Journal of Chemical Ecology, Vol. 15, No. 1, 1989
(Z)-ll-EICOSENYL ACETATE, AN AGGREGATION PHEROMONE IN Drosophila malerkotliana
ANGELA
M . S C H A N E R , L A R R Y L. J A C K S O N , a n d R U S S E L L D. L E U
K A T E J. G R A H A M ,
Biochemistry Group Chenffstry Department Montana State UniversitT Bozeman, Montana 59717
(Received August 3. 1987; accepted November 16, 1987) Abstract--(Z)-ll-Eicosenyl acetate (Zll-20:Ac) was identified as the aggregation pheromone in Drosophila malerkotliana. The pheromone (200300 ng/fly) was isolated from hexane extracts of the ejaculatory bulb of sexually mature male flies. Males released very little, if any, Z11-20: Ac to the food at any time. During mating there was a transfer of ca. 100 ng of ZI 120:Ac to the female's reproductive tract. The mated female fly transferred the ZI 1-20: Ac to the surrounding surfaces in just a few hours after mating. In bioassay in a wind-tunnel olfactometer, ZI 1-20:Ac was not attractive alone, but was synergistic with fermenting food or with acetone. Although D. malerkotliana has no (Z)- 1 l-octadecenyl acetate (Z 11-18 : Ac), it was as attracted to Z 11-18 : Ac as to an equal quantity of Z11-20 : Ac. D. melanogaster and D. simulans, however, responded to the ZI 1-18:Ac that they produced and did not respond to Z I 1-20: Ac. Key Words--Drosophila malerkotliana, Diptera, Drosophilidae, aggregation pheromone, (Z)- 1 l-eicosenyl acetate.
INTRODUCTION A g g r e g a t i o n p h e r o m o n e s h a v e b e e n d e m o n s t r a t e d in 11 species o f Drosophila: D. virilis ( B a r t e l t a n d J a c k s o n , 1984; B a r t e l t et al., 1985a); D. a. americana, D. a. texana, D. novamexicana, a n d D. lummei (Bartelt et al., 1986); D. borealis a n d D. littoralis ( B a r t e l t et al., 1988); D. hydei ( M o a t s et al., 1987); D. melanogaster ( B a r t e l t et al., 1985b), D. simulans ( S c h a n e r et al,, 1987); and D. mulleri ( B a r t e l t et al., 1989). In m a n y o f the c l o s e l y related s p e c i e s , the 265 0098-0331/8910100-0265$06.00/0 ~) 1989 Plenum Publishing Corporation
26 6
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aggregation pheromones had the same or similar chemical structures, but between some species groups there appeared to be considerable differences in the chemical structures of the aggregation pheromones. D. melanogaster and D. simulans of the melanogaster species group and melanogaster subgroup both used (Z)-I 1-octadecenyl acetate (Z11-18 : Ac, cisvaccenyl acetate, cVA) as their aggregation pheromone (Bartelt et al., 1985b; Schaner et al., 1987). No other fly-derived coattractant was necessary, but food or food-derived odors were synergistic with the Z11-18 : Ac. In D. melanogaster and D. simulans the Z11-18 : Ac was stored in the ejaculatory bulb of sexually mature male flies, transferred to females during mating, and released by mated female flies to the food media within hours after mating. D. malerkotliana was chosen for aggregation pheromone investigation because it was in the melanogaster group but in the ananassae subgroup, and we could test whether male-derived compound(s) could function as an aggregation pheromone and whether the compound(s) were the same or differed from the Z 11-18 : Ac used by members of the melanogaster subgroup.
