Journal of Chemical Ecology, Vol. 15, No. 1. 1989
A G G R E G A T I O N P H E R O M O N E C O M P O N E N T S IN
Drosophila multeri A Chiral Ester and an Unsaturated Ketone
ROBERT J. BARTELT, j ANGELA M. SCHANER, and LARRY L. JACKSON Department of Chemistr3, Montana State University Bozeman, Montana 59717 (Received October 19, 1987; accepted December 2I, 1987)
Abstract--Existence of an aggregation pheromone was demonstrated in Drosophila mulleri. Mature males produce at least two compounds that are lacking from females and newly emerged males and that attract both males and females in a wind-tunnel bioassay. These compounds are (S)-(+)-2-tridecanol acetate and (Z)-10-heptadecen-2-one. Both were synthesized, and the flies responded to the synthetic compounds as well as to fly-derived preparations. The flies also responded to racemic 2-tridecanol acetate but not to the pure R enantiomer. A more polar, very volatile attractant was also extracted from both sexes of D. mulleri but was not identified. Key Words--Drosophila mulleri, Diptera, Drosophilidae, aggregation, pheromone, chirality, enantiomers, ester, ketone, (S)-(+)-2-tridecanol acetate, (Z)- 10-heptadecen-2-one.
INTRODUCTION
Aggregation pheromones have been demonstrated in a number of species of the genus Drosophila (Diptera: Drosophilidae) (Moats et al., 1987; Schaner et al., 1987; Bartelt et al., 1986, 1987; and references therein). In these species, sexually mature males possess compounds that attract both males and females in a laboratory wind-tunnel bioassay. These compounds are lacking in females and in newly emerged males. The pheromone components include hydrocarbons, ~Present address: USDA-ARS, Northern Regional Research Center, Peoria, Illinois 61604.
399 0098-0331/89/0100-0399506.00/0 ~' 1989PlenumPublishingCorporation
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BARTELT ET AL.
esters, and ketones, and mixtures of compounds are often required for optimal responses. Volatiles from fermenting food media usually synergize the pheromones. Research on pheromones of Drosophila species was continued with D. mulleri Patterson. A comparison between D. mulleri and D. hydei (see Moats et al., 1987) was of special interest because both belong to the same (repleta) species group. D. mulleri was found to possess pheromone components not previously encountered in other Drosophila.
METHODS AND MATERIALS
Flies, Extracts, and Bioassays. The D. mulleri culture was obtained from the National Drosophila Resource Center, in Bowling Green, Ohio (stock number 15081-1371). The flies were reared on Formula 4-24 Instant Drosophila Medium (Carolina Biological Supply, Burlington, North Carolina). The age of maturity of male and female flies was determined as described by Moats et al. (1987), so that appropriate ages for extraction and bioassay could be chosen. We found that males ofD. mulleri become mature at 5-6 days and females at 2-3 days. In other Drosophila species we have studied, pheromone production begins in males as sexual maturity is approached, but flies of any age have usually responded to the pheromones. Thus, to obtain pheromone, flies were not extracted until 6-7 days of age, but 1- to 4-day-old flies were used for most bioassays because the immature males would be less likely to possess pheromones that could compete with bioassay treatments. Flies to be extracted were segregated by sex within one day of emergence and extracted by soaking them overnight in hexane at room temperature. Bioassays were performed in a wind-tunnel olfactometer essentially as described earlier (Bartelt and Jackson, 1984). About 16 hr before bioassay tests began, ca. 1000 flies were added to the olfactometer. As with the previously studied species, a starvation period was necessary before flies would respond to pheromones. Each bioassay treatment was applied to a filter paper strip lining the lip of a glass vial. A drop of water was added to the bottom of each bioassay vial to arrest responding flies (Bartelt and Jackson, 1984). Two such vials, with treatments to be compared, were placed in the upwind end of the olfactometer. After 3 min the vials were capped, and the captured flies were counted and, in selected experiments, sexed. Tests were performed throughout the day at ca. 10-min intervals. In experiments with more than two treatments, they were tested in all possible pairs (a balanced incomplete block design). Statistical analysis followed the method of Yates (1940) and was done on data transformed as log(X + 1) to stabilize variance. Chromatography and Spectra. Crude hexane extracts were chromato-
Drosophila mulleri AGGREGATIONPHEROMONE
401
graphed initially on open columns of silicic acid and eluted with two void volumes of each of the following solvents: hexane; 5%, 10%, and 50% ether in hexane; and 10% methanol in methylene chloride. All gas chromatography (GC) was conducted on a Varian 3700 using He as the carrier gas. For preparative GC, a 1.3-m x 4-mm 5% SE-52 column was used, and the effluent was monitored with a thermal conductivity detector. Three columns were utilized for capillary GC: a 30-m x 0.25-mm DB-1, a 15m x 0.32-mm DB-5, and a 30-m x 0.25-mm DB-225 (J & W Scientific, Rancho Cordova, California). Peaks were monitored with a flame ionization detector. Various temperature programs were used, as described below. High-performance liquid chromatography (HPLC) was conducted isocratically with a Waters 6000A pump and a Waters 401 differential refractometer. Columns included a 25-cm x 4.6-ram ID Adsorbosphere silicic acid column (Applied Science, Deerfield, Illinois), eluted with 5% ether in hexane, and a 25-cm x 4.6-mm ID silicic acid column, coated with AgNO3 as described by Heath and Sonnet (1980), and eluted with toluene. Electron impact mass spectra were obtained on a VG-MMI6F mass spectrometer. Samples were introduced through a capillary GC column. Synthetic Esters. (+)-2-Tridecanol acetate was prepared by treating dodecanal with methylmagnesium bromide in ether, followed by acetylation of the resulting alcohol with acetic anhydride in pyridine. After chromatography on an open column of silicic acid and preparative GC, the ester was > 9 8 % pure by capillary GC. The ester was diluted with hexane to 10 ng/t~l for bioassays. A portion of the (+)-2-tridecanol was set aside for chromatographic studies. (S)-2-Tridecanol was synthesized (Figure 1) from a commercially available, optically active starting material, ethyl (S)-lactate (I) (Sigma Chemical Co., St. Louis, Missouri). Synthetic steps were performed as described in the literature for analogous reactions: After conversion to a tetrahydropyranyl (THP) ether (II) (Miyashita et al., 1977), the ester function was reduced with lithium aluminum hydride (Moil, 1976), and the resulting primary alcohol (III) was esterified with p-toluenesulfonyl chloride to form the tosylate (IV) (Moil, 1976). Decylmagnesium bromide was linked to this tosylate in the presence of dilithium tetrachlorocuprate to form the THP ether of (S)-2-tildecanol (V) (Suguro and Moil, 1979). Finally, the tetrahydropyranyl protecting group was removed (Miyashita et al., 1977). The crude (S)-2-tridecanol (VI) crystallized at ca. - 1 0 ° C from a 20% solution in pentane. By GC, the purity of the recovered product was 88%. All reactions were monitored by GC and mass spectrometry. All were essentially quantitative except for the alkylation of the tosylate, for which the yield was ca. 50%. Intermediate products were not purified before subsequent reactions, except to dry (Na2SO4) and to remove solvents.
