Journal of Chemical Ecology, Vol. 17, No. 12, 1991
CHEMISTRY OF VENOM ALKALOIDS IN THE ANT Megalomyrmex foreli (MYRMICINAE) FROM COSTA RICA
T . H . J O N E S , I'* P.J. D E V R I E S , z and P. E S C O U B A S 3
ILaboratory of Biophysical Chemistry National Heart, Lung, and Blood Institute Bethesda, Maryland 20892 2Department of Zoology University of Texas Austin, Texas 78712 3Plant Ecochemicals Project Research Development Corporation of Japan Megumino Kita 3-1-1 Eniwa-shi 061-13, Japan (Received June 26, 1991; accepted August 19, 1991) Abstract--Chemical analysis of the venom of the myrmicine ant Megalomyrmexforeli from Costa Rica revealed the presence of four major alkaloidal components. Two of these, 2-butyl-5-(E, 1-heptenyl)-5-pyrroline (3) and 2-butyl-5-(E, E, 1,3-heptadienyl)-5-pyrroline (4), constitute a new functional class of ant venom alkaloids, whose structures were assigned from their spectral and chemical behavior and unambiguous syntheses. The function of these compounds is suggested by field observations of the behavior of M. foreli, its sting morphology, and the relative toxicity of 3 and 4 against termite workers. Key Words--Megalomyrmex, Hymenoptera, Myrmicinae, venom, alkaloids, ants, pyrrolines, alarm behavior, stridulation, toxicity.
INTRODUCTION T h e v e n o m s o f m a n y species o f the t w o related m y r m i c i n e genera Solenopsis and Monomorium h a v e b e e n s h o w n to contain 2,5-dialkylpyrrolidines. T h e s e c o m p o u n d s h a v e b e e n f o u n d in s o m e Solenopsis (Diplorhoptrum) species and in nearly e v e r y species o f Monomorium w h e r e alkaloids h a v e b e e n detected * To whom correspondence should be addressed. 2507 0098q)331/91/1200-2507506.50/0 9 1991 Plenum Publishing Corporation
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(Jones et al., 1982). Recently, we have also found 2,5-dialkylpyrrolidines in two Megalomyrmex species (Jones et al., 1991). In a number of cases, smaller amounts of 1-pyrrolines may also be also present (Fales et al., 1980; Pedder et al., 1976; Jones et al., 1982, 1988a,b) although they may be instrumental artifacts. The only other unsaturation detected in the naturally occurring 2,5dialkylpyrrolidines has been the presence of one or more terminal double bonds on the alkyl groups (Jones et al., 1982). Other species in the genus Megalomyrmex have been found to produce saturated 2,5-dialkylpyrrolidines, and in one case, a 3,5-dialkylpyrrolizidine (Jones et al., 1991). In the present paper, we report the elucidation of two novel 1-pyrrolines with conjugated unsaturation, produced by workers of M. foreli. Some insight into the chemical ecology of this species has been gained from field observations and consideration of its sting morphology and the topical toxicities of its venom alkaloids.
METHODS AND MATERIALS
Chemical Analyses. Gas chromatographic analyses were carried out using a Shimadzu GC-9A equipped with a 30 m • 0.5 mm i.d. open DB-17 column (1 /~m film thickness). The temperature for the analyses was programed from 60-215~ at 10~ and the carrier gas flow rate was 15 mL/min. On a given day, retention temperatures were reproducable to I~ Preparative gas chromatography was conducted with a Varian model 1400 gas chromatograph equipped with a 2 m x 5 mm i.d. column packed with 10% OV-17 on 100120 mesh Supelcoport. IR spectra were obtained using a Hewlett-Packard model 5965A FTIR. IH and 13C NMR spectra were obtained from CDC13 solutions using a Varian XL-200 spectrometer. 13C assignments were made on the basis of standard 2D COSY and HETCOR experiments. Electron impact mass spectra were obtained using a LKB-9000 GC/MS equipped with a 30 m x 0.52 mm i.d. open DB-17 column (1 /zm film thickness), a LKB-2091 equipped with a 25 m x 0.31 mm i.d. HP-5 column, or a Finnigan ion trap model 800 equipped with a 25 m x 0.31 mm i.d. HP-5 column. High resolution mass spectra were obtained using a VG 7070F instrument in the E1 mode at an ionizing voltage of 70 eV. Ants. Collections and observations on Megalomyrmexforeli were made at Jardin Botanico Wilson, San Vito de Java, Puntarenas Province, 1100 m in southwestern Costa Rica. Intermittent observations on M. foreli behavior were made from February 1990 through March 1991 on nine separate colonies. These were: five colonies with ca. 100 individuals/nest found in the sheathing stems of Calathea spp. (Marantaceae) from 0.5-1 m above the ground in secondary forest, and four subterranean nests in open second growth: three at the base of
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shrubs and one in a rock wall. None of the subterranean nests had any tumulus at the entrance, and overall colony sizes were estimated to range from 100-300 individuals. Ants were immediately placed in glass vials containing 1-2 ml of CH2C12. Specimens of M. foreli were deposited in the collection of the Los Angeles County Museum of Natural History, Los Angeles, California, and have been accessed by Roy R. Snelling. For analysis, these mixtures were reduced to ca. 0.2 ml with a slow stream of nitrogen. GC/MS and GC/FTIR analyses revealed the presence of four (1-4) nitrogen containing components in a 2 : 1 : 7 :5 ratio with the following spectral data: 1: IR 1644 cm-J; MS m/z (relative intensity) 223 (M § 2), 222 (2), 208 (2), 194 (4), 180 (7), 166 (14), 152 (23), 139 (44), 124 (4), 110 (7), 96 (14), 83 (13), 82 (100), 69 (6), 68 (6), 67 (8), 55 (23), 41 (34). 2: MS m/z (relative intensity) 225 (M +, 2), 169 (3), 168 (65), 127 (10), 126 (100), 109 (10), 95 (10), 82 (15), 67 (30), 55 (40). 3: IR 1648, 1594, 1459, 1331, and 973 cm -~; MS m/z (relative intensity) 221 (25, M+), 179 (24), 178 (72), 166 (14), 165 (87), 164 (100), 150 (29), 122 (37), 109 (15), 108 (77), 106 (18), 94 (58), 80 (33), 79 (21), 67 (32), 55 (52), 54 (34), 53 (23), 41 (97). 4: IR 1622, 1578, 1459, 1330, and 987 cm -~; MS m/z (relative intensity) 219 (25, M§ 190 (29), 177 (26), 176 (99), 163 (45), 162 (45), 148 (22), 134 (44), 132 (18), 121 (22), 120 (100), 119 (29), 118 (29), 107 (18), 106 (27), 105 (12), 94 (13), 93 (18), 91 (26), 80 (24), 79 (31), 77 (25), 67 (19), 65 (18), 55 (25), 53 (17), 41 (60). In addition, a small quantity ( < 5 % of 4) of an earlier eluting isomer of 4 was also detected, with a mass spectrum identical to that of 4, and the following infrared spectrum: IR 1642, 1458, 1320, 989, and 887 cm -l Hydrogenation. A slow stream of hydrogen was passed through a small portion of the extract containing ca. 5 mg of PtO2 for 5 min. Reexamination by GC/MS revealed only two major nitrogen containing components whose gas chromatographic retention times, and infrared and mass spectra were identical to those of a sample of synthetic cis/trans 2. 2-Butyl-5-methyl-5-pyrroline (5). A solution containing 5.26 g (40 retool) of 1-nitropentane and 3.7 ml of methyl vinyl ketone in 70 ml of benzene was cooled to 0~ and treated with 0.2 g of tributylphosphine. The mixture was stirred overnight, treated with 0.5 ml of iodomethane, and filtered through a short overnight, treated with 0.5 ml of iodomethane, and filtered through a short Florisil column. Gas chromatographic analysis revealed the presence of one product. The solution was heated to reflux under a Dean-Stark trap with 5 ml of ethylene glycol and 0.3 g of p-toluenesulfonic acid for 3 hr until the formation of water ceased. The mixture was cooled, washed with saturated NaHCO3, dried over anhydrous MgSO4, and filtered. GC/MS analysis showed the presence of one major long retention time component, 2, MS m/z 206 (8, M-CH3), 99 (11), 88 (5), 87 (100), 55 (10), and 43 (58). After the solvent was removed, the residue was taken up in EtOH and hydrogenated over 5 g of 10%
