Naturwissenschaften 85, 275–277 (1998) Springer-Verlag 1998 were excised and extracted with hex- ane. These extracts (25 ml, 0.5 gland equivalents) were applied to artificial Trail Pheromone from the Pavan Gland of the trails and tested against a control (hexane) trail. Two 15-cm intercross- Ant Dolichoderus thoracicus (Smith) ing curves that originated from a Pheromones, 108 [1] common point were employed for this dual-choice bioassay. One arm of A.B. Attygalle the artificial trail was streaked with a Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, test substance and the other with hex- NY 14853, USA ane. Of the 200 test ants that were ob- served, 144 individuals followed the A. Mutti, W. Rohe, U. Maschwitz artificial trails treated with Pavan Zoologisches Institut, UniversitÄt Frankfurt, Siesmayerstrasse 70, gland extracts while the other test D-60054 Frankfurt, Germany substances were virtually ineffective (Fig. 1). In a similar experiment we W. Garbe, H.J. Bestmann observed that the activity of a mixture Institut für Organische Chemie, Universität Erlangen-Nürnberg, of Pavan and poison gland extracts is Henkestrasse 42, D-91054 Erlangen, Germany more or less similar to that of Pavan gland alone. Gas chromatography–mass spectros- Received: 4 December 1997 / Accepted in revised form: 8 April 1998 copy (GC/MS) analysis of Pavan gland extracts showed the presence of trace quantities of many volatile con- Efficient communication mechanisms Gombak research station of Univer- stituents. The mass spectra corre- are vital for the social behavior of sity of Malaya. These ants are arbor- sponding to most of these consti- ants. Complex interactions that take eal and make nests usually among tuents appeared to be congruent with place among nestmates in an ant colo- leaves of plants. In the laboratory the those of unsaturated aldehydes or al- ny are mediated essentially by semio- colonies were sheltered on Ficus ben- cohols. A subsequent derivatization chemicals. Of the variety of chemical jamini plants. In order to determine of a glandular extract with 1,1-dime- signals employed, trail pheromones the glandular origin of the trail phe- thylhydrazine (DMH) followed by play an eminent role in coordinating romone, the pygidial, Dufour, Pavan mass spectrometric analysis of the food-retrieval and colony-emigration and poison glands, the rectal bladder, reaction mixture confirmed that most behavior of ants. The chemistry and and glandular epithelia associated constituents are indeed aldehydes biology of a number of trail phero- with abdominal sternites V and VI (Fig. 2). The mass spectra of 1,1-di- mones of ants have been investigated [1–4]. However, the chemistry of trail pheromones in the subfamily Doli- choderinae is one of the poorly inves- tigated areas. The only substance that is known to be effective in evoking trail following in a dolichoderine spe- cies is (Z)-9-hexadecenal [5, 6]. Al- though this compound elicits trail-fol- lowing behavior in Iridomyrmex hu- milis, it is reported that the activity is inferior to that evoked by glandular extracts [7, 8]. Although an earlier re- port indicated that dolichoderine trail pheromones are highly species specif- ic [9], our recent investigations show that a reasonable degree of cross-ac- tivity exists even among species of Dolichoderinae. In this communica- tion we report the first chemical and behavioral characterization of a mul- ticomponent trail pheromone from the subfamily Dolichoderinae. Several colonies of Dolichoderus tho- Fig. 1. Trail-following activity released by tissue extracts of Dolichoderus thoracicus and hex- racicus were collected near the Ulu ane. The responses of 200 ants to artificial trails streaked with each extract were recorded Naturwissenschaften 85 (1998) Q Springer-Verlag 1998 275 methylhydrazones of aldehydes are particularly useful since they show not only significant molecular ions but also a very prominent and diag- nostic McLafferty rearrangement peak at m/z 86 [10–12]. Therefore the m/z 86 signal was used to locate 1,1- dimethylhydrazone peaks by the se- lected ion retrieval procedure (Fig. 2). In this way the major volatile component of the Pavan gland was identified as an octadecenal. The double-bond position of the ma- jor monounsaturated aldehydes was determined by the dimethyl disulfide (DMDS) derivatization procedure [13]. After the derivatization, for ex- ample, the major product that was obtained yielded a mass spectrum with a molecular ion at m/z 360 (17%) and two fragment ions at m/z 173 (100%) and 187 (85%). This re- sult indicated the presence of a dime- Fig. 2. A selected-ion retrieval chromatogram for m/z 86 obtained by GC/MS analysis of the thyl disulfide derivative of 