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Flo. 1. Synthetic scheme for ( S ) - ( + ) - 2 - t r i d e c a n o l , its acetate, and its (S)-2-(acetyloxy)propanoate. Abbreviations: T H P = tetrahydropyranyl group, PPTS = pyridinium p-toluenesulfonate, T O S = p-toluenesulfonyl group, A P C = (S)-2-(acetyloxy)propanoyl chloride.
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Drosophila mulleri AGGREGATIONPHEROMONE
403
(R)-2-Tridecanol was prepared in an identical manner except that the starting material was methyl (R)-lactate (Sigma). (S)-2-Tridecanol was either converted to an acetate for bioassays or was derivatized as described below for GC studies of optical purity and stereochemical configuration. The acetate (Figure 1, VII) was formed by treatment with acetic anhydride in pyridine. After purification by chromatography on silicic acid and preparative GC, the ester's purity was > 9 9 % , by capillary GC. Approximately 10 mg of pure ester were prepared. The ester had an identical mass spectrum and GC retention (DB-1, DB-5) as the racemic synthetic (+_)2-tridecanol acetate. The specific rotation was determined: [O~]D 3 = +4.6 ° (C = 0.57%, hexane) (literature value: [~]~o = +4.63 o, absolute configuration not given, Pickard and Kenyon, 1914). (R)-(-)-2-Tridecanol acetate was likewise prepared from the (R)-2-tridecanol. The measured purity of the ester was > 99 % and the optical rotation, [O/]D3 ~--- - 4 . 9 ° (e = 0.57%, hexane). GC retention and mass spectrum were as for the S isomer. Both enantiomeric esters were diluted with hexane for bioassays. Studies of Optical Purity and Absolute Configuration. Diastereomeric esters (Figure 1, VIII) were formed from the synthetic 2-tridecanols so that optical purity could be analyzed on an achiral GC column. The (S)-(2-acetyloxy)propanoates (acetyl lactates) were prepared essentially as described by Slessor et al. (1985). The derivatizing reagent was (S)-(2-acetyloxy)propanoyl chloride (APC). GC analysis was on the DB-5 capillary column, temperature programmed from 120°C to 150°C at 5°C/min. To derivatize the 2-tridecanol acetate from D. mulleri, the ester was first reduced with LiAIH4. To ca. 10 #g of purified D. mulIeri ester in 20/xl ether was added 15 /zl LiAIH4 reagent (1.0 M in ether). After 10 min at room temperature, the vial was cooled over ice and 2 drops of H20 were added to decompose excess reagent. The organic layer was transferred to a clean vial and stripped to dryness under N2; then the alcohol was derivatized with APC. Synthesis ofKetones. (Z)-9-Hexadecenoic acid (50 mg, 0.2 mmol, Sigma) in 2 ml dry ether was cooled to 0°C. Methyllithium in ether (0.8 mmol, 0.6 ml) was added in one portion. The mixture was warmed to room temperature, refluxed for 20 min, then cooled to 0°C, after which 1 ml water was added very slowly. The ether layer was washed with dilute HC1, water, and NaHCO3 solution, then dried over Na2SO4. The resulting (Z)-10-heptadecen-2-one was purified on an open column of silicic acid and diluted with hexane for bioassay. The purity was 9 9 + % by capillary GC. 2-Heptadecanone was similarly prepared in 99 + % purity from palmitic (hexadecanoic) acid. Preliminary Experiments with Volatile, Polar Attractants. Apparatus was assembled to allow evaporation of a sample (up to 500/zl) under a gentle stream of nitrogen, so that the vapors passed through a 2.5 x 25-mm column of 20/
404
BARTELT ET AL.
35 mesh Tenax porous polymer (Applied Science). Both the fly-derived 10% MeOH-CH2CI 2 fraction and a CHzCI2 rinse of rearing medium (inoculated with yeast but without Drosophila) were treated in this way. Each sample was reconstituted with an equal volume of fresh solvent immediately as the sample went to dryness, and the Tenax column was eluted with an equivalent volume of pentane. The reconstituted samples and Tenax column rinses were compared to the original samples by bioassay. The Tenax column rinses were examined by GC and mass spectrometry.
RESULTS AND DISCUSSION
Bioassay Characteristics. When the flies were first put into the olfactometer, they formed tight aggregations low on the sides and moved about very little. After starvation for 14-16 hr at a temperature of 24-25°C, these groups dispersed and the flies became more active, usually with ca. 20-30 flies in flight at any given time. Response to an active treatment was usually by an upwind, zigzag, hovering flight, followed by alighting on the vial. Bioassays of Crude Extracts and Fractions. The crude hexane extract of mature male D. mulleri was active in the bioassay (Table 1A), and three fractions derived from this extract were significantly active. Based on standards, the first (eluted with 5% ether-hexane) had the polarity of esters while the second (eluted with 10% ether-hexane) had the polarity of ketones. The last was considerably more polar (eluted with 10% methanol-methylene chloride). Surprisingly, the female-derived extract was also very active (Table IB), and this activity was located in the most polar fraction. Thus D. mulleri was like the previously studied species in that mature males had attractive nonpolar compounds that were lacking in mature females, but D. mulleri was unlike the other species in that very active, relatively polar compounds(s) were present in both sexes. Further purification of the male-derived 5% and 10% ether-hexane fractions was accomplished by preparative GC. The activity from the 5 % etherhexane fraction was retained slightly longer than an alkane of 16 carbons on the nonpolar preparative GC column, and the activity from the 10% etherhexane fraction was similar in retention to an alkane of 19 carbons. No other GC fractions had significant bioassay activity. Subsequent HPLC on silicic acid produced only one active region from each GC fraction. The activity originally from the 5 % ether-hexane silicic acid fraction eluted 5.5-6.5 ml after injection, while that from the 10% ether-hexane fraction eluted at 10-11 ml. Based on standards, these retentions supported that the activity in the former fraction was due to an ester, and the latter, a ketone. Ester Component. After preparative GC and HPLC, the active ester corn-
Drosophila mulleri AGGREGATION PHEROMONE
405
TABLE 1. ACTIVITY OF SILICIC ACID FRACTIONS RELATIVE TO CONTROLS AND TO MALE-DER/VED CRUDE EXTRACT
Mean bioassay catch (N -> 8)I'
Treatment"
Treatment
Control
Male-derived crude extract
A. Male-derived fractions Hexane 5% Ether-hexane 10% Ether-hexane 50% Ether-hexane 10% Methanol-methylene chloride
8.5 13.6"** 8.0*** 5.5 23.0***
8.7 3.8 2.8 6.0 1.0
57.5*** 23.4*** 33.4*** 49. I *** 23.8***
B. Female-derived extract and fractions Crude hexane extract Hexane fraction 5% Ether-hexane 10% Ether-hexane 50% Ether-hexane 10% Methanol-methylene chloride
29.1 *** 4.4 3,2 6.3 2.3 27.5***
5.0 4.0 4.2 4.4 4.3 2.9
21.7*** 52.2*** 40.9*** 42.6*** 22.9*** 22.0***
"One fly equivalent per test, Each row is a balanced incomplete block experiment, in which the test treatment, the control, and the male-derived crude extract are tested in pairs, in all possible combinations. /'In each row, means followed by *** are significantly different from the control at the 0.001 level (t tests). No other differences were significant at even the 0.05 level.