METHODS AND MATERIALS
Flies, Extraction, Chromatography, and Bioassay. D. malerkotliana malerkotliana (strain 14024-0391.0) was obtained from the National Drosophila Species Resource Center at Bowling Green, Ohio. It was originally collected at Mysore, India. D. melanogaster (wild type, Canton S strain) and D. simulans (strain 14021-0251.0) were as reported previously (Bartelt et al., 1985b; Schaher et al., 1987). Details of rearing, extraction, chromatography, and bioassay of the flies have been described previously (Bartelt and Jackson, 1984, Bartelt et al., 1985b; Schaner et al., 1987). Briefly, flies were separated by sex at 0-6 hr old and extracted at 6-7 days of age by soaking the flies in hexane at room temperature for 24 hr. Fractionation of the hexane extracts was on open columns of silicic acid eluted with hexane, 10% ether in hexane, 50% ether in hexane, and 10% methanol in methylene chloride. The 10% ether in hexane fraction was purified further by preparative gas chromatography (GC). Bioassays were conducted in a wind-tunnel olfactometer containing ca. 1000 (0- to 2-day-old) flies that had been without food and water for approximately 2 hr. An extract, fraction, synthetic compound, or control solvent was applied to a filter paper strip inserted around the lip of the glass vial. Each bioassay test consisted of placing two differently treated vials, to be compared, into the olfactometer for 3 min. The bioassay data was transformed to the log (x + 1) scale before analysis to stabilize variance and analysis was done by the method of Yates' (1940). Identification. Identification of the pheromone was done by comparing
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retention times on GC, by gas chromatography-mass spectrometry (GC-MS) of the pheromone on a VG-MM16 GC-MS and by GC-MS of the dimethyl disulfide reaction product (Nichols et al., 1986). The determination of the isomer of the double-bond was done by comparison of synthetic Z 1 1 - 2 0 : A c (Sigma Chemical, St. Louis, Missouri) and the fly-derived I 1-20:Ac on a 30-m DB1 capillary GC column with a temperature program of 140-220°C with a 4-min initial hold and a rate of 4°C/min. With this method the Z and E isomers of 918 : Ac had previously been separated by a difference in the retention times of 0.11 min. The isomer of the fly-derived 11-20 : Ac was determined by a coinjection of this compound with the synthetic Z 11-20 : Ac. Production of Z11-20:Ac with Age. Since Z 11-18:Ac was found in the ejaculatory bulb in D. melanogaster (Brieger and Butterworth, 1970), the ejaculatory bulbs of D. malerkotliana were removed, extracted with hexane, and analyzed by GC. To remove ejaculatory bulbs, the males were placed in the freezer for approximately 10 min and then placed, ventral side up, on a cork board with a dissecting pin through the thorax. The genital region was teased loose with forceps and placed in a drop of Drosophila Ringer's solution (Ephrussi and Beadle, 1936). Using two dissecting pins, the ejaculatory bulb was removed from the genital region under 45 x magnification. Two sets of five bulbs for age groups from 2 hr to 8 days were placed in 1-ml conical vials that contained 500 ng nonadecane as an internal standard in 10 #1 of hexane. The bulbs were smashed with the head of a dissecting pin and the hexane extract analyzed by GC. For comparison, we also analyzed hexane extracts of whole flies (3 × 20 flies) at each age. The flies were extracted at room temperature for 24 hr, and the hexane extracts were fractionated on open silicic acid columns described above. Z 11-20 : Ac was quantified by GC relative to the internal standard, Z11-16: Ac. Transfer during Mating. The transfer of Z 11-20 : Ac from males to females and the subsequent release of the ester from the females was investigated. Virgin females (6-7 days old) were mated with virgin males of the same age. Immediately after completion of mating, the females were either extracted or placed into an empty 8-ml vial for 6 hr and then extracted. The vials were rinsed with I ml of hexane to recover any Z 11-20 : Ac that had been released. The mated males were treated in a similar fashion and, as controls, comparable sets of extracts were obtained from males and females that had not been allowed to mate. Each extract represented 10 fly equivalents and there were four replications per extract. The Z 11-20 : Ac in the extracts was quantitated by GC relative to an internal standard. Female Reproductive Tract Removal. The female reproductive tract was also removed and the extract analyzed for the presence of Z11-20 : Ac. Immediately upon completion of mating, the female was placed in the freezer for approximately 10 min. Viewed through a 10 × dissecting scope, the female was
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held ventral side up with forceps pressed laterally on the abdomen. The ovipositor was clasped with another pair of forceps and teased until the reproductive tract, without the ovaries, was pulled away from the fly. Two sets of five reproductive tracts were placed in conical vials with an intemal standard and the extract analyzed as described previously for the ejaculatory bulbs. The hexane extract of female flies minus the reproductive tracts were analyzed by GC after they were soaked in hexane for 1 hr.