8- and/or volatiles extracted from five Pavan glands after derivatization with 1,1-dimethylhydrazine 9-octadecenal in the reaction mixture. (30 m!0.22 mm SE-30 coated capillary column; 607 C for 4 min, 107 C/min to 2607 C, and Although low-resolution mass spectra 7 15 min at 260 C; spectra were acquired at a rate of approximately 1 s/scan). Peak numbers refer of these two DMDS derivatives do to those given in Table 1 not differentiate between the two iso- mers, a comparison of gas chromato- graphic retention times obtained on a Table 1. Aldehydes detected in the Pavan gland of Dolichoderus thoracicus Noa Compound Relative 70-eV electron-ionization mass spectrum of amountd DMHf derivative [m/z(%)] DMDSg derivative [m/z(%)] 1(Z)-9-hexadecenal 1–9 280(Mc, 100), 86(87) 322(Mc, 20), 145(100), 187(95) 2 hexadecenal 3–5 282(Mc, 48), 238(5), 86(100) 3 heptadecenalb ~0.5 294(Mc, 75), 86(100) 4 heptadecanal ~0.5 296(Mc, 70), 86(100) 5 linoleyl aldehydee 6 306(Mc, 100), 262(8), 86(100) 6(Z)-9-octadecenal 100 308(Mc, 98), 264(10), 86(100) 360(Mc, 17), 313(4), 187(85), 173(100) 7 octadecanal ~0.5 310(Mc, 100), 266(12), 86(72) 8 eicosadienalb ~0.5 334(Mc, 68), 290(5), 86(100) 9(Z)-11-eicosenal 8–11 336(Mc, 100), 292(5), 86(95) 388(Mc, 10), 341(17), 215(100), 173(72) 10 eicosenalb, c ~0.5 11 eicosanal ~0.5 338(Mc, 100), 294(5), 86(55) 12 docosadienalb ~0.5 362(Mc, 100), 86(92) 13 (Z)-13-docosenal 14–25 364(Mc, 100), 320(7), 86(72) 416(Mc, 10), 243(100), 173(60) 14 docosenalb ~0.5 364(Mc, 100), 320(7), 86(43) 15 docosanal ~0.5 366(Mc, 82), 322(8), 86(100) 16 tetracosadienalb ~0.5 390(Mc, 100), 86(58) 17 tetracosenalb ~0.5 392(Mc, 100), 348(6), 86(42) 18 tetracosanal ~0.5 394(Mc, 100), 86(70) a Numbers refer to Gas chromatographic peaks in Figure 2. b The from DMH derivatives indicated the presence of minute traces of a double bond positions of trace constituents were not determined. octadecadetrienal which eluted immediately before the GC peak of c Presumably, this trace constituent is (Z)-13-eicosenal. d Amounts the linoleyl aldehyde derivative. f DMH, 1,1-dimethylhydrazine. are reported relative to that of (Z)-9-octadecenal, the most abundant g DMDS, dimethyl disulfide. component. e A mass chromatographic search in the data obtained 276 Naturwissenschaften 85 (1998) Q Springer-Verlag 1998 Table 2. Trail-following activity of Dolichoderus thoracicus workers evoked by synthetic sub- synthetic, material is comparable with stance that of the natural trails. Although there was no opportunity to Test material Percentage trail- test cross-activities of Dolichoderus following activitya thoracicus and Iridomymex humilis, it (Z)-7-tetradecenal 0 would not be surprising if these two (Z)-9-hexadecanal 55 species respond to trails of each other (Z)-11-hexadecenal 0 since (Z)-9-hexadecenal is a common hexadecenal 0 component in the trail pheromone of (Z)-9-octadecenal 75 the two species. Furthermore, our (E)-9-octadecenal 0 preliminary behavioral bioassays us- (Z)-11-eicosenal 25 ing several species of Dolichoderus (Z)-13-docosenal 20 suggest that dolichoderine trails are (Z)-15-tetracosenal 0 linolenyl aldehyde 0 not highly species specific, as envis- aged by the initial reports [9]. In fact, (Z)-9-hexadecenalc(Z)-9-octadecenal 90 a considerable degree of trail follow- c (Z)-9-octadecenal (Z)-11-eicosenal 80 ing was observed when workers of (Z)-9-octadecenalc(Z)-13-docosenal 80 several species of Dolichoderus were (Z)-9-hexadecenalc(Z)-9-octadecenal 95 presented with artificial trails made of c(Z)-11-eicosenalc(Z)-13-docosenal (Z)-9-octadecenal and Pavan gland extract (1 gland equivalent) 100 extracts which were not conspecific. hexane 0 We thank the Alexander von Hum- a Responses of 20 ants to artificial trails (20 cm) streaked with each test substance (1 ng/cm) or boldt Stiftung and Deutsche For- mixture (1 ng/cm; equal proportions of each component) were recorded. schungsgemeinschaft for fellowships. polar stationary phase (SP-2340) with (Z)-9-hexadecenal plus (Z)-9-octade- 1. Bestmann HJ, Übler E, Hölldobler B those from authentic standards con- cenal, (Z)-9-octadecenal plus (Z)-11- (1997) Pheromones, 107. Angew Chem firmed not only the position but also eicosenal, and (Z)-9-octadecenal plus Int Ed Engl 36:395 Z 2. Hölldobler B, Wilson EO (1990) The the configuration of the double bond. ( )-13-docosenal showed much high- Ants. Belknap, Cambridge In this way the major component was er activities (Table 2). Evidently ants 3. Attygalle AB, Morgan ED (1985) Adv characterized as (Z)-9-octadecenal. are able to distinguish the position Insect Physiol 18:1 The other aldehydes were identified and configuration of the double 4. Janssen E, Übler E, Bauriegel F, Kern F, in a similar manner (Table 1).