ponent was > 98% pure, by capillary GC. Mature males possessed ca. 300 ng of this compound, but it was lacking in females and newly emerged males. In the mass spectrum, no molecular ion was seen, but fragments at m/z 87 (40%) and 182 (15%, M-60) suggested an acetate of the secondary alcohol, 2-tridecanol. The base peak was m/z 43, and peaks also occurred at 198 and 199 (both ca. 1%, M-44 and M-43, respectively). Racemic 2-tridecanol acetate and the D. mulleri compound had identical GC retention times (DB-1) and mass spectra. Both reacted with methanolic KOH, resulting in a GC peak with the same retention as authentic 2-tridecanol. Racemic 2-tridecanol acetate was active in the bioassay (Table 2A). The fly-derived 2-tridecanol acetate, having one asymmetric center, was analyzed for optical purity and absolute configuration (summarized in Figure 2). The two diastereomers derived from racemic 2-tridecanol were easily separated on the DB-5 capillary column. Based on the synthetic standards, the first of these peaks (Kovats index = 1966) corresponded to the R configuration, while the peak at KI = 1982 represented the S configuration. The derivative
406
BARTELT ET AL.
TABLE 9_. KEY BIOASSAY RESULTS RELATING TO IDENTITIES OF NONPOLAR PHEROMONE COMPONENTS
Treatment
Mean bioassay catch"
A. Preliminary identification of ester component (N = 74). (+)-2-Tridecanol acetate (50 ng) Control
26.5a 4.9b
B. Determination of active enantiomer (N = 40). (S)-(+)-2-Tridecanol acetate (25 ng) (R)-(-)-2-Tridecanol acetate (25 ng) Control
22.9a 5.6b 6.3b
C. Comparison of male-derived "ester" fraction and synthetic ester (N = |2). (Both treatments had 300 ng 2-tridecanol acetate per test). (S)-( + )-2-Tridecanol acetate Male-derived 5% ether-hexane fraction Control
14.2a 14.2a 3.1b
D. Comparison of male-derived "'ketone" fraction and synthetic ketone (N = 16). [Both treatments had 0.7 ng (Z)-10-heptadecen-2-one per test.] (Z)- 10-Heptadecen-2-one 10% Ether-hexane fraction Control
14. I a 19.9a 2.9b
E. Activity of two 17-carbon ketones, alone and in combination (N = 12). (Each ketone used at 8 ng per test) (Z)- 10-Heptadecen-2-one 2-Heptadecanone (Z)-10-Heptadecen-2-one + 2-heptadecanone Control
14.9b 4.9c 25.4a 4.4c
"In each experiment, means followed by the same letter not significantly different at the 0.05 level (LSD method).
prepared f r o m the D. mulleri p h e r o m o n e had a Kovats index o f 1982 and a high degree o f optical purity (enantiomeric e x c e s s calculated from peak integrations was 9 8 . 7 % ) . By c o m p a r i s o n to the synthetic standards, the D. mulleri pherom o n e has the S configuration. T h e e n a n t i o m e r i c excess represents a m i n i m u m , as the departure f r o m 100% c o u l d be due to optical impurity o f the derivatizing reagent a n d / o r slight racemization o c c u r r i n g during c h e m i c a l procedures. D. mulleri r e s p o n d e d in the bioassay only to the e n a n t i o m e r o f 2-tridecanol acetate which they produce (Table 2B). T h e S i s o m e r was clearly active, w h i l e the R i s o m e r was not. That the R i s o m e r was not repellent was consistent with
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RETENTION TIME (MIN) FIG. 2. GC analysis of optical purity and absolute configuration of the 2-tridecanol acetate produced by D. mulleri. Shaded peaks represent (S)-2-(acetyloxy)propanoate derivatives of synthetic and fly-derived 2-tridecanol. Kovats indices are indicated at peak apices. The peaks at ca. 4.3 and 5.8 min are n-alkanes of 19 and 20 carbons, respectively, coinjected as retention standards. The DB-5 capillary column was 120°C initially and was increased at 5°C/min.
the activity of the racemic synthetic ester. (S)-2-Tildecanol acetate adequately accounts for the activity of the male-derived 5 % ether-hexane fraction (Table 2C). In this experiment both treatments had equivalent amounts of the ester. Chiral pheromones have been demonstrated in a number of insect groups. One common pattern is that insects respond only to the optical isomer that they produce but that the other enantiomer is not repellent (Moil, 1985), a pattern which has now been demonstrated in the Drosophila also. Ketone Component. The active "ketone" fraction, after preparative GC
408
BARTELT ET AL.
and HPLC on silicic acid, contained two peaks, by capillary GC. Both were small peaks, representing ca. 1 ng/mature male. The latter of the two peaks was identical in GC retention and mass spectrum with 2-heptadecanone [key spectral peaks were at m/z 43 (100%), 58 (80%), and 254 (4%)]. The other peak eluted slightly earlier on the nonpolar DB-1 capillary column, and its mass spectrum indicated a molecular weight of 252 (2%), 2 mass units less than 2heptadecanone. Fragments still appeared at m/z 43 (100%) and 58 (26%); thus an unsaturated methyl ketone seemed likely. (Z)-10-Heptadecen-2-one was synthesized as a standard for comparative purposes. This ketone was accessible in one step from the readily available (Z)-9-hexadecenoic (palmitoleic) acid. Fortuitously, this ketone had the same retention on three GC columns as the D. mulleri ketone (KI = 1860, 1873, and 2243 on DB-1, DB-5, and DB-225, respectively), and the mass spectra and retentions on the AgNO3 HPLC column were identical also. Since these chromatographic methods are sensitive to double-bond position and configuration, the data strongly supported (Z)-10-heptadecen-2-one as the structure of the unknown compound. Because of the minute amount of ketone in each male, confirmation of double bond location by ozonolysis was not attempted. (Z)-10-Heptadecen-2-one was active in bioassay and accounts fairly well for the activity of the male-derived 10% ether-hexane fraction (Table 2D). 2Heptadecanone, also found in D. mulleri males, was not active in the bioassay by itself, although a mixture of 2-heptadecanone + (Z)-10-heptadecen-2-one was somewhat more active than the unsaturated ketone alone (Table 2E). The extracts were subsequently examined for 2-tridecanone and 2-pentadecanone, pheromone components in the related species, D. hydei (Moats et al., 1987). Traces of both ketones were found (2-4 ng/mature male), but the synthetic ketones were not active in the bioassay. None of the ketones were detected in extracts of female flies.