RESULTS AND DISCUSSION
Demonstration, Purification, and Identification of Pheromone. Initial attempts to demonstrate an aggregation pheromone in D. malerkotliana were patterned after previous experiments with D. melanogaster (Bartelt et al., 1985b) and D. simulans (Schaner et al., 1987). The hexane extract of mature males, with a food synergist, was clearly active in bioassay, and the hexane extract of mature females was, with food, also about twice as attractive as the food (Table 1, A). The male fly hexane fraction alone was not attractive. Although food was an effective coattractant, we felt that using a synergist that was less attractive might clarify pheromonal effects and make pheromone identification more rapid. Acetone was the preferred coattractant, which lead to the identification of the aggregation pheromone in D. melanogaster (Bartelt et al., 1985b). Compounds such as acetone and ethyl acetate are known to be produced by molds and yeasts (Fogleman, 1982) and could well influence the behavior of the flies under natural conditions. When acetone was used as a coattractant in D. malerkotliana (Table 1, B), the male fly hexane extract plus acetone was about 10 times more attractive than the female fly hexane extract plus acetone. Acetone alone was not attractive. Both sexes of flies respond nearly equally to the hexane extracts of mature male flies. Since the attractive nature of the male fly extracts was much greater than that of female fly extracts, this paper will concentrate on the male fly-derived pheromone. After fractionation of the hexane extract of male flies on silicic acid, only the 10% ether-hexane fraction was active. The remaining fractions from the extract of male flies were not different in activity from the control solvent. By capillary GC, the male 10% ether-hexane fraction was predominantly one component. GC-MS of the major peak in the male 10% ether-hexane fraction indicated that the component was an acetate ester of a monounsaturated alcohol with 20 carbons. GC-MS of the dimethyl disulfidetreated fraction revealed that the double bond in the 20-carbon alcohol of the ester was at 11. Synthetic (Z)-I 1-eicosenyl acetate (abbreviated Z 11-20 : Ac) compared identically with the fly-derived component in GC and GC-MS. GC comparison of the fly-derived compound and the synthetic Z 11-20 : Ac identified the fly-derived 11-20 : Ac as the Z isomer. Z 11-20 : Ac was present in the
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TABLE 1. FOUR SERIES OF BIOASSAY EXPERIMENTS WITH EXTRACTS OF O.
materkotliana TO ISOLATE AND CHARACTERIZE AGGREGATION PHEROMONE. Mean bioassay catchh
Treatment~ A:
Synergism of food" by hexane extracts of flies Fermenting food Female fly hexane extract + food Male fly hexane extract + food Male fly hexane extract B: Comparison of hexane extracts from mate and female flies with acetone as a coattractant Male fly hexane extract + acetonea Female fly hexane extract + acetone Acetone control C: Test for equivalence of synthetic Z 11-20 : Ac with extracts, fractions and purified compounds Synthetic Z11-20: Ac + acetone Male-derived Z 11-20 : Ac + acetone 10% ether in bexane fraction + acetone Male fly hexane extract + acetone Acetone control D: Synergism between Z I 1-20 : Ac and fermenting food Synthetic Z 11-20 : Ac + food Fermenting food Z 11-20 : Ac Hexane control
(N = 18) 12.3d 26.8c 56.5b 0.7a (N = 8) 13.0b 1.2a 0.4a (N = 16) 11.0c 6.1 b 9.5bc 6.1b 1.0a (N = 24) 73.3c 5.3b 1.2a 0.7a
"All fly-derived fractions and their synthetic counterparts were used at 1 fly equivalent per test (220 ng of Z 11-20 : Ac). ~'In any experiment, means followed by the same letter were not significantly different at the 5% level (LSD). 'Fermenting food is Formula 4-24 Instant Drosophila Medium (Carolina Biological Supply, Burlington, North Carolina) to which active yeast has been added at least 24 hr previous to testing. aAcetone, 10 #1, was added to the filter paper with the extracts.
h e x a n e e x t r a c t o f m a t u r e m a l e flies at a c o n c e n t r a t i o n o f ca. 2 0 0 rig/fly, a n d there was no indication of the presence of Z 11-18:Ac
in t h e e x t r a c t . T h i s is
t h e first r e p o r t o f Z 1 1 - 2 0 : A c as an a g g r e g a t i o n p h e r o m o n e in Drosophila. The male hexane extract, 10% ether-hexane fraction, synthetic Zll20 : A c , a n d m a l e - d e r i v e d Z 1 1 - 2 0 : A c w e r e c o m p a r e d b y b i o a s s a y (all at 2 2 0 ng Z 1 1 - 2 0 : A c ; T a b l e 1, C ) . A l l w e r e s i g n i f i c a n t l y g r e a t e r t h a n t h e c o n t r o l ( P
I-> I.-- 300
> 200 ..J W I00
20
200 ZII-20:AC
2000 (ng)
20000
FiG. 1. Activity of D. malerkotliana towards different doses of Z 11-20 : Ac. Relative activities are 100% for the 200-rig dose of Z l l - 2 0 : A c ( - 1 fly eq) and 0% for the controls (N = 8). In each case, 10 /zl of acetone was used as a coattractant. Spacing along the x axis is logarithmic.
SCHANER ET AL.
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increase in Z 11-20 : Ac. Over the next few days there is a gradual increase to a plateau o f about 300 ng/fly (Figure 2). The assays o f ejaculatory bulb contents are somewhat higher than the 24-hr hexane extracts o f whole flies. The same trend in Z 11-20 : Ac production is observed from the whole fly and ejaculatory bulb extracts. Although the whole fly extracts are much easier to perform, they do not provide a reliable quantitative estimation. Virgin females possess no Z I 1-20 : Ac. However, males transferred over half o f the Z l 1-20 : Ac they possessed to the female during mating. Immediately after mating about 85% o f the male-transferred Z 1 1 - 2 0 : A c is in the female's reproductive tract. Within 6 hr after mating, the female transferred approximately one third o f the Z I 1-20 : Ac into an empty vial. Males, virgin or mated, emitted no Z I 1 - 2 0 : A c into an empty vial that they occupied for 6 hr. Therefore, as in D. melanogaster and D. simulans (Bartelt et al., 1985b; Schaner et al., 1987), it is the mated female that emits the pheromone into the environment although it is male-produced. In summary, sexually mature D. malerkotliana males produce Z 11-20: Ac,
A
~- 3 0 0 E ¢-
c~ 2 0 0
/
(5 cxl !