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Pd/C at 3 atm pressure for 3 days. After filtration, the solvent was removed, and the residue was stirred with 70 ml of 10% HC1 containing 1 ml of 60% HC104 for 2 hr. The aqueous solution was washed with ether, and carefully made alkaline with 10% NaOH. The aqueous solution was extracted with ether (4 x 50 ml), and the combined ether layers were dried over anhydrous K2CO 3. Distillation provided 3.2 g (58% from 1-nitropentane) of 5, bp 65-68~ mm Hg: IR 1652 c m - l ; [IH] NMR ~ = 3.9 (1H, m), 2.48 (2H, m), 2,05 (1H, m), 2.02 (3H, s), 1.7 (1H, m), 1.5-1.3 (4H, m), 0.9 (3H, br t), 13C NMR 6 = 173.57, 72.93, 38.84, 36.52, 29.17, 29.02, 22.89, 19.81, 14.08; MS m/z (relative intensity) 139 (6, M+), 124 (3), 97 (20), 96 (37), 83 (78), 82 (100), 81 (8), 69 (10), 68 (46), 55 (38), 54 (13), 53 (11), 43 (6), 42 (82), 41 (59), 39 (39); HRMS m/z 139.1355 (C9HI7N M +, calcd. 139.1361). 2-(Diethylphosphonomethyl)-5-butyl-2-pyrroline (6). A solution containing 0.14 g of 5 in 2 ml of THF was added over 15 min to a solution containing 2 mmol of diisopropylamide at - 7 8 ~ in 5 ml of THF'(from 0.28 ml of diisopropylamine and 1.3 ml of 1.6 M n-butyllithium) under a nitrogen atmosphere. After 1 hr, a solution containing 0.14 ml of diethyl chlorophosphate in THF was added slowly and the mixture was stirred for 3 hr. The reaction was quenched with 1 ml of saturated NaHCO3 and warmed to room temperature. After the addition of 10 ml of ether, the aqueous layer was separated, and the organic mixture was dried over anhydrous MgSO4. Kugelrohr distillation (210220 ~ mm Hg) of the filtered mixture provided 0.25 g (90 %) of 6 as a yellow oil: IR 3316, 1619, 1279, 1040, 950, and 787 cm -l; iH NMR 6 = 7.13 (br s), 4.12 (m), 3.95 (m), 3.6 (m), 3.5 (d, J = 14.3 Hz), 3.0 (d, J = 22.3 Hz), 2.55 (m), 2.0 (m), 1.5-1.2 (m), 0.8 (m); 13C NMR 6 = 168.3, 167.4, 72.90, 64.30, 62.12, 60.69, 60.35, 59.36, 38.18, 36.04,.33.42, 33.06, 31.46, 29.08, 28.92, 28.68, 28.47, 22.71, 16.35, 16.16, 13.91; MS m/z (relative intensity) 275 (7, M+), 219 (26), 218 (76), 190 (23), 162 (22), 144 (28), 138 (30), 137 (32), 136 (16), 122 (27), 82 (86), 81 (26), 80 (100), 55 (19); HRMS m/z 275.1657 (CI3Hz6NO3P M +, calcd. 275.1650). 2-Butyl-5-(E, 1-heptenyl)-5-pyrroline (3). A solution containing 0.4 g (1.45 mmol) of 6 in 10 ml of THF was cooled to - 7 8 ~ under a nitrogen atmosphere and treated with 0.9 ml of 1.6 M n-butyllithium in hexane. After 30 min, a solution containing 0.145 g of hexanal in 2 ml of THF was added and the mixture was allowed to warm to room temperature over 12 hr. The mixture was acidified with 10% HCI, and after separation, the aqueous phase was washed once with ether. The aqueous solution was then made alkaline with 10% NaOH and extracted 2 • 10 ml with ether. The combined ether extracts were dried, filtered, and the solvent removed in vacuo, to provide 0.14 g (45 % yield) of 3 as a single component by GC analysis. The IR and mass spectra, and gas chromatographic retention time of 3 were identical to those observed for natural 3 from M. foreli. IH NMR 6 = 6.34 (1H, d, J = 16 Hz), 6.12 (1H, d of t, J =
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16, 6.6 Hz), 3.92 (1H, br m), 2.6 (2H, complex m), 2.1 (2H, m), 1.95 (1H, m), 1.7 (1H, m), 1.5-1.2 (13H, m), 0.85 (6H, m); 13C[ NMR 6 = 172.33 (C=N), 141.99 (CH2-CH=), 127.55 ( = C H - C = N ) , 72.65 (C=N-CH), 36.47 (N-CH-CH2-n-propyl), 33.59 (C__H2-C= N), 32.76 (CH2-CH =), 31.34 (CH2CH2-CH=), 29.02, 28.46 (CHz-CH2-C =N), 28.32, 22.83 (CH2-CH3), 22.44 (CHz-CH3), 13.99 (CH3), 13.90 (CH3); HRMS m/z 221.2155 (CIsH27N M +, calcd. 221.2144). 2-Butyl-5-(E,E,1,3-heptadienyl)-5-pyrroline (4). In the same manner as described for 3, a solution containing 0.4 g (1.47 mmol) of 6 in 10 ml of THF was treated sequentially with n-butyllithium and E-2-hexenal, and the reaction was worked up as described above to give 0.12 g (37% yield) of 4 as a single component by GC analysis. The IR and mass spectra, and gas chromatographic retention time of 4 were identical to those of natural 4 from M. foreli. 1H NMR = 6.56 (1H, d of d, J = 15.8, 9.8 Hz), 6.38 (1H, d, J = 15.8 Hz), 6.17 (1H, d o f d o f t , J = 15.0, 9.8, 1.2 Hz), 5.90 (1H, d o f t , J = 15.0, 7.1 Hz), 4.0 (1H, m), 2.6 (2H, m), 2.1 (2H, m), 1.7 (1H, m), 1.5-1.3 (9H, m), 0.9 (6H, br t); 13C NMR 172.4 (C=N), 139.49 (CH2-CH=), 138.92 (CH2C H = C H - C H = ) , 130.13 (CH2-CH=CH), 126.68 (=CC_H-C=N), 72.91 ( C = N - C H ) , 36.47 (N-CH-CH2-n-propyl), 34.92 (CHz-CH=), 33.58 (C__H2C = N ) , 29.00, 28.53, 22.86 (CH2-CH3), 22.25 (CH2-CH3), 14.02 (CH3), 13.62 (CH3); HRMS m/z 219.1989 (CI5H25N M +, calcd. 219.1987). Toxicity Determinations. The toxicities of 3 and 4 were determined from topical application bioassays against Reticulitermes speratus collected near Yokkaichi Mie prefecture, Japan, as previously described (Jones et al., 1988a). The LDs0 of 3 was 1.06 ___0.93/~g/mg termite, and the LDso of 4 was 2.39 ___ 0.69 #g/mg termite. RESULTS
Field Observations. The diet of M. foreli consisted mainly of secretions produced by plants and insects. Workers were observed consistently to harvest secretions from at least three species of Membracidae (Homoptera), extrafloral nectar of Cassia sp., lnga spp. (Leguminoseae), and secretions of the (both Riodinidae). Just after dawn, some workers also occasionally harvested dead arthropods that had collected under a porch light. Although workers foraged throughout a 24-hr cycle, typically the greatest abundance of individual workers per colony was at night, and there was no indication that any foraged more than 3-5 m from the nest entrance. When nests located in Calthea stems were broken into, there was a rapid scatter response; individuals ran excitedly over the stems, dropped to the ground, or retreated further into the stem. While in this state, individuals produced stridulations ranging from 500 to 2100 Hz at 20-25 pulses/ sec (DeVries, 1991a).