Synergistic Activity of (S )-(+ )-2-Tridecanol Acetate and (Z )-lO-Heptadecen-2-one. Dose-response data for various mixtures of (S)-2-tridecanol acetate and (Z)-10-heptadecen-2-one are given in Table 3. The response generally increased with increases in either component, although there was little difference between the 300- and 3000-ng levels of the ester. On a weight-for-weight basis, the ketone was the more active compound. For example, 10 ng of ketone elicited a stronger response than even 3000 ng of ester. Response by Sex. Both sexes responded readily to all preparations tested. For example, in a paired comparison experiment with the crude hexane extract of mature males (1 equivalent), (S)-2-tridecanol acetate (300 ng), (Z)-10-heptadecen-2-one (10 ng), and controls, the mean captures (and percent females) for the treatments were 38.7 (45%), 11.8 (47%), 29.5 (45%), and 2.0 (67%), respectively (N = 6). Polar Attractant. Despite concerted efforts, the polar attractant(s) in the
Drosophila multeri AGGREGATIONPHEROMONE
409
TABLE 3. RELATIVEACTIVITIESFOR VARIOUS MIXTURESOF (S)-(+)-2-TRIDECANOL ACETATEAND (Z)- 10-HEPTADECEN-2-ONEa Amount of (Z)10-heptadecen-2one (ng) 0.0 0.1 1
10 100
Amount of ( S)-( +)-2-tridecanol acetate (ng) 0
3
30
300
3000
0h 3~ 14 92 258
8 28 39 151 364
19 35 58 170 387
50 74 100a 226 504
57 58 140 294 555
"Each test mixture compared to solvent controls and the standard mixture (300 ng (S)-(+)-2tridecanol acetate plus 1 ng (Z)-10-heptadecen-2-one)in a balanced incompleteblock experiment (N = 8). Relative activity (RA) based on means from these experiments: RA = 100 x (test mixture - control)/(standardmixture - control). "By definition, RA for control = 0. Overall, the mean bioassaycatch for controls was 4.5 flies per 3-min test. 'All samples except this one attracted significantlymore flies than the control (P < 0.01). aBy definition. RA for standard mixture (ca. 1 male equivalent)was 100. Overall, the mean bioassay catch for this standard was 34.8 flies per 3-min test.
10% MeOH-CHzC12 fraction was (were) not identified. The activity was extremely volatile, compared to the ester and ketone pheromone components. Evaporating the polar fraction carefully to dryness under N2 and immediately reconstituting the fraction with fresh solvent resulted in the loss of ca. 80% of the bioassay activity (Table 4). When the vapors from the evaporating fraction were directed through a column of Tenax, the activity could be recovered by rinsing the column with pentane. (Evaporation of control solvent through the Tenax trap did not produce a trap rinse that was active in bioassay). Examination of the active trap rinse by GC revealed many minute peaks, but there was little consistency among runs and there was not enough material for definitive mass spectral analysis. It is likely that a mixture of compounds is involved in the response. The polar attractant was unlike any of the previously studied Drosophila pheromone fractions in that those from both sexes were highly active, instead of only those from males. It is possible that this activity is related to the diet medium rather than being pheromonal, since the flies could pick up compounds from their food. A CH2C12 rinse of diet medium, which was never exposed to Drosophila, was similarly attractive in bioassay, and when it was evaporated through Tenax, the rinse from the Tenax column was also active (Table 4). The involvement of the volatile attractants in the ecology of this fly species will require additional research.
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BARTELT ET AL.
TABLE 4. VOLATILE ATTRACTANT IN FLY-DERIVED POLAR FRACTION AND EXTRACT OF REARING MEDIUM Mean bioassay catch"
D. mulleri Treatments compared
polar fractionI'
A. Loss of activity upon evaporation of solvent from samplea Original sample 35.1a (N = 16) Evaporated, reconstituted 7.3b Solvent control 1.6c B. Transfer of activity to Tenax as sample evaporated" Original sample 25.2a (N = 8) Rinse of Tenax trap 26. la Solvent control 3.4b
CH2CI2 extract of rearing medium
22.1a (N = 4) 5.0b 1.0c
21.4a (N = 8) 13.9a 0.9b
"Each group of three means represents a balanced incomplete block experiment. Values followed by the same letter not significantly different (LSD, 0.05). hResults for fractions from males and females nearly identical; combined data presented. "Rearing medium inoculated with yeast, but no Drosophila present. aSamples evaporated gently under stream of N,; samples reconstituted with fresh solvent immediately after going to dryness. "Volatiles from evaporating sample passed through Tenax trap; collection terminated when sample went to dryness. Tenax trap rinsed with pentane.
D. mulleri is like the o t h e r , p r e v i o u s l y s t u d i e d Drosophila s p e c i e s in that a t t r a c t a n t s exist that are f o u n d o n l y in the m a t u r e m a l e s . D. mulleri is s i m i l a r to D. hydei (also o f the repleta g r o u p ) in that b o t h s p e c i e s h a v e e s t e r a n d k e t o n e p h e r o m o n e c o m p o n e n t s . In D. hydei, h o w e v e r , the e s t e r is e t h y l tiglate ( M o a t s et al., 1987), w h i l e D. mulleri u s e s ( S ) - ( + ) - 2 - t r i d e c a n o l a c e t a t e . I n d e e d , the e s t e r o f D. mulleri is p r o b a b l y b i o s y n t h e t i c a l l y related to the k e t o n e c o m p o n e n t o f D. hydei, 2 - t r i d e c a n o n e . Y e t D. mulleri still r e t a i n s a m e t h y l k e t o n e c o m p o n e n t , a l t h o h g h it is f o u r c a r b o n s l a r g e r t h a n that f r o m D. hydei a n d is u n s a t urated. Acknowledgments--This research was supported by NSF grant DCB-8509976 and is published as paper No. J-2062 in the Montana Agricultural Experiment Station Journal Series. REFERENCES BARTELT, R.J., and JACKSON, L.L. 1984. Hydrocarbon component of the Drosophila virilis (Diptera: Drosophilidae)aggregation pheromone: (Z)-10-heneicosene. Ann. Entomol. Soc. Am. 77:364-371.
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BARTELT, R.J., SCHANER, A.M., and JACKSON,L.L. 1986. Aggregation pheromones in five taxa of the Drosophila virilis species group. Physiol. Entomol. 11:367-376. BARTELT, R.J., SCHANER, A.M., and JACKSON,L.L. 1988. Aggregation pheromones in Drosophila borealis and Drosophila littoralis. J. Chem. Ecol. 14:1319-1327. HEATH, R.R., and Sonnet, P.E. 1980. Technique for in situ coating of Ag + onto silica gel in HPLC columns for the separation of geometrical isomers. J. Liq. Chromatogr. 3:1129-1135. MIYASHITA, M., YOSHIKOSHI, A., and GRIECO, P.A. 1977. Pyridinium p-toluenesulfonate: A mild and efficient catalyst for the tetrahydropyranylation of alcohols. J. Org. Chem. 42:3772-3774. MOATS, R.A.. BARTELT, R.J., JACKSON, L.L., and SCHANER, A.M. 1987. Ester and ketone components of the aggregation pheromone of Drosophila hydei. J. Chem. Ecol. 13:451-462. MoRI, K. 1976. Synthesis of optically active forms of ipsenol, the pheromone of lps bark beetles. Tetrahedron 32: I 10 I- I 106. MORt, K. 1985. Chiral synthesis: Examples in the pheromone field, pp. 293-306, in N.F. Janes (ed.). Recent Advances in the Chemistry of Insect Control. Royal Society of Chemists, London. PICKARD, R.H., and KENYON, J. 1914. Investigations on the dependence of rotatory power on chemical constitution. Part V. The simpler esters of the carbinols CH3-CH(OH)-R. J. Chem. Soc. 105:830-898. SCHANER, A.M., BARTELT, R.J., and JACKSON, L.L. 1987. (Z)-ll-Octadecenyl acetate as an aggregation pheromone in Drosophila simulans. J. Chem. Ecol. 13:1777-1786. SLESSOR, K.N., KING, G.G.S., MILLER, D.R., W~NSTON, M.L., and CUTFORTH, T.L. 1985. Determination of chirality of alcohol or latent alcohol semiochemicals in individual insects. J. Chem. Ecol. 11:1659-1667. SUGURO, T., and MORI, K. 1979. Synthesis of optically active forms of 10-methyldodecyl acetate, a minor component of the pheromone complex of the smaller tea tortrix moth. Agric. Biol. Chem. 43:869-870. YATES, F. 1940. The recovery of interblock information in balanced incomplete block designs. Ann. Eugen. 10:317-325.
A G G R E G A T I O N P H E R O M O N E C O M P O N E N T S IN
Drosophila multeri A Chiral Ester and an Unsaturated Ketone
ROBERT J. BARTELT, j ANGELA M. SCHANER, and LARRY L. JACKSON Department of Chemistr3, Montana State University Bozeman, Montana 59717 (Received October 19, 1987; accepted December 2I, 1987)
Abstract--Existence of an aggregation pheromone was demonstrated in Drosophila mulleri. Mature males produce at least two compounds that are lacking from females and newly emerged males and that attract both males and females in a wind-tunnel bioassay. These compounds are (S)-(+)-2-tridecanol acetate and (Z)-10-heptadecen-2-one. Both were synthesized, and the flies responded to the synthetic compounds as well as to fly-derived preparations. The flies also responded to racemic 2-tridecanol acetate but not to the pure R enantiomer. A more polar, very volatile attractant was also extracted from both sexes of D. mulleri but was not identified. Key Words--Drosophila mulleri, Diptera, Drosophilidae, aggregation, pheromone, chirality, enantiomers, ester, ketone, (S)-(+)-2-tridecanol acetate, (Z)- 10-heptadecen-2-one.
INTRODUCTION
Aggregation pheromones have been demonstrated in a number of species of the genus Drosophila (Diptera: Drosophilidae) (Moats et al., 1987; Schaner et al., 1987; Bartelt et al., 1986, 1987; and references therein). In these species, sexually mature males possess compounds that attract both males and females in a laboratory wind-tunnel bioassay. These compounds are lacking in females and in newly emerged males. The pheromone components include hydrocarbons, ~Present address: USDA-ARS, Northern Regional Research Center, Peoria, Illinois 61604.
399 0098-0331/89/0100-0399506.00/0 ~' 1989PlenumPublishingCorporation
400
BARTELT ET AL.
esters, and ketones, and mixtures of compounds are often required for optimal responses. Volatiles from fermenting food media usually synergize the pheromones. Research on pheromones of Drosophila species was continued with D. mulleri Patterson. A comparison between D. mulleri and D. hydei (see Moats et al., 1987) was of special interest because both belong to the same (repleta) species group. D. mulleri was found to possess pheromone components not previously encountered in other Drosophila.
METHODS AND MATERIALS
Flies, Extracts, and Bioassays. The D. mulleri culture was obtained from the National Drosophila Resource Center, in Bowling Green, Ohio (stock number 15081-1371). The flies were reared on Formula 4-24 Instant Drosophila Medium (Carolina Biological Supply, Burlington, North Carolina). The age of maturity of male and female flies was determined as described by Moats et al. (1987), so that appropriate ages for extraction and bioassay could be chosen. We found that males ofD. mulleri become mature at 5-6 days and females at 2-3 days. In other Drosophila species we have studied, pheromone production begins in males as sexual maturity is approached, but flies of any age have usually responded to the pheromones. Thus, to obtain pheromone, flies were not extracted until 6-7 days of age, but 1- to 4-day-old flies were used for most bioassays because the immature males would be less likely to possess pheromones that could compete with bioassay treatments. Flies to be extracted were segregated by sex within one day of emergence and extracted by soaking them overnight in hexane at room temperature. Bioassays were performed in a wind-tunnel olfactometer essentially as described earlier (Bartelt and Jackson, 1984). About 16 hr before bioassay tests began, ca. 1000 flies were added to the olfactometer. As with the previously studied species, a starvation period was necessary before flies would respond to pheromones. Each bioassay treatment was applied to a filter paper strip lining the lip of a glass vial. A drop of water was added to the bottom of each bioassay vial to arrest responding flies (Bartelt and Jackson, 1984). Two such vials, with treatments to be compared, were placed in the upwind end of the olfactometer. After 3 min the vials were capped, and the captured flies were counted and, in selected experiments, sexed. Tests were performed throughout the day at ca. 10-min intervals. In experiments with more than two treatments, they were tested in all possible pairs (a balanced incomplete block design). Statistical analysis followed the method of Yates (1940) and was done on data transformed as log(X + 1) to stabilize variance. Chromatography and Spectra. Crude hexane extracts were chromato-
Drosophila mulleri AGGREGATIONPHEROMONE
401
graphed initially on open columns of silicic acid and eluted with two void volumes of each of the following solvents: hexane; 5%, 10%, and 50% ether in hexane; and 10% methanol in methylene chloride. All gas chromatography (GC) was conducted on a Varian 3700 using He as the carrier gas. For preparative GC, a 1.3-m x 4-mm 5% SE-52 column was used, and the effluent was monitored with a thermal conductivity detector. Three columns were utilized for capillary GC: a 30-m x 0.25-mm DB-1, a 15m x 0.32-mm DB-5, and a 30-m x 0.25-mm DB-225 (J & W Scientific, Rancho Cordova, California). Peaks were monitored with a flame ionization detector. Various temperature programs were used, as described below. High-performance liquid chromatography (HPLC) was conducted isocratically with a Waters 6000A pump and a Waters 401 differential refractometer. Columns included a 25-cm x 4.6-ram ID Adsorbosphere silicic acid column (Applied Science, Deerfield, Illinois), eluted with 5% ether in hexane, and a 25-cm x 4.6-mm ID silicic acid column, coated with AgNO3 as described by Heath and Sonnet (1980), and eluted with toluene. Electron impact mass spectra were obtained on a VG-MMI6F mass spectrometer. Samples were introduced through a capillary GC column. Synthetic Esters. (+)-2-Tridecanol acetate was prepared by treating dodecanal with methylmagnesium bromide in ether, followed by acetylation of the resulting alcohol with acetic anhydride in pyridine. After chromatography on an open column of silicic acid and preparative GC, the ester was > 9 8 % pure by capillary GC. The ester was diluted with hexane to 10 ng/t~l for bioassays. A portion of the (+)-2-tridecanol was set aside for chromatographic studies. (S)-2-Tridecanol was synthesized (Figure 1) from a commercially available, optically active starting material, ethyl (S)-lactate (I) (Sigma Chemical Co., St. Louis, Missouri). Synthetic steps were performed as described in the literature for analogous reactions: After conversion to a tetrahydropyranyl (THP) ether (II) (Miyashita et al., 1977), the ester function was reduced with lithium aluminum hydride (Moil, 1976), and the resulting primary alcohol (III) was esterified with p-toluenesulfonyl chloride to form the tosylate (IV) (Moil, 1976). Decylmagnesium bromide was linked to this tosylate in the presence of dilithium tetrachlorocuprate to form the THP ether of (S)-2-tildecanol (V) (Suguro and Moil, 1979). Finally, the tetrahydropyranyl protecting group was removed (Miyashita et al., 1977). The crude (S)-2-tridecanol (VI) crystallized at ca. - 1 0 ° C from a 20% solution in pentane. By GC, the purity of the recovered product was 88%. All reactions were monitored by GC and mass spectrometry. All were essentially quantitative except for the alkylation of the tosylate, for which the yield was ca. 50%. Intermediate products were not purified before subsequent reactions, except to dry (Na2SO4) and to remove solvents.
