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3 Biosynthesis of Monoterpenes in an Ant (Acanthomyops claviger) G. M. HAPP and J. MEINWALD

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1

Department of Chemistry and Division of Biology, Cornell University, Ithaca, Ν. Y.

Terpenoid substances are of broad distribution and di verse function in insects. One set, elaborated by the mandibular glands of A c a n t h o m y o p s claviger, acts both as a defensive secretion and as an alarm releaser. When fed C -labeled acetate or mevalonate, laboratory colo nies of these ants produce radioactive citronellal and citral, providing unambiguous evidence for de n o v o syn thesis of these terpenes by the ant. The incorporations of these precursors implicate the mevalonic acid pathway as the likely biosynthetic route. 14

n p h e defensive glands of arthropods produce a variety of chemical substances, among w h i c h are f o r m i c acid, p-benzoquinones, and aliphatic aldehydes (14,16). O f t e n the particular molecular species employed as toxins are representatives of chemical classes o f w i d e biological distribution. A l t h o u g h B r o w e r and B r o w e r (2) have sug gested that many natural insect toxicants are derived f r o m secondary plant substances, at present it is far f r o m clear whether most must come f r o m such dietary sources or whether the majority can be synthesized de novo b y insects. A s defensive secretions can be isolated relatively easily f r o m their capacious integumentary reser voirs, they offer especially favorable material f o r biosynthetic studies. Aside f r o m the w o r k of Waterhouse and coworkers (10) w h i c h demonstrates the incorporation of C - l a b e l e d acetate, propionate, caproate, or decanoate into all major aliphatic constituents of the 14

Present address: Washington, D. C. 1

Department of Biology, Catholic University of America,

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Crosby; Natural Pest Control Agents Advances in Chemistry; American Chemical Society: Washington, DC, 1966.

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defensive secretion of a pentatomid bug, little data is available o n their biogenesis. T h e monoterpenes, w h i c h include citronellal and citral identified i n the mandibular gland secretion of the ant Acanthomyops claviger (Roger) (3), comprise one of the more interesting classes of defensive substances. These and related isoprenoid molecules serve as physio logical or behavioral messengers i n a variety of insect groups (19,23, 24). I n spite o f a f e w exceptional cases (4, 6,15), as a rule insects do not manufacture steroids (5,11); thus ecdysone, the m o l t i n g hormone, appears t o be derived f r o m ingested cholesterol (12). I n contrast, Schmialek (27) has s h o w n that after the injection of 2 - C - m e v a l o n i c acid into s i l k w o r m caterpillars, radioactive farnesol can be recovered. In the present study, w e are concerned w i t h the biogenesis o f the monoterpene aldehydes w h i c h serve as alarm releasers and defensive substances f o r Acanthomyops (9). 14

Table I. Experimental Results Sodium A cetate Mevalonic A cid Lactone i-C Compound fed Amount, mg. A c t i v i t y , d.p.m. Derivatives isolated Citronellal

2-C

1 4

0.18 2χΐ0?

Total activity, d.p.m. % incorporation Specific activity, d.p.m./mmole Citral

0.048 3χΐ0

2.5X10 0.1 3.3X10

T o t a l activity, d.p.m.

2.5X10

%

0.001

incorporation

Specific activity, d.p.m./mmole

4

6

2

3.3X10

2-C

1 4

4

1 4

10 -

1X10 0.1

4

3.3X10

6

-

1.4X10

6

1.5X10

3

0

6.0X10

2

-

0.006

-

1X10

0.005

5

2-C

6.2 2.32 1X10? 1X10?

7

2.5X10 0.07

1 4

2.7 X 1 0

6

6

L a b o r a t o r y colonies o f w o r k e r ants were f e d sugar-water c o n taining l-C -acetate, 2-C -acetate, or i n a carefully controlled simul taneous feeding, 1 - C - o r 2-C -mevalonate. A f t e r an appropriate period, the ants were frozen and extracted w i t h methylene chloride and the terpene aldehydes (citronellal and citral) isolated b y t h i n layer chromatography. These were then converted into their d i n i 14

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Biosynthesis of Monoterpenes

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trophenylhydrazones, w h i c h were further purified to constant radio activity. Results and

Discussion

Acetate, labeled at either the methyl or carboxyl position, was significantly incorporated into both citronellal and citral. 2 - C Mevalonate was similarly w e l l incorporated, but, i n contrast, 1 - C mevalonate produced o n l y v e r y slight radioactive labeling i n the terpenes. T h e results of these experiments are summarized i n T a b l e I. It is immediately clear that Acanthomyops need not rely o n dietary sources of terpenes but can synthesize citronellal and citral f r o m either acetate or mevalonate. T h e higher total activity of the citronellal as compared w i t h the citral probably reflects the natural preponderance of citronellal (ca. 90%) i n the ant secretion. A s the specific activities show, these results are consistent w i t h a c o m m o n biogenetic origin of both terpenes. In the mevalonic acid pathway as described f r o m other organisms (13), the radioactive carbon of l - C - m e v a l o n a t e is lost u p o n formation of isopentenyl pyrophos phate. 14

14

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- v \ / \ H0 *C; C ^ CH2OH * ' cu{ ? b H C

H

C

2

H

s e v e r a l

steps *co 2

+ >

C

H

\

P\ H

C

CH OP20eH 2

3

CVk

κ

In Acanthomyops, the strikingly different incorporations f o l l o w i n g the t w o mevalonate feedings indicate that mevalonate is not degraded before being built into terpenes but rather is decarboxylated, as i n the classical mevalonic acid pathway. T h e presence of the mevalonic acid pathway f o r terpene biosyn thesis suggested that these ants might also manufacture steroids. T o investigate this possibility, a n e w sample of ants f r o m the l-C -acetate experiment were examined f o r the presence of radioactive cholesterol or other β-hydroxysteroids. T h e ants were extracted w i t h ethanol and ether, this l i p i d extract was saponified, and the nonsaponifiable material (plus a f e w milligrams of nonradioactive carrier cholesterol) chromatographed o n a deactivated alumina c o l u m n (18). Upon elution w i t h a solvent series of increasing polarity (see F i g u r e 1 ) the cholesterol was recovered in fractions 96 to 104 (benzene). It was further purified b y thin-layer chromatography and b y preparation 14


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of its digitonide. T o make certain that other β-hydroxysteroids of Acanthomyops w o u l d not be missed, the remaining fractions were pooled under their respective solvents and the digitonides o f each were prepared. W h e n assayed f o r C - i n c o r p o r a t i o n , none of the 14

Α

Β

20

C

40

60

80

FRACTION

Figure 1. Chromatography

O

E

100

120

F

140

160

NUMBER

of nonsaponifiable

lipids

from

A.

claviger after feeding of 1-C ~acetate u

Absorbent:

20 grams of deactivated Merck alumina eluted with: A. B. C. D. E. F.

Petroleum ether Petroleum ether-benzene (10 to 1) Petroleum ether-benzene (4 to 1) Benzene Diethyl ether Methanol Weight recovered C.p.m. recovered

digitonin-precipitable material was radioactive. A p p a r e n t l y the meva l o n i c acid p a t h w a y is employed b y Acanthomyops f o r the synthesis of monoterpenes but not f o r the formation of steroids. Experimental Assay of Radioactive Compounds. T h e radioactive samples were counted o n steel planchets i n a N u c l e a r C h i c a g o M o d e l D-47 l o w - b a c k g r o u n d gas-flow c o u n t i n g chamber w i t h an absolute count ing efficiency (estimated b y comparison w i t h a standard) of about 20%.


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A N D MEINWALD

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Purification of Carrier Compounds. Citronellal (b.p. 9 9 99.5°/25 mm.) and citral (b.p. 92-93°/4.2 mm.) were purified by distillation, and the purity was checked by vapor-phase chromatog raphy. Cholesterol (m.p. 1 4 8 . 5 - 4 9 . 5 ° ) was purified via its dibromide (8). Administration of Tracers. Workers of Acanthomyops clavi ger (Roger) were freshly collected near Ithaca, Ν . Y . , for each ex periment. Over the course of each feeding, 1000 to 1500 ants were maintained in a two-chamber Lucite Wilson nest. One chamber was filled with moist earth and shielded from light while the other served as a foraging area. C -labeled acetate and mevalonate were fed in glucose solution and were distributed throughout the colony by regurgitative feeding (22,24). In the experiments with 1 - C - and 2-C -mevalonate, all workers were collected from a single natural colony and were individually sorted into one or the other laboratory colonies to avoid any possible bias due to physiological caste differ ences analogous to those reported in other ant species ( 7 ) . Isolation of Citronellal and C i t r a l . A t the close of each ex periment (7 to 10 days), the nests were frozen intact. Groups of 200 workers were placed in a micro-Soxhlet apparatus and extracted for 8 hours with methylene chloride. A few milligrams of carrier citronellal and citral were added and the mixture was applied to a thin-layer chromatoplate (silica gel G ) which was developed with hexane-ethyl acetate (92 to 8) to separate citronellal and citral (3). The aldehydes were detected by spraying with a solution of 2,4-dinitrophenylhydrazine in tetrahydrofuran (20) and the citronellal and citral peaks were scraped off and allowed to react with excess dinitrophenylhydrazine reagent for a further 12 hours. The dinitrophenylhydrazones were separated from the reaction mixture by thin-layer chromatography (silica gel G developed with benzene) and further purified by thin-layer chromatography on aluminum oxide G (petroleum ether-diethyl ether (96 to 4 ) , silica gel G (chloroform),, and silica gel G (diethyl ether)). In all cases, the specific activities of the dinitrophenylhydrazones remained con stant over the course of the last two purifications. Collection of Nonsaponifiable L i p i d s . T w o hundred ants from the 2-C -acetate feeding were ground with sand and the resulting brei refluxed for 4 hours in 25 ml. of ethanol, ethanol-diethyl ether (3 to 1), and diethyl ether (twice). T h e extracts were pooled, the solvents were evaporated, and the residue was saponified by refluxing for 1 hour with 20 ml. of methanolic potassium hydroxide ( 1 0 % potassium hydroxide in 6 0 % aqueous methanol). A n equal quantity of water was added and the aqueous solution extracted three 14

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times w i t h diethyl ether (100 m l . total) to isolate the nonsaponifiable fraction. T h e ether extract was then shaken w i t h 100 m l . of 1 % aqueous potassium hydroxide to remove any free fatty acids. T h e activity of the total extract was about 8 . 0 X 1 0 d.p.m. and that of the nonsaponifiable material 3 . 0 X 1 0 d.p.m. 6

5

Chromatography of Nonsaponifiable Lipids. T h e nonsaponi fiable residue plus 4.5 m g . of carrier cholesterol was applied t o the top of a 7.5X1.7 c m . c o l u m n containing 20 grams of M e r c k alumina (suitable f o r chromatographic adsorption) w h i c h had been previously deactivated b y m i x i n g w i t h 7 % aqueous acetic acid ( 1 0 % glacial acetic acid i n distilled water) (18). T h e c o l u m n was packed i n petroleum ether (redistilled, b.p. 6 0 - 7 0 ° C.) and 10 m l . fractions were collected. T h e eluting solvents are shown in T a b l e II. Table II. Fraction 1-20 21-65 66-85 86-115 116-135 136-160

Eluting Solvents Solvent Petroleum ether Petroleum ether—benzene Petroleum ether—benzene Benzene Diethyl ether Methanol

(10:1) (4:1 )

T h e o n l y white solid recovered was i n fractions 97 to 104. T h i n layer chromatography o n silica gel G impregnated w i t h Rhodamine 6 G showed that o n l y this fraction contained cholesterol (1). A f t e r a second chromatography using c h l o r o f o r m as the solvent, 3.25 m g . of white crystalline material w i t h an activity of 2 X 1 0 d.p.m. was recovered. T h i s cholesterol was further purified b y preparation of its digitonide (21). A l l other fractions were pooled under their respec tive solvents, 1 m g . of carrier cholesterol was added to each, and the digitonides were prepared. N o activity above background was de tected i n any of the digitonin precipitates. 2

Literature Cited (1) Avigan, J., Goodman, D e W . S., Steinberg, D . , J. Lipid Res. 4, 100 (1963). (2) Brower, L . P., Brower, J . van Z., Zoologica 49, 137 (1964). (3) Chadha, M. S., Eisner, T . , Monro, Α., Meinwald J., J. Insect Physiol. 8, 175 (1962). (4) Clayton, R. B., J. Biol. Chem. 235, 3421 (1960). (5) Clayton, R. B., J. Lipid Res. 5, 3 (1964).


3. (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24)

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Clayton, R. B., Edwards, A. M., Bloch, K . , Nature 195, 1125 (1962). Ehrhardt, S., Z. Morphol. Ökol. Tiere 20, 755 (1931). Fieser, L . F . , J. Am. Chem. Soc. 75, 5421 (1953). Ghent, R . L., P h . D . thesis, Cornell University, Ithaca, Ν . Y . , 1961. Gordon, H. T., Waterhouse, D . F., G i l b y , A. R., Nature 197, 818 (1963). House, H. L., Ann. Rev. Biochem. 31, 653 (1962). Karlson, P., Hoffmeister, H., Z. Physiol. Chem. 331, 298 (1963). Richards, J . H., Hendrickson, J . B., " T h e Biosynthesis of Steroids, Terpenes, and Acetogenins," W. A. Benjamin, N e w Y o r k , 1964. Roth, L. M., Eisner, T . , Ann. Rev. Entomol. 7, 107 (1962). Saito, M., Yamazaki, M., Kobayashi, M., Nature 198, 1324 (1963). Schildknecht, H., Angew. Chem. 75, 762 (1963). Schmialek, P., Z. Naturforsch. 18b, 462 (1963). Schneider, P . B., Clayton, R . B., Bloch, K . , J. Biol. Chem. 224, 175 (1957). Schneiderman, Η. Α., Gilbert, L. I., Science 143, 325 (1964). Shine, H. J., J. Org. Chem. 24, 252, 1790 (1959). Sperry, W. M., W e b b , K . , J. Biol. Chem. 187, 97 (1950). Wallis, D . I., Behaviour 17, 17 (1961). W i l s o n , E . O . , Bossert, W. H., Recent Prog. Hormone Res. 19, 673 (1963). Wilson, E . O . , Eisner, T . , Insectes Sociaux 4, 157 (1957).

RECEIVED M a y 13, 1965, G. M. H a p p was supported b y a National Insti tutes of Health Postdoctoral Fellowship ( G M - 1 1 873-01). W o r k assisted by Grant AI-02908 (National Institutes of Health).


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