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FIG. 1. The spatulate sting of Megalomyrmexforeli (Bottom: x 50; top x 200). The easily visible sting was angled slightly dorsally and was extruded when an individual was held between two fingers, although restrained individuals of M. foreli did not sting readily. Apparently, the sting could not pierce the callous tissue at the fingertips. A microscopic examination of the sting revealed the spatulate nature of its lancets (Fig. 1). Alkaloid Analysis. Analysis of the methylene chloride extracts from all of the collections of Megalomyrmex foreli revealed the presence of four major alkaloidal components in a 2 : 1 : 7 : 5 ratio with parent ions at m/z = 223,225 221, and 219, respectively. Comparison of their mass and infrared spectra and retention times to those of authentic samples showed that the first two components were 2-butyl-5-heptyl-5-pyrroline (1) and trans-2-butyl-5-heptylpyrrolidine (2) (Jones et al., 1982). Hydrogenation of a portion of the extract over
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PtO2 gave an isomeric mixture of cis/trans 2, demonstrating that the carbonnitrogen skeleton of 2 was common to the remaining alkaloids. The longer retention times and the pair of strong bands between 1650 and 1550 cm ~ in the infrared spectra of the third and fourth eluting alkaloids indicated the presence of one or two units of conjugated unsaturation in these compounds. In addition, strong out-of-plane bending bands at 973 and 987 cm -1, respectively, were evidence for the E geometry of the added double bonds in these compounds. The mass spectra of the third and fourth eluting compounds both showed ions attributable to loss of propene (M-42) + and the loss of Call 9 that would be expected if they were unsaturated analogues of 1 such as 2-butyl-5-(E, 1-heptenyl)-5-pyrroline (3) and 2-butyl-5-(E,E, 1,3-heptadienyl)-5-pyrroline (4) (Fig.
2). In order to confirm these assignments, pyrroline 3 and 4 were synthesized as shown in Scheme I. The ethylene ketal of the nitro ketone formed by the addition of 1-nitropentane to methylvinylketone (Miyakoshi, 1986) was hydrogenated, and following acidification, this sequence provided 2-butyl-5-methyl5-pyrroline (5) in nearly 60% yield. Reaction of the kinetic enolate of 5 with diethyl chlorophosphate gave a good yield of the pyrroline phosphonate 6. The condensation of the anion of 6 (n-butyllithium, - 7 8 ~ with hexanal or E,2NO2
O
NO2
(Et~ 6
6
+
~
C
H
O
~
3
6
+
~
C
H
O
~
4
SCHEME 1.
JONES ET AL.
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H 2
3
4
FIG. 2. The major alkaloids from the venom of Megalomyrmex foreli. hexenal in a Wadsworth-Emmons-type reaction formed 3 and 4, respectively, in moderate yield. In their IH NMR spectra, the olefinic coupling constants of 16 Hz in 3 and 15.8 Hz and 15.0 Hz in 4 confirm the E geometry of the side chain double bonds in these products. The retention times as well as the infrared and mass spectra of 3 and 4 were identical to those of the third and fourth eluting alkaloids from M. foreli. Finally, a minor component with a retention time between that of 3 and 4 and a mass spectrum identical to that of 4 was detected. The band at 887 c m - 1 in the infrared spectrum of this compound along with its retention time suggested that it was very likely a geometrical isomer of one of the double bonds of 4. Since 3 and 4 are new ant venom alkaloids, their toxicity against termites (Reticulitermes spp.) in topical application bioassays was determined. At 1.06 /~g/mg and 2.39 #g/mg of termite, respectively, the LDso'S of 3 and 4 were within the range of those found for other ant-venom-derived l-pyrrolines (Escoubas et al., 1988; Jones et al., 1988a). For comparison, the LDso of nicotine is 0.5/~g/mg termite. No "knock-down" effect was observed, even when the mortality became 100%.
DISCUSSION
The venom alkaloids 1-4 (Fig. 2) present in M. foreli share a common carbon-nitrogen skeleton, and the presence of pyrrolidine 2 suggests employment of the venom as a repellent, since this compound has been shown to have a strong repellent effect on other ants (Blum et al., 1980). M. leoninus produces only 2 in its venom (Jones et al., 1991), which may emphasize its taxonomic
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relationship to M. foreli, as M. foreli is a member of the M. leoninus species group (Brandao, 1990). Saturated 2,5-dialkylpyrrolidines, including 2, are frequently encountered venom components in other myrmicine species (Jones et al., 1982), and it may be that the pyrrolines 1, 3, and 4 are the result of an incomplete or modified biosynthetic pathway to 2. The unsaturated pyrrolines 3 and 4 constitute a new functional class of ant venom alkaloids. Their conjugated imine groups are reminiscent of the vinyl ketones produced in the defensive secretions of some termites, e.g., Schedorhinotermes spp., whose toxicity has been attributed to their strongly, electrophilic nature (Quennedey et al., 1973; Prestwich and Collins, 1982; Prestwich et al., 1975). The toxicities of 3 and 4 in topical application against termites (Reticulitermes spp.) are similar to those found for other ant-derived 1-pyrrolines, and are somewhat less than those reported for the corresponding pyrrolidines (Escoubas et al., 1988; Jones et al., 1988a, 1989). It is not surprising that the more polar 3 and 4 are not exceptionally toxic to termites compared to other more lipophilic ant venom alkaloids, since topical activity requires cuticular penetration. Our observations suggest that termites and M. foreli seldom, if ever, have strong interactions. Thus, it is unclear if these results can be taken as an indication that M. foreli evolved as a defense against termites. On the other hand, the functionally analogous vinyl ketones, produced by termites (Schedorhinotermes) for defense, have demonstrable in vivo toxicity (Spanton and Prestwich, 1981; Prestwich and Collins, 1982), and are inhibitors of in vitro pheromone degradation (Prestwich et al., 1989; Tasayco and Prestwich, 1990a,b). As defensive chemicals, the unsaturated pyrrolines 3 and 4, functioning as irreversible electrophiles, may be used by M. foreli as a defense by disrupting the enzymatic degradation of chemical signals in the antennal sensilla of would be predators in a similar fashion. The reluctance of M. foreli to sting led to an examination of the sting apparatus. The terminal lobate portion of the sting (Fig. 1) indicates some function other than piercing, and fortifies the suggestion that Megalomyrmex stings may be used as a chemical applicator (Kugler, 1979). Indeed, as has been observed in the genus Tetraponera, a characteristic of ants with well-developed chemical defenses may be replacement of stinging by some other mode of defense (Kugler, 1979; Braekman et al., 1987). The alarm-defense behaviors in M. foreli parallel those of other ant taxa. The rapid scatter response and stridulations produced by these ants when they are disturbed are similar to such responses commonly observed in the genera Azteca, Dolichoderus, Tapinoma (Dolichoderinae), OecophyUa, Camponotus (Formicinae), and in the myrmicine genus Crematogaster (DeVries, personal observation), all taxa with vestigal stings and highly evolved chemical defenses (H611dobler and Wilson, 1990). Additionally, it is well established that alkaloid producing ants can utilize these compounds in the form of an aerosol (Obin and Vander Meer, 1985).
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Taken together, the rapid scatter response, apparently non-penetrating sting, and relatively low topical toxicity of 3 and 4, along with their electrophilic functionality, suggest that the venom of M. foreli may be deployed in much the same way. The most recent review (H/511dobler and Wilson, 1990) indicates that little is published on the natural history of Megalomyrmex. It has been observed that some species of Megalomyrmex are apparently xenobiotic parasites of its nearest relatives, Cyphomyrmex and Sericomyrmex, and that in some species in the M. leoninus group, reproduction, or the queen caste, has been taken over by the workers (Brandao, 1987; H6lldobler and Wilson, 1990). The present observations of M. foreli suggest that the unique venom chemistry and modified sting play a role in the foraging and defense behaviors of these ants. Furthermore, these behaviors converge upon other, distantly related ant taxa that posses highly developed chemical defenses. Since plants and insects that form associations with ants receive survival benefit from the symbiotic interaction (Way, 1963; Beattie, 1985; Pierce, 1987; DeVries and Baker, 1989; DeVries, 1991b), it is likely that the E. lycisca and N. cachrus caterpillars reported here benefit from forming symbioses with M. foreli. Thus, although the defensive chemistry in M. foreli undoubtedly evolved in response to specific selective pressures, totally unrelated organisms like plants and other insects may benefit from the unique venom alkaloids of this ant.
Acknowledgments--We wish to thank Dr. H.M. Garraffo and Mr. Noel F. Whittaker of the laboratory of Bioorganic Chemistry, NIDDK for FTIR and high resolution mass spectra, respectively, and Dr. R.J. Highet of the laboratory of biophysical chemistry, NHLBI for the NMR spectra. We also thank Mr. R. Dreyfuss of the medical arts and photography branch, NIH, for the photomicroscopy. PJD would like to thank J. Longino, B. Hawkins, J. Clark, and the MacArthur Foundation for field assistance, and dedicate this paper to the work of P. Martino.
REFERENCES
BEATTIE,A.J., 1985. The Evolutionary Ecology of Ant-Plant Mutualisms. Cambridge University Press, Cambridge. BLUM, M.S., JONES, T.H., HOLLDOBLER,B., FALES, H.M., and JAOUNI,T. 1980. Alkaloidal venom mace: Offensive use by a thief ant. Naturwissenschaften. 67:144-145. BRAEKMAN,J.C., DALOZE, D., PASTEELS, J.M., VAN HECKE, P., DECLERCQ, J.P., SINNWELL, V., and FRANCKE, W. 1987. Tetraponerine-8, an alkaloidal contact poison in a Neoguinean pseudomyrmecine ant, Tetraponera sp. Z. Naturforsch. 42c:627-630. BRANDAO, C.R.F. 1987. Queenlessness in Megalomyrmex (Formicidae: Myrmicinae) with a discussion of the effects of the loss of true queens in ants, pp. 111-1112, in J. Eder and H. Rembold (eds.). Chemistry and Biology of Social Insects. Proc. 10th IUSSI, Munich 1986, Verlag J. Peperny, Munich.
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BRANDAO, C.R.F. 1990. Systematic revision of the neotropical ant genus Megalomyrmex Forel (Hymenoptera: Formicidae: Myrmicinae). Arquivos de Zoologica 3 l: 411-481. DEVRIES, P.J. 1991a. Detecting and recording the calls produced by butterfly caterpillars and ants. J. Lep. Res. In press. DEVRIES, P.J. 199 lb. Mutnalism between Thisbe irenea larvae and ants, and the role of ant ecology in the evolution of myrmecophilous butterflies. Biol. J. Linn. Soc. 43:178-195. DEVRIES, P.J., and BAKER,I., 1989. Butterfly exploitation of a plant-ant mutualism: Adding insult to herbivory. J. N. E Entomol. Soc. 97:332-340. ESCOUBAS, P., CLEMENT, J.L., BLUM, M.S., JONES, T.H., LHOMMET, G., and CELERIER, J.P. 1988. Toxicit6 des alcaloides de fourmis. Actes Coll. Insectes Sociatcc. 4:51-58. FALLS, H.M., COMSTOCK,W., and JONES, T.H. 1980. Test for dehydrogenation in gas chromatography-mass spectrometry systems. Anal. Chem. 52:980-982. Ht)LLDOBLER, B., and WILSON, E.O. 1990. The Ants. Harvard University Press, Cambridge. JONES, T.H., BLUM, M.S., and FALLS, H.M. 1982. Ant venom alkaloids from Solenopsis and Monomorium species. Tetrahedron 38:1949-1958. JONES, T.H., BLUM, M.S., ANDERSEN, A.N., FALLS, H.M., and ESCOUBAS, P. 1988a. Novel 2-ethyl-5-alkylpyrrolidines in the venom of an Australian ant of the genus Monomorium. J. Chem. Ecol. 14:3545. JONES, T.H., STAHLY.,S.M., DON, A.W., and BLUM, M.S. 1988b. Chemotaxonomic implications of the venom chemistry of some Monomorium "'antarcticum" populations. J. Chem. Ecol. 14:2197-2212. JONES, T.H., BLUM, M.S., ESCOUBAS,P., and MUSTHAKALI, T.M. 1989. Novel pyrrolidines in the venom of the ant Monomorium indicum. J. Natl. Prod. 52:779-784. JONES, T.H., BLUM, M.S., FALLS, H.M., BRANDAO,C.R.F., and LATTKE,J. 1991. Chemistry of venom alkaloids in the ant genus Megalomyrmex. J. Chem. Ecol. 17:1897-1908. KUGLER, C. 1979. Evolution of the sting apparatus in the myrmicine ants. Evolution 33:117-130. MIYAKOSHI, T. 1986. A convenient synthesis of 4-oxoalkanals. Synthesis 1986:766-768. OE1N, M.S., and VANDERMEER, R.K., 1985. Gaster flagging by fire ants (Solenopsis spp.): Functional significance of venom dispersal behavior. J. Chem. Ecol. 11:1757-1768. PEDDER, D.J., FALLS,H.M., JAOUNI,T., BLUM,M.S., MACCONNELL,J., and CREWE,R.M. 1976. Constituents of the venom of a South African fire ant (Solenopsis punctaticeps). Tetrahedron. 32:2275-2279. PIERCE, N.E. 1987. The evolution and biogeography of associations between lycaenid butterflies and ants. Oxford Surveys Evolutionary Biol. 4: 89-116. PRESTWICH,G.D., and COLLINS,M.S. 1982. Chemical defense secretions of the termite soldiers of Acorhinotermes and Rhinotermes (lsoptera: Rhinoterminae). J. Chem. Ecol. 8:147-161. PRESTWICH, G.D., KAIB, M., WOOD, W.F., and MEINWALD,J. 1975. 1,13-Tetradecadien-3-one and homologs: New natural products isolated from Schedorhinotermes soldiers. Tetrahedron Lett. 4701-4704. PRESTWICH, G.D., GRAHAM,S. McG., HANDLEY,M., LATLI, B., STREINZ, L., and TASAYCO,J., M.L. 1989. Enzymatic processing of pheromones and pheromone analogs. Experientia 45:263270. QUENNEDEY, A., BRULE, G., RIGAUD,J., DUBOlS, P., and BROSSUT, R. 1973. La glande frontale des soldats de Shedorhinotermes putorius (Isoptera): Analyse chimique et fonctionnement. Insect Biochem. 3:67-74. SPANTON,S.G., and PRESTWICH,G.D. 1981. Chemical self-defenseby termite workers: Prevention of autotoxication in two rhinotermitids. Science 214:1363-1365. TASAYCO, J., M.L. and PRESTW~CH,G.D. 1990a. Aldehyde-oxidizing enzymes in an adult moth:
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In vitro study of aldehyde metabolism in Heliothis virescens. Arch. Biochem. Biophys.
278:444-451. TASAVCO, J., M.L., and PRESTWlCH, G.D. 1990b. A specific affinity reagent to distinguish aldehyde dehydrogenases and oxidases. J. Biol. Chem. 265:3094-3101. WAY, M.J. 1963. Mutualism between ants and honeydew producing Homoptera. Annu. Rev. Entomol. 8:307-344.
CHEMISTRY OF VENOM ALKALOIDS IN THE ANT Megalomyrmex foreli (MYRMICINAE) FROM COSTA RICA
T . H . J O N E S , I'* P.J. D E V R I E S , z and P. E S C O U B A S 3
ILaboratory of Biophysical Chemistry National Heart, Lung, and Blood Institute Bethesda, Maryland 20892 2Department of Zoology University of Texas Austin, Texas 78712 3Plant Ecochemicals Project Research Development Corporation of Japan Megumino Kita 3-1-1 Eniwa-shi 061-13, Japan (Received June 26, 1991; accepted August 19, 1991) Abstract--Chemical analysis of the venom of the myrmicine ant Megalomyrmexforeli from Costa Rica revealed the presence of four major alkaloidal components. Two of these, 2-butyl-5-(E, 1-heptenyl)-5-pyrroline (3) and 2-butyl-5-(E, E, 1,3-heptadienyl)-5-pyrroline (4), constitute a new functional class of ant venom alkaloids, whose structures were assigned from their spectral and chemical behavior and unambiguous syntheses. The function of these compounds is suggested by field observations of the behavior of M. foreli, its sting morphology, and the relative toxicity of 3 and 4 against termite workers. Key Words--Megalomyrmex, Hymenoptera, Myrmicinae, venom, alkaloids, ants, pyrrolines, alarm behavior, stridulation, toxicity.
INTRODUCTION T h e v e n o m s o f m a n y species o f the t w o related m y r m i c i n e genera Solenopsis and Monomorium h a v e b e e n s h o w n to contain 2,5-dialkylpyrrolidines. T h e s e c o m p o u n d s h a v e b e e n f o u n d in s o m e Solenopsis (Diplorhoptrum) species and in nearly e v e r y species o f Monomorium w h e r e alkaloids h a v e b e e n detected * To whom correspondence should be addressed. 2507 0098q)331/91/1200-2507506.50/0 9 1991 Plenum Publishing Corporation
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(Jones et al., 1982). Recently, we have also found 2,5-dialkylpyrrolidines in two Megalomyrmex species (Jones et al., 1991). In a number of cases, smaller amounts of 1-pyrrolines may also be also present (Fales et al., 1980; Pedder et al., 1976; Jones et al., 1982, 1988a,b) although they may be instrumental artifacts. The only other unsaturation detected in the naturally occurring 2,5dialkylpyrrolidines has been the presence of one or more terminal double bonds on the alkyl groups (Jones et al., 1982). Other species in the genus Megalomyrmex have been found to produce saturated 2,5-dialkylpyrrolidines, and in one case, a 3,5-dialkylpyrrolizidine (Jones et al., 1991). In the present paper, we report the elucidation of two novel 1-pyrrolines with conjugated unsaturation, produced by workers of M. foreli. Some insight into the chemical ecology of this species has been gained from field observations and consideration of its sting morphology and the topical toxicities of its venom alkaloids.
METHODS AND MATERIALS
Chemical Analyses. Gas chromatographic analyses were carried out using a Shimadzu GC-9A equipped with a 30 m • 0.5 mm i.d. open DB-17 column (1 /~m film thickness). The temperature for the analyses was programed from 60-215~ at 10~ and the carrier gas flow rate was 15 mL/min. On a given day, retention temperatures were reproducable to I~ Preparative gas chromatography was conducted with a Varian model 1400 gas chromatograph equipped with a 2 m x 5 mm i.d. column packed with 10% OV-17 on 100120 mesh Supelcoport. IR spectra were obtained using a Hewlett-Packard model 5965A FTIR. IH and 13C NMR spectra were obtained from CDC13 solutions using a Varian XL-200 spectrometer. 13C assignments were made on the basis of standard 2D COSY and HETCOR experiments. Electron impact mass spectra were obtained using a LKB-9000 GC/MS equipped with a 30 m x 0.52 mm i.d. open DB-17 column (1 /zm film thickness), a LKB-2091 equipped with a 25 m x 0.31 mm i.d. HP-5 column, or a Finnigan ion trap model 800 equipped with a 25 m x 0.31 mm i.d. HP-5 column. High resolution mass spectra were obtained using a VG 7070F instrument in the E1 mode at an ionizing voltage of 70 eV. Ants. Collections and observations on Megalomyrmexforeli were made at Jardin Botanico Wilson, San Vito de Java, Puntarenas Province, 1100 m in southwestern Costa Rica. Intermittent observations on M. foreli behavior were made from February 1990 through March 1991 on nine separate colonies. These were: five colonies with ca. 100 individuals/nest found in the sheathing stems of Calathea spp. (Marantaceae) from 0.5-1 m above the ground in secondary forest, and four subterranean nests in open second growth: three at the base of
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shrubs and one in a rock wall. None of the subterranean nests had any tumulus at the entrance, and overall colony sizes were estimated to range from 100-300 individuals. Ants were immediately placed in glass vials containing 1-2 ml of CH2C12. Specimens of M. foreli were deposited in the collection of the Los Angeles County Museum of Natural History, Los Angeles, California, and have been accessed by Roy R. Snelling. For analysis, these mixtures were reduced to ca. 0.2 ml with a slow stream of nitrogen. GC/MS and GC/FTIR analyses revealed the presence of four (1-4) nitrogen containing components in a 2 : 1 : 7 :5 ratio with the following spectral data: 1: IR 1644 cm-J; MS m/z (relative intensity) 223 (M § 2), 222 (2), 208 (2), 194 (4), 180 (7), 166 (14), 152 (23), 139 (44), 124 (4), 110 (7), 96 (14), 83 (13), 82 (100), 69 (6), 68 (6), 67 (8), 55 (23), 41 (34). 2: MS m/z (relative intensity) 225 (M +, 2), 169 (3), 168 (65), 127 (10), 126 (100), 109 (10), 95 (10), 82 (15), 67 (30), 55 (40). 3: IR 1648, 1594, 1459, 1331, and 973 cm -~; MS m/z (relative intensity) 221 (25, M+), 179 (24), 178 (72), 166 (14), 165 (87), 164 (100), 150 (29), 122 (37), 109 (15), 108 (77), 106 (18), 94 (58), 80 (33), 79 (21), 67 (32), 55 (52), 54 (34), 53 (23), 41 (97). 4: IR 1622, 1578, 1459, 1330, and 987 cm -~; MS m/z (relative intensity) 219 (25, M§ 190 (29), 177 (26), 176 (99), 163 (45), 162 (45), 148 (22), 134 (44), 132 (18), 121 (22), 120 (100), 119 (29), 118 (29), 107 (18), 106 (27), 105 (12), 94 (13), 93 (18), 91 (26), 80 (24), 79 (31), 77 (25), 67 (19), 65 (18), 55 (25), 53 (17), 41 (60). In addition, a small quantity ( < 5 % of 4) of an earlier eluting isomer of 4 was also detected, with a mass spectrum identical to that of 4, and the following infrared spectrum: IR 1642, 1458, 1320, 989, and 887 cm -l Hydrogenation. A slow stream of hydrogen was passed through a small portion of the extract containing ca. 5 mg of PtO2 for 5 min. Reexamination by GC/MS revealed only two major nitrogen containing components whose gas chromatographic retention times, and infrared and mass spectra were identical to those of a sample of synthetic cis/trans 2. 2-Butyl-5-methyl-5-pyrroline (5). A solution containing 5.26 g (40 retool) of 1-nitropentane and 3.7 ml of methyl vinyl ketone in 70 ml of benzene was cooled to 0~ and treated with 0.2 g of tributylphosphine. The mixture was stirred overnight, treated with 0.5 ml of iodomethane, and filtered through a short overnight, treated with 0.5 ml of iodomethane, and filtered through a short Florisil column. Gas chromatographic analysis revealed the presence of one product. The solution was heated to reflux under a Dean-Stark trap with 5 ml of ethylene glycol and 0.3 g of p-toluenesulfonic acid for 3 hr until the formation of water ceased. The mixture was cooled, washed with saturated NaHCO3, dried over anhydrous MgSO4, and filtered. GC/MS analysis showed the presence of one major long retention time component, 2, MS m/z 206 (8, M-CH3), 99 (11), 88 (5), 87 (100), 55 (10), and 43 (58). After the solvent was removed, the residue was taken up in EtOH and hydrogenated over 5 g of 10%
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Pd/C at 3 atm pressure for 3 days. After filtration, the solvent was removed, and the residue was stirred with 70 ml of 10% HC1 containing 1 ml of 60% HC104 for 2 hr. The aqueous solution was washed with ether, and carefully made alkaline with 10% NaOH. The aqueous solution was extracted with ether (4 x 50 ml), and the combined ether layers were dried over anhydrous K2CO 3. Distillation provided 3.2 g (58% from 1-nitropentane) of 5, bp 65-68~ mm Hg: IR 1652 c m - l ; [IH] NMR ~ = 3.9 (1H, m), 2.48 (2H, m), 2,05 (1H, m), 2.02 (3H, s), 1.7 (1H, m), 1.5-1.3 (4H, m), 0.9 (3H, br t), 13C NMR 6 = 173.57, 72.93, 38.84, 36.52, 29.17, 29.02, 22.89, 19.81, 14.08; MS m/z (relative intensity) 139 (6, M+), 124 (3), 97 (20), 96 (37), 83 (78), 82 (100), 81 (8), 69 (10), 68 (46), 55 (38), 54 (13), 53 (11), 43 (6), 42 (82), 41 (59), 39 (39); HRMS m/z 139.1355 (C9HI7N M +, calcd. 139.1361). 2-(Diethylphosphonomethyl)-5-butyl-2-pyrroline (6). A solution containing 0.14 g of 5 in 2 ml of THF was added over 15 min to a solution containing 2 mmol of diisopropylamide at - 7 8 ~ in 5 ml of THF'(from 0.28 ml of diisopropylamine and 1.3 ml of 1.6 M n-butyllithium) under a nitrogen atmosphere. After 1 hr, a solution containing 0.14 ml of diethyl chlorophosphate in THF was added slowly and the mixture was stirred for 3 hr. The reaction was quenched with 1 ml of saturated NaHCO3 and warmed to room temperature. After the addition of 10 ml of ether, the aqueous layer was separated, and the organic mixture was dried over anhydrous MgSO4. Kugelrohr distillation (210220 ~ mm Hg) of the filtered mixture provided 0.25 g (90 %) of 6 as a yellow oil: IR 3316, 1619, 1279, 1040, 950, and 787 cm -l; iH NMR 6 = 7.13 (br s), 4.12 (m), 3.95 (m), 3.6 (m), 3.5 (d, J = 14.3 Hz), 3.0 (d, J = 22.3 Hz), 2.55 (m), 2.0 (m), 1.5-1.2 (m), 0.8 (m); 13C NMR 6 = 168.3, 167.4, 72.90, 64.30, 62.12, 60.69, 60.35, 59.36, 38.18, 36.04,.33.42, 33.06, 31.46, 29.08, 28.92, 28.68, 28.47, 22.71, 16.35, 16.16, 13.91; MS m/z (relative intensity) 275 (7, M+), 219 (26), 218 (76), 190 (23), 162 (22), 144 (28), 138 (30), 137 (32), 136 (16), 122 (27), 82 (86), 81 (26), 80 (100), 55 (19); HRMS m/z 275.1657 (CI3Hz6NO3P M +, calcd. 275.1650). 2-Butyl-5-(E, 1-heptenyl)-5-pyrroline (3). A solution containing 0.4 g (1.45 mmol) of 6 in 10 ml of THF was cooled to - 7 8 ~ under a nitrogen atmosphere and treated with 0.9 ml of 1.6 M n-butyllithium in hexane. After 30 min, a solution containing 0.145 g of hexanal in 2 ml of THF was added and the mixture was allowed to warm to room temperature over 12 hr. The mixture was acidified with 10% HCI, and after separation, the aqueous phase was washed once with ether. The aqueous solution was then made alkaline with 10% NaOH and extracted 2 • 10 ml with ether. The combined ether extracts were dried, filtered, and the solvent removed in vacuo, to provide 0.14 g (45 % yield) of 3 as a single component by GC analysis. The IR and mass spectra, and gas chromatographic retention time of 3 were identical to those observed for natural 3 from M. foreli. IH NMR 6 = 6.34 (1H, d, J = 16 Hz), 6.12 (1H, d of t, J =
ANT VENOM
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16, 6.6 Hz), 3.92 (1H, br m), 2.6 (2H, complex m), 2.1 (2H, m), 1.95 (1H, m), 1.7 (1H, m), 1.5-1.2 (13H, m), 0.85 (6H, m); 13C[ NMR 6 = 172.33 (C=N), 141.99 (CH2-CH=), 127.55 ( = C H - C = N ) , 72.65 (C=N-CH), 36.47 (N-CH-CH2-n-propyl), 33.59 (C__H2-C= N), 32.76 (CH2-CH =), 31.34 (CH2CH2-CH=), 29.02, 28.46 (CHz-CH2-C =N), 28.32, 22.83 (CH2-CH3), 22.44 (CHz-CH3), 13.99 (CH3), 13.90 (CH3); HRMS m/z 221.2155 (CIsH27N M +, calcd. 221.2144). 2-Butyl-5-(E,E,1,3-heptadienyl)-5-pyrroline (4). In the same manner as described for 3, a solution containing 0.4 g (1.47 mmol) of 6 in 10 ml of THF was treated sequentially with n-butyllithium and E-2-hexenal, and the reaction was worked up as described above to give 0.12 g (37% yield) of 4 as a single component by GC analysis. The IR and mass spectra, and gas chromatographic retention time of 4 were identical to those of natural 4 from M. foreli. 1H NMR = 6.56 (1H, d of d, J = 15.8, 9.8 Hz), 6.38 (1H, d, J = 15.8 Hz), 6.17 (1H, d o f d o f t , J = 15.0, 9.8, 1.2 Hz), 5.90 (1H, d o f t , J = 15.0, 7.1 Hz), 4.0 (1H, m), 2.6 (2H, m), 2.1 (2H, m), 1.7 (1H, m), 1.5-1.3 (9H, m), 0.9 (6H, br t); 13C NMR 172.4 (C=N), 139.49 (CH2-CH=), 138.92 (CH2C H = C H - C H = ) , 130.13 (CH2-CH=CH), 126.68 (=CC_H-C=N), 72.91 ( C = N - C H ) , 36.47 (N-CH-CH2-n-propyl), 34.92 (CHz-CH=), 33.58 (C__H2C = N ) , 29.00, 28.53, 22.86 (CH2-CH3), 22.25 (CH2-CH3), 14.02 (CH3), 13.62 (CH3); HRMS m/z 219.1989 (CI5H25N M +, calcd. 219.1987). Toxicity Determinations. The toxicities of 3 and 4 were determined from topical application bioassays against Reticulitermes speratus collected near Yokkaichi Mie prefecture, Japan, as previously described (Jones et al., 1988a). The LDs0 of 3 was 1.06 ___0.93/~g/mg termite, and the LDso of 4 was 2.39 ___ 0.69 #g/mg termite. RESULTS
Field Observations. The diet of M. foreli consisted mainly of secretions produced by plants and insects. Workers were observed consistently to harvest secretions from at least three species of Membracidae (Homoptera), extrafloral nectar of Cassia sp., lnga spp. (Leguminoseae), and secretions of the (both Riodinidae). Just after dawn, some workers also occasionally harvested dead arthropods that had collected under a porch light. Although workers foraged throughout a 24-hr cycle, typically the greatest abundance of individual workers per colony was at night, and there was no indication that any foraged more than 3-5 m from the nest entrance. When nests located in Calthea stems were broken into, there was a rapid scatter response; individuals ran excitedly over the stems, dropped to the ground, or retreated further into the stem. While in this state, individuals produced stridulations ranging from 500 to 2100 Hz at 20-25 pulses/ sec (DeVries, 1991a).
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FIG. 1. The spatulate sting of Megalomyrmexforeli (Bottom: x 50; top x 200). The easily visible sting was angled slightly dorsally and was extruded when an individual was held between two fingers, although restrained individuals of M. foreli did not sting readily. Apparently, the sting could not pierce the callous tissue at the fingertips. A microscopic examination of the sting revealed the spatulate nature of its lancets (Fig. 1). Alkaloid Analysis. Analysis of the methylene chloride extracts from all of the collections of Megalomyrmex foreli revealed the presence of four major alkaloidal components in a 2 : 1 : 7 : 5 ratio with parent ions at m/z = 223,225 221, and 219, respectively. Comparison of their mass and infrared spectra and retention times to those of authentic samples showed that the first two components were 2-butyl-5-heptyl-5-pyrroline (1) and trans-2-butyl-5-heptylpyrrolidine (2) (Jones et al., 1982). Hydrogenation of a portion of the extract over
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PtO2 gave an isomeric mixture of cis/trans 2, demonstrating that the carbonnitrogen skeleton of 2 was common to the remaining alkaloids. The longer retention times and the pair of strong bands between 1650 and 1550 cm ~ in the infrared spectra of the third and fourth eluting alkaloids indicated the presence of one or two units of conjugated unsaturation in these compounds. In addition, strong out-of-plane bending bands at 973 and 987 cm -1, respectively, were evidence for the E geometry of the added double bonds in these compounds. The mass spectra of the third and fourth eluting compounds both showed ions attributable to loss of propene (M-42) + and the loss of Call 9 that would be expected if they were unsaturated analogues of 1 such as 2-butyl-5-(E, 1-heptenyl)-5-pyrroline (3) and 2-butyl-5-(E,E, 1,3-heptadienyl)-5-pyrroline (4) (Fig.
2). In order to confirm these assignments, pyrroline 3 and 4 were synthesized as shown in Scheme I. The ethylene ketal of the nitro ketone formed by the addition of 1-nitropentane to methylvinylketone (Miyakoshi, 1986) was hydrogenated, and following acidification, this sequence provided 2-butyl-5-methyl5-pyrroline (5) in nearly 60% yield. Reaction of the kinetic enolate of 5 with diethyl chlorophosphate gave a good yield of the pyrroline phosphonate 6. The condensation of the anion of 6 (n-butyllithium, - 7 8 ~ with hexanal or E,2NO2
O
NO2
(Et~ 6
6
+
~
C
H
O
~
3
6
+
~
C
H
O
~
4
SCHEME 1.
JONES ET AL.
2514
H 2
3
4
FIG. 2. The major alkaloids from the venom of Megalomyrmex foreli. hexenal in a Wadsworth-Emmons-type reaction formed 3 and 4, respectively, in moderate yield. In their IH NMR spectra, the olefinic coupling constants of 16 Hz in 3 and 15.8 Hz and 15.0 Hz in 4 confirm the E geometry of the side chain double bonds in these products. The retention times as well as the infrared and mass spectra of 3 and 4 were identical to those of the third and fourth eluting alkaloids from M. foreli. Finally, a minor component with a retention time between that of 3 and 4 and a mass spectrum identical to that of 4 was detected. The band at 887 c m - 1 in the infrared spectrum of this compound along with its retention time suggested that it was very likely a geometrical isomer of one of the double bonds of 4. Since 3 and 4 are new ant venom alkaloids, their toxicity against termites (Reticulitermes spp.) in topical application bioassays was determined. At 1.06 /~g/mg and 2.39 #g/mg of termite, respectively, the LDso'S of 3 and 4 were within the range of those found for other ant-venom-derived l-pyrrolines (Escoubas et al., 1988; Jones et al., 1988a). For comparison, the LDso of nicotine is 0.5/~g/mg termite. No "knock-down" effect was observed, even when the mortality became 100%.
DISCUSSION
The venom alkaloids 1-4 (Fig. 2) present in M. foreli share a common carbon-nitrogen skeleton, and the presence of pyrrolidine 2 suggests employment of the venom as a repellent, since this compound has been shown to have a strong repellent effect on other ants (Blum et al., 1980). M. leoninus produces only 2 in its venom (Jones et al., 1991), which may emphasize its taxonomic
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relationship to M. foreli, as M. foreli is a member of the M. leoninus species group (Brandao, 1990). Saturated 2,5-dialkylpyrrolidines, including 2, are frequently encountered venom components in other myrmicine species (Jones et al., 1982), and it may be that the pyrrolines 1, 3, and 4 are the result of an incomplete or modified biosynthetic pathway to 2. The unsaturated pyrrolines 3 and 4 constitute a new functional class of ant venom alkaloids. Their conjugated imine groups are reminiscent of the vinyl ketones produced in the defensive secretions of some termites, e.g., Schedorhinotermes spp., whose toxicity has been attributed to their strongly, electrophilic nature (Quennedey et al., 1973; Prestwich and Collins, 1982; Prestwich et al., 1975). The toxicities of 3 and 4 in topical application against termites (Reticulitermes spp.) are similar to those found for other ant-derived 1-pyrrolines, and are somewhat less than those reported for the corresponding pyrrolidines (Escoubas et al., 1988; Jones et al., 1988a, 1989). It is not surprising that the more polar 3 and 4 are not exceptionally toxic to termites compared to other more lipophilic ant venom alkaloids, since topical activity requires cuticular penetration. Our observations suggest that termites and M. foreli seldom, if ever, have strong interactions. Thus, it is unclear if these results can be taken as an indication that M. foreli evolved as a defense against termites. On the other hand, the functionally analogous vinyl ketones, produced by termites (Schedorhinotermes) for defense, have demonstrable in vivo toxicity (Spanton and Prestwich, 1981; Prestwich and Collins, 1982), and are inhibitors of in vitro pheromone degradation (Prestwich et al., 1989; Tasayco and Prestwich, 1990a,b). As defensive chemicals, the unsaturated pyrrolines 3 and 4, functioning as irreversible electrophiles, may be used by M. foreli as a defense by disrupting the enzymatic degradation of chemical signals in the antennal sensilla of would be predators in a similar fashion. The reluctance of M. foreli to sting led to an examination of the sting apparatus. The terminal lobate portion of the sting (Fig. 1) indicates some function other than piercing, and fortifies the suggestion that Megalomyrmex stings may be used as a chemical applicator (Kugler, 1979). Indeed, as has been observed in the genus Tetraponera, a characteristic of ants with well-developed chemical defenses may be replacement of stinging by some other mode of defense (Kugler, 1979; Braekman et al., 1987). The alarm-defense behaviors in M. foreli parallel those of other ant taxa. The rapid scatter response and stridulations produced by these ants when they are disturbed are similar to such responses commonly observed in the genera Azteca, Dolichoderus, Tapinoma (Dolichoderinae), OecophyUa, Camponotus (Formicinae), and in the myrmicine genus Crematogaster (DeVries, personal observation), all taxa with vestigal stings and highly evolved chemical defenses (H611dobler and Wilson, 1990). Additionally, it is well established that alkaloid producing ants can utilize these compounds in the form of an aerosol (Obin and Vander Meer, 1985).
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Taken together, the rapid scatter response, apparently non-penetrating sting, and relatively low topical toxicity of 3 and 4, along with their electrophilic functionality, suggest that the venom of M. foreli may be deployed in much the same way. The most recent review (H/511dobler and Wilson, 1990) indicates that little is published on the natural history of Megalomyrmex. It has been observed that some species of Megalomyrmex are apparently xenobiotic parasites of its nearest relatives, Cyphomyrmex and Sericomyrmex, and that in some species in the M. leoninus group, reproduction, or the queen caste, has been taken over by the workers (Brandao, 1987; H6lldobler and Wilson, 1990). The present observations of M. foreli suggest that the unique venom chemistry and modified sting play a role in the foraging and defense behaviors of these ants. Furthermore, these behaviors converge upon other, distantly related ant taxa that posses highly developed chemical defenses. Since plants and insects that form associations with ants receive survival benefit from the symbiotic interaction (Way, 1963; Beattie, 1985; Pierce, 1987; DeVries and Baker, 1989; DeVries, 1991b), it is likely that the E. lycisca and N. cachrus caterpillars reported here benefit from forming symbioses with M. foreli. Thus, although the defensive chemistry in M. foreli undoubtedly evolved in response to specific selective pressures, totally unrelated organisms like plants and other insects may benefit from the unique venom alkaloids of this ant.
Acknowledgments--We wish to thank Dr. H.M. Garraffo and Mr. Noel F. Whittaker of the laboratory of Bioorganic Chemistry, NIDDK for FTIR and high resolution mass spectra, respectively, and Dr. R.J. Highet of the laboratory of biophysical chemistry, NHLBI for the NMR spectra. We also thank Mr. R. Dreyfuss of the medical arts and photography branch, NIH, for the photomicroscopy. PJD would like to thank J. Longino, B. Hawkins, J. Clark, and the MacArthur Foundation for field assistance, and dedicate this paper to the work of P. Martino.
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