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Drosophila mulleri AGGREGATIONPHEROMONE
403
(R)-2-Tridecanol was prepared in an identical manner except that the starting material was methyl (R)-lactate (Sigma). (S)-2-Tridecanol was either converted to an acetate for bioassays or was derivatized as described below for GC studies of optical purity and stereochemical configuration. The acetate (Figure 1, VII) was formed by treatment with acetic anhydride in pyridine. After purification by chromatography on silicic acid and preparative GC, the ester's purity was > 9 9 % , by capillary GC. Approximately 10 mg of pure ester were prepared. The ester had an identical mass spectrum and GC retention (DB-1, DB-5) as the racemic synthetic (+_)2-tridecanol acetate. The specific rotation was determined: [O~]D 3 = +4.6 ° (C = 0.57%, hexane) (literature value: [~]~o = +4.63 o, absolute configuration not given, Pickard and Kenyon, 1914). (R)-(-)-2-Tridecanol acetate was likewise prepared from the (R)-2-tridecanol. The measured purity of the ester was > 99 % and the optical rotation, [O/]D3 ~--- - 4 . 9 ° (e = 0.57%, hexane). GC retention and mass spectrum were as for the S isomer. Both enantiomeric esters were diluted with hexane for bioassays. Studies of Optical Purity and Absolute Configuration. Diastereomeric esters (Figure 1, VIII) were formed from the synthetic 2-tridecanols so that optical purity could be analyzed on an achiral GC column. The (S)-(2-acetyloxy)propanoates (acetyl lactates) were prepared essentially as described by Slessor et al. (1985). The derivatizing reagent was (S)-(2-acetyloxy)propanoyl chloride (APC). GC analysis was on the DB-5 capillary column, temperature programmed from 120°C to 150°C at 5°C/min. To derivatize the 2-tridecanol acetate from D. mulleri, the ester was first reduced with LiAIH4. To ca. 10 #g of purified D. mulIeri ester in 20/xl ether was added 15 /zl LiAIH4 reagent (1.0 M in ether). After 10 min at room temperature, the vial was cooled over ice and 2 drops of H20 were added to decompose excess reagent. The organic layer was transferred to a clean vial and stripped to dryness under N2; then the alcohol was derivatized with APC. Synthesis ofKetones. (Z)-9-Hexadecenoic acid (50 mg, 0.2 mmol, Sigma) in 2 ml dry ether was cooled to 0°C. Methyllithium in ether (0.8 mmol, 0.6 ml) was added in one portion. The mixture was warmed to room temperature, refluxed for 20 min, then cooled to 0°C, after which 1 ml water was added very slowly. The ether layer was washed with dilute HC1, water, and NaHCO3 solution, then dried over Na2SO4. The resulting (Z)-10-heptadecen-2-one was purified on an open column of silicic acid and diluted with hexane for bioassay. The purity was 9 9 + % by capillary GC. 2-Heptadecanone was similarly prepared in 99 + % purity from palmitic (hexadecanoic) acid. Preliminary Experiments with Volatile, Polar Attractants. Apparatus was assembled to allow evaporation of a sample (up to 500/zl) under a gentle stream of nitrogen, so that the vapors passed through a 2.5 x 25-mm column of 20/
404
BARTELT ET AL.
35 mesh Tenax porous polymer (Applied Science). Both the fly-derived 10% MeOH-CH2CI 2 fraction and a CHzCI2 rinse of rearing medium (inoculated with yeast but without Drosophila) were treated in this way. Each sample was reconstituted with an equal volume of fresh solvent immediately as the sample went to dryness, and the Tenax column was eluted with an equivalent volume of pentane. The reconstituted samples and Tenax column rinses were compared to the original samples by bioassay. The Tenax column rinses were examined by GC and mass spectrometry.
RESULTS AND DISCUSSION
Bioassay Characteristics. When the flies were first put into the olfactometer, they formed tight aggregations low on the sides and moved about very little. After starvation for 14-16 hr at a temperature of 24-25°C, these groups dispersed and the flies became more active, usually with ca. 20-30 flies in flight at any given time. Response to an active treatment was usually by an upwind, zigzag, hovering flight, followed by alighting on the vial. Bioassays of Crude Extracts and Fractions. The crude hexane extract of mature male D. mulleri was active in the bioassay (Table 1A), and three fractions derived from this extract were significantly active. Based on standards, the first (eluted with 5% ether-hexane) had the polarity of esters while the second (eluted with 10% ether-hexane) had the polarity of ketones. The last was considerably more polar (eluted with 10% methanol-methylene chloride). Surprisingly, the female-derived extract was also very active (Table IB), and this activity was located in the most polar fraction. Thus D. mulleri was like the previously studied species in that mature males had attractive nonpolar compounds that were lacking in mature females, but D. mulleri was unlike the other species in that very active, relatively polar compounds(s) were present in both sexes. Further purification of the male-derived 5% and 10% ether-hexane fractions was accomplished by preparative GC. The activity from the 5 % etherhexane fraction was retained slightly longer than an alkane of 16 carbons on the nonpolar preparative GC column, and the activity from the 10% etherhexane fraction was similar in retention to an alkane of 19 carbons. No other GC fractions had significant bioassay activity. Subsequent HPLC on silicic acid produced only one active region from each GC fraction. The activity originally from the 5 % ether-hexane silicic acid fraction eluted 5.5-6.5 ml after injection, while that from the 10% ether-hexane fraction eluted at 10-11 ml. Based on standards, these retentions supported that the activity in the former fraction was due to an ester, and the latter, a ketone. Ester Component. After preparative GC and HPLC, the active ester corn-
Drosophila mulleri AGGREGATION PHEROMONE
405
TABLE 1. ACTIVITY OF SILICIC ACID FRACTIONS RELATIVE TO CONTROLS AND TO MALE-DER/VED CRUDE EXTRACT
Mean bioassay catch (N -> 8)I'
Treatment"
Treatment
Control
Male-derived crude extract
A. Male-derived fractions Hexane 5% Ether-hexane 10% Ether-hexane 50% Ether-hexane 10% Methanol-methylene chloride
8.5 13.6"** 8.0*** 5.5 23.0***
8.7 3.8 2.8 6.0 1.0
57.5*** 23.4*** 33.4*** 49. I *** 23.8***
B. Female-derived extract and fractions Crude hexane extract Hexane fraction 5% Ether-hexane 10% Ether-hexane 50% Ether-hexane 10% Methanol-methylene chloride
29.1 *** 4.4 3,2 6.3 2.3 27.5***
5.0 4.0 4.2 4.4 4.3 2.9
21.7*** 52.2*** 40.9*** 42.6*** 22.9*** 22.0***
"One fly equivalent per test, Each row is a balanced incomplete block experiment, in which the test treatment, the control, and the male-derived crude extract are tested in pairs, in all possible combinations. /'In each row, means followed by *** are significantly different from the control at the 0.001 level (t tests). No other differences were significant at even the 0.05 level.
ponent was > 98% pure, by capillary GC. Mature males possessed ca. 300 ng of this compound, but it was lacking in females and newly emerged males. In the mass spectrum, no molecular ion was seen, but fragments at m/z 87 (40%) and 182 (15%, M-60) suggested an acetate of the secondary alcohol, 2-tridecanol. The base peak was m/z 43, and peaks also occurred at 198 and 199 (both ca. 1%, M-44 and M-43, respectively). Racemic 2-tridecanol acetate and the D. mulleri compound had identical GC retention times (DB-1) and mass spectra. Both reacted with methanolic KOH, resulting in a GC peak with the same retention as authentic 2-tridecanol. Racemic 2-tridecanol acetate was active in the bioassay (Table 2A). The fly-derived 2-tridecanol acetate, having one asymmetric center, was analyzed for optical purity and absolute configuration (summarized in Figure 2). The two diastereomers derived from racemic 2-tridecanol were easily separated on the DB-5 capillary column. Based on the synthetic standards, the first of these peaks (Kovats index = 1966) corresponded to the R configuration, while the peak at KI = 1982 represented the S configuration. The derivative
406
BARTELT ET AL.
TABLE 9_. KEY BIOASSAY RESULTS RELATING TO IDENTITIES OF NONPOLAR PHEROMONE COMPONENTS
Treatment
Mean bioassay catch"
A. Preliminary identification of ester component (N = 74). (+)-2-Tridecanol acetate (50 ng) Control
26.5a 4.9b
B. Determination of active enantiomer (N = 40). (S)-(+)-2-Tridecanol acetate (25 ng) (R)-(-)-2-Tridecanol acetate (25 ng) Control
22.9a 5.6b 6.3b
C. Comparison of male-derived "ester" fraction and synthetic ester (N = |2). (Both treatments had 300 ng 2-tridecanol acetate per test). (S)-( + )-2-Tridecanol acetate Male-derived 5% ether-hexane fraction Control
14.2a 14.2a 3.1b
D. Comparison of male-derived "'ketone" fraction and synthetic ketone (N = 16). [Both treatments had 0.7 ng (Z)-10-heptadecen-2-one per test.] (Z)- 10-Heptadecen-2-one 10% Ether-hexane fraction Control
14. I a 19.9a 2.9b
E. Activity of two 17-carbon ketones, alone and in combination (N = 12). (Each ketone used at 8 ng per test) (Z)- 10-Heptadecen-2-one 2-Heptadecanone (Z)-10-Heptadecen-2-one + 2-heptadecanone Control
14.9b 4.9c 25.4a 4.4c
"In each experiment, means followed by the same letter not significantly different at the 0.05 level (LSD method).
prepared f r o m the D. mulleri p h e r o m o n e had a Kovats index o f 1982 and a high degree o f optical purity (enantiomeric e x c e s s calculated from peak integrations was 9 8 . 7 % ) . By c o m p a r i s o n to the synthetic standards, the D. mulleri pherom o n e has the S configuration. T h e e n a n t i o m e r i c excess represents a m i n i m u m , as the departure f r o m 100% c o u l d be due to optical impurity o f the derivatizing reagent a n d / o r slight racemization o c c u r r i n g during c h e m i c a l procedures. D. mulleri r e s p o n d e d in the bioassay only to the e n a n t i o m e r o f 2-tridecanol acetate which they produce (Table 2B). T h e S i s o m e r was clearly active, w h i l e the R i s o m e r was not. That the R i s o m e r was not repellent was consistent with
Drosophila mulleri AGGREGATIONPHEROMONE C'q IX3 03 t.O r-tD O~ v-.
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RETENTION TIME (MIN) FIG. 2. GC analysis of optical purity and absolute configuration of the 2-tridecanol acetate produced by D. mulleri. Shaded peaks represent (S)-2-(acetyloxy)propanoate derivatives of synthetic and fly-derived 2-tridecanol. Kovats indices are indicated at peak apices. The peaks at ca. 4.3 and 5.8 min are n-alkanes of 19 and 20 carbons, respectively, coinjected as retention standards. The DB-5 capillary column was 120°C initially and was increased at 5°C/min.
the activity of the racemic synthetic ester. (S)-2-Tildecanol acetate adequately accounts for the activity of the male-derived 5 % ether-hexane fraction (Table 2C). In this experiment both treatments had equivalent amounts of the ester. Chiral pheromones have been demonstrated in a number of insect groups. One common pattern is that insects respond only to the optical isomer that they produce but that the other enantiomer is not repellent (Moil, 1985), a pattern which has now been demonstrated in the Drosophila also. Ketone Component. The active "ketone" fraction, after preparative GC
408
BARTELT ET AL.
and HPLC on silicic acid, contained two peaks, by capillary GC. Both were small peaks, representing ca. 1 ng/mature male. The latter of the two peaks was identical in GC retention and mass spectrum with 2-heptadecanone [key spectral peaks were at m/z 43 (100%), 58 (80%), and 254 (4%)]. The other peak eluted slightly earlier on the nonpolar DB-1 capillary column, and its mass spectrum indicated a molecular weight of 252 (2%), 2 mass units less than 2heptadecanone. Fragments still appeared at m/z 43 (100%) and 58 (26%); thus an unsaturated methyl ketone seemed likely. (Z)-10-Heptadecen-2-one was synthesized as a standard for comparative purposes. This ketone was accessible in one step from the readily available (Z)-9-hexadecenoic (palmitoleic) acid. Fortuitously, this ketone had the same retention on three GC columns as the D. mulleri ketone (KI = 1860, 1873, and 2243 on DB-1, DB-5, and DB-225, respectively), and the mass spectra and retentions on the AgNO3 HPLC column were identical also. Since these chromatographic methods are sensitive to double-bond position and configuration, the data strongly supported (Z)-10-heptadecen-2-one as the structure of the unknown compound. Because of the minute amount of ketone in each male, confirmation of double bond location by ozonolysis was not attempted. (Z)-10-Heptadecen-2-one was active in bioassay and accounts fairly well for the activity of the male-derived 10% ether-hexane fraction (Table 2D). 2Heptadecanone, also found in D. mulleri males, was not active in the bioassay by itself, although a mixture of 2-heptadecanone + (Z)-10-heptadecen-2-one was somewhat more active than the unsaturated ketone alone (Table 2E). The extracts were subsequently examined for 2-tridecanone and 2-pentadecanone, pheromone components in the related species, D. hydei (Moats et al., 1987). Traces of both ketones were found (2-4 ng/mature male), but the synthetic ketones were not active in the bioassay. None of the ketones were detected in extracts of female flies.
Synergistic Activity of (S )-(+ )-2-Tridecanol Acetate and (Z )-lO-Heptadecen-2-one. Dose-response data for various mixtures of (S)-2-tridecanol acetate and (Z)-10-heptadecen-2-one are given in Table 3. The response generally increased with increases in either component, although there was little difference between the 300- and 3000-ng levels of the ester. On a weight-for-weight basis, the ketone was the more active compound. For example, 10 ng of ketone elicited a stronger response than even 3000 ng of ester. Response by Sex. Both sexes responded readily to all preparations tested. For example, in a paired comparison experiment with the crude hexane extract of mature males (1 equivalent), (S)-2-tridecanol acetate (300 ng), (Z)-10-heptadecen-2-one (10 ng), and controls, the mean captures (and percent females) for the treatments were 38.7 (45%), 11.8 (47%), 29.5 (45%), and 2.0 (67%), respectively (N = 6). Polar Attractant. Despite concerted efforts, the polar attractant(s) in the
Drosophila multeri AGGREGATIONPHEROMONE
409
TABLE 3. RELATIVEACTIVITIESFOR VARIOUS MIXTURESOF (S)-(+)-2-TRIDECANOL ACETATEAND (Z)- 10-HEPTADECEN-2-ONEa Amount of (Z)10-heptadecen-2one (ng) 0.0 0.1 1
10 100
Amount of ( S)-( +)-2-tridecanol acetate (ng) 0
3
30
300
3000
0h 3~ 14 92 258
8 28 39 151 364
19 35 58 170 387
50 74 100a 226 504
57 58 140 294 555
"Each test mixture compared to solvent controls and the standard mixture (300 ng (S)-(+)-2tridecanol acetate plus 1 ng (Z)-10-heptadecen-2-one)in a balanced incompleteblock experiment (N = 8). Relative activity (RA) based on means from these experiments: RA = 100 x (test mixture - control)/(standardmixture - control). "By definition, RA for control = 0. Overall, the mean bioassaycatch for controls was 4.5 flies per 3-min test. 'All samples except this one attracted significantlymore flies than the control (P < 0.01). aBy definition. RA for standard mixture (ca. 1 male equivalent)was 100. Overall, the mean bioassay catch for this standard was 34.8 flies per 3-min test.
10% MeOH-CHzC12 fraction was (were) not identified. The activity was extremely volatile, compared to the ester and ketone pheromone components. Evaporating the polar fraction carefully to dryness under N2 and immediately reconstituting the fraction with fresh solvent resulted in the loss of ca. 80% of the bioassay activity (Table 4). When the vapors from the evaporating fraction were directed through a column of Tenax, the activity could be recovered by rinsing the column with pentane. (Evaporation of control solvent through the Tenax trap did not produce a trap rinse that was active in bioassay). Examination of the active trap rinse by GC revealed many minute peaks, but there was little consistency among runs and there was not enough material for definitive mass spectral analysis. It is likely that a mixture of compounds is involved in the response. The polar attractant was unlike any of the previously studied Drosophila pheromone fractions in that those from both sexes were highly active, instead of only those from males. It is possible that this activity is related to the diet medium rather than being pheromonal, since the flies could pick up compounds from their food. A CH2C12 rinse of diet medium, which was never exposed to Drosophila, was similarly attractive in bioassay, and when it was evaporated through Tenax, the rinse from the Tenax column was also active (Table 4). The involvement of the volatile attractants in the ecology of this fly species will require additional research.
410
BARTELT ET AL.
TABLE 4. VOLATILE ATTRACTANT IN FLY-DERIVED POLAR FRACTION AND EXTRACT OF REARING MEDIUM Mean bioassay catch"
D. mulleri Treatments compared
polar fractionI'
A. Loss of activity upon evaporation of solvent from samplea Original sample 35.1a (N = 16) Evaporated, reconstituted 7.3b Solvent control 1.6c B. Transfer of activity to Tenax as sample evaporated" Original sample 25.2a (N = 8) Rinse of Tenax trap 26. la Solvent control 3.4b
CH2CI2 extract of rearing medium
22.1a (N = 4) 5.0b 1.0c
21.4a (N = 8) 13.9a 0.9b
"Each group of three means represents a balanced incomplete block experiment. Values followed by the same letter not significantly different (LSD, 0.05). hResults for fractions from males and females nearly identical; combined data presented. "Rearing medium inoculated with yeast, but no Drosophila present. aSamples evaporated gently under stream of N,; samples reconstituted with fresh solvent immediately after going to dryness. "Volatiles from evaporating sample passed through Tenax trap; collection terminated when sample went to dryness. Tenax trap rinsed with pentane.
D. mulleri is like the o t h e r , p r e v i o u s l y s t u d i e d Drosophila s p e c i e s in that a t t r a c t a n t s exist that are f o u n d o n l y in the m a t u r e m a l e s . D. mulleri is s i m i l a r to D. hydei (also o f the repleta g r o u p ) in that b o t h s p e c i e s h a v e e s t e r a n d k e t o n e p h e r o m o n e c o m p o n e n t s . In D. hydei, h o w e v e r , the e s t e r is e t h y l tiglate ( M o a t s et al., 1987), w h i l e D. mulleri u s e s ( S ) - ( + ) - 2 - t r i d e c a n o l a c e t a t e . I n d e e d , the e s t e r o f D. mulleri is p r o b a b l y b i o s y n t h e t i c a l l y related to the k e t o n e c o m p o n e n t o f D. hydei, 2 - t r i d e c a n o n e . Y e t D. mulleri still r e t a i n s a m e t h y l k e t o n e c o m p o n e n t , a l t h o h g h it is f o u r c a r b o n s l a r g e r t h a n that f r o m D. hydei a n d is u n s a t urated. Acknowledgments--This research was supported by NSF grant DCB-8509976 and is published as paper No. J-2062 in the Montana Agricultural Experiment Station Journal Series. REFERENCES BARTELT, R.J., and JACKSON, L.L. 1984. Hydrocarbon component of the Drosophila virilis (Diptera: Drosophilidae)aggregation pheromone: (Z)-10-heneicosene. Ann. Entomol. Soc. Am. 77:364-371.
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