison gland and Dufour gland possess a common excretory duct, and the possibility always exists that a little of the duct is excised with the gland and that cross-contamination of the contents occurs. This is easily recognized in gas chromatography and occurs from time to time. The profile of the gas chromatograms of the votatiles from the two glands are very different (MORGAN and TYLER, 1977). But in order to check whether the Dufour gland produced any trail-following effect in workers, the less volatile compounds identified here have been tested on a circular trail. The compounds tested, together with the results, are given in Table. 3. Butenone (methyl vinyl ketone) potymerizes readily on keeping, and the polymer so formed was also tested. The results show that the substances were highly significantly different from the poison gland contents, and contamination with these substances could not account for the activity shown by poison gland secretion. The workers approaching the circle impregnated with the chemicals tested here moved slowly, touching the paper with their antennae, sometimes stopping or walking along the circumference for a few cm. They never ran towards the circle or along its circumference for long distances as they usually did when it had been impregnated with poison gland extract. A chemical analysis of the volatile fraction of the poison gland contents has already been described (CAMMAERTS-TRICOTet al., 1977). Methanol, ethanal, ethanol, acetone, and butanone are present in both secretions but in different proportions, and the total concentration is about 5 times higher in the Dufour gland. This study confirms that the secretion considered as emitted by the Dufour gland is effectively produced by that gland.
E. D. MORGAN, R. C. TYLER ANt) M. C. CAMMAERT$
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Dufour gland secretion of M. rubra CONCLUSIONS AND DISCUSSION The Dufour gland of M. rubra workers emits a mixture of straight chain hydrocarbons (C13 to C~91 and sesquiterpenoid alkenes (C15 to C~ 7i in which volatile oxygenated compounds are dissolved, and there is essentially no water present, into which the volatile compounds might partition preferentially. The volume of the gland found, by chemical methods, to be an average value of 7.5 nl, confirms accurately that found from observation under the microscope of the volume of older workers" Dufour glands (CAMMAERTSTRICOT and VERHAEGHE. 1974). A freshly removed Dufour gland is known to ato'act the workers and induces in them a rapid and rather sinuous activity (CAMMAERTS-TRICOT, 1973). These ethological activities can be attributed to the volatile substances emitted by the gland. Ethanal, the major volatile compound, is a true attractant for worker ants. Acetone, the second major volatile compound, incites the ants to move quickly. Ethanol, together with butanone, induces a side-to-side, wandering or sinuous movement. Methanol and butenone hardly affect the ants" movement: 2-methylpropanal and 1-butanol increase the wandering or sinuous motion when presented in relatively large quantities. but since many unfamiliar chemicals, if "presented in sufficient concentration, will also alarm the ants and induce them to walk sinuously this is not considered particularly significant. The ethologically active volatile compounds have been shown to act when presented in the very low concentrations that would be expected to be emitted by the ants (CAMMAERTSTRICOT et al.. 1976). The non-volatile hydrocarbons m the Dulour gland apparently have no ethological effect directly, and in the absence of more direct information are presumed to act as a non-volatile solvent for the active volatile portion, and would have the effect of lowering their vapour pressures and causing them to be released more slowly into the atmosphere. It therefore is entirely appropriate that there should be no water present as well. otherwise the small molecules such as ethanal and ethanol would partition themselves into the aqueous (more volatile solvent) portion to a large extent and the 'keeping' property of the hydrocarbons would be lost. The components of the Dufour gland secretion have therefore been analysed in a very careful man-
515
ner. and the ethological effect of a freshly removed gland explained. Whether there is a less volatile substance, still active after the very volatile materials have evaporated is still under investigation. Glands removed from the ants for a time lose some of their activity, but retain an effect. We suspect that the less volatile portion still contains an active compound or compounds. Acknowledgement--E D. M. wishes to thank the Science Research Council for their support of this work through the purchase of equipment.
REFERENCES CAMMAERTS-TRICOTM. C. (1973) Pheromones agregeant les ouvrieres de Myrmica rubra. J. Insect Physiol. 19, 1299-1315. CAMMAERTSoTRICOT M. C. (1974) Recrutement d'Ouvrieres chez Myrmica ruhra par les pheromones de l'appareil /t venin. Behaviour 50, 111-122. CAMMAERTS-TRICOT M. C.. MORGAN E. D.. TYLER R. C..
and BRAEKMANJ.-C. (1976) Dufour's gland secretion of Myrmica rubra: chemical, electrophysiological and ethological studies. J. Insect Physiol. 22, 927-932. CAMMAERTS-TRICOTM. C.. MORGANE. D., and TYLER R. C. (]977) Isolation of the Trail pheromone of M. rubra. d. Insect Physiol. 23, In press. CAMMAER'FS-TmcoTM. C. and VERHAEGHE J. C. (19741 Ontogenesis of trail pheromone production and trail following behaviour in the workers of Myrmica ruhra L. (Formicidae). Insectes Soc. 21, 275-282. JENTSCHJ. (19691 A procedure for purification of Myrmica venom: the isolation of the convulsive components, l'Ith Congr. int. Union Study Soc. Insects 6, 69-75. MORGAN E. D. and TYLER R. C. (1977) Microchemical methods for the identification of volatile pheromones. d. Chromalog. 134, 174-177. MORGANE. D. and WADHAMSL. J. (1972a) Chemical constituents of Dufours gland in the ant M)~'mica rubra. J. Insect. Physiol. 18. 1125-1135. MORGAN E. D. and WADHAMSL. J. (1972b1 Gas chromatography of volatile compounds in small samples of biological materials. J. Chromato#. Sci. 10, 528-529. PASTEELSJ-M. and VERHAEGHEJ-C. (1974) Dosage biologique de la pheromone de piste chez les fourrageuses et les reines de Myrmica rubra. Insectes Soc. 21, 167-180. TRICOT M. C.. PASTEELS J. M., and TURSCH B. 11972) Pheromones stimulant et inhibant l'argressivite chez Myrmica rubra, d. Insect Physiol. 18, 499-509.