N
/
~ I00 0 I-Z
0 0
I
I
i
I
I
I
I
I
I
0
I
2
3
4
5
6
7
8
AGE OF ADULT FLIES (days) FIG. 2. Amounts of ZI 1-20 : Ac extracted from virgin males D. malerkotliana of various ages. Solid circles indicate the Z 11-20 : Ac from a 24-hr hexane soak of the whole fly, and open circles indicate the Z I 1-20 : Ac within the hexane extract of ejaculatory bulbs. The bars indicate standard deviation.
AGGREGATION PHEROMONE IN Drosophila
273
w h i c h is s t o r e d in the e j a c u l a t o r y b u l b , t r a n s f e r r e d to the r e p r o d u c t i v e tract o f f e m a l e s d u r i n g m a t i n g , a n d r e l e a s e d f r o m the f e m a l e s to the s u r r o u n d i n g surfaces a f t e r m a t i n g . A l o n g w i t h food o d o r s , Z 1 1 - 2 0 : A c acts as an a g g r e g a t i o n p h e r o m o n e to attract D. m a l e r k o t l i a n a o f e i t h e r sex to food a n d p o t e n t i a l m a t i n g sites. Acknowledgments--The research was supported by NSF grant DCB-8509976. The paper is contribution No. J-2044 in the Montana Agricultural Experiment Station Scientific 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. 77:364-37 I. BARTELT, R.J., JACKSON, L.L., and SCHANER, A.M. 1985a. Ester components of the aggregation pheromone of Drosophila virilis (Diptera: Drosophila). J. Chem. Ecol. 11 : 1197-1208. BARTELT, R.J., SCHANER,A.M., and JACKSON, L.L. 1985b. cis-Vaccenyl acetate as an aggregation pheromone in Drosophila melanogaster. J. Chem. Ecol. 11:1747-1756. BARTELT, R.J., SCHANER, A.M., and JACKSON, L.L. 1986. Aggregation pheromone in five taxa of the Drosophila virilis species group. Physiol. Entomol. I 1:367-376. 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. BARTELT, R.J., SCHANER, A.M., and JACKSON, L.L. 1989. Aggregation pheromone compounds in Drosophila mulleri: A chirat ester and an unsaturated ketone. J. Chem. Ecol. 15:399-412. BRtEGER. G., and BU'rTERWORTH,G.M. 1970. Drosophila melanogaster: Identity of male lipid in reproductive system. Science 167:1262. EPHRUSSJ, B., and BEADLE, G.W. 1936. A technique of transplantation in Drosophila. Am. Nat. 70:218-225. FOGLEMAN,J.C. 1982. The role of volatiles in the ecology of catophilic Drosophila, pp. 191-206, in J.S.F. Barker and W.T. Starmer (eds.). Ecological Genetics and Evolution: The CactusYeast Drosophila Model System. Academic Press, Sydney, Australia. MOATS, R.A., BAaTEL'r, 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. NICHOLS, P.D., GUCKERT, J.B., and WroTE, D.C. 1986. Determination of monounsaturated fatty acid double-bond position and geometry for microbial monocultures and complex consortic by capillary GC-MS of their dimethyl disulphide adducts. J. Microbiol. Methods 5:49-55. SCHANER, A.M., BARTELT, R.J., and JACKSON, L.L. t987. Z-I 1-Octadecenyl acetate is an aggregation pheromone in Drosophila simulans. J. Chem. Ecol. 13:1777-1786. YATES, F. 1940. The recovery of interblock information in balanced incomplete block designs. Amz. Eugen. 10:317-325.
(Z)-ll-EICOSENYL ACETATE, AN AGGREGATION PHEROMONE IN Drosophila malerkotliana
ANGELA
M . S C H A N E R , L A R R Y L. J A C K S O N , a n d R U S S E L L D. L E U
K A T E J. G R A H A M ,
Biochemistry Group Chenffstry Department Montana State UniversitT Bozeman, Montana 59717
(Received August 3. 1987; accepted November 16, 1987) Abstract--(Z)-ll-Eicosenyl acetate (Zll-20:Ac) was identified as the aggregation pheromone in Drosophila malerkotliana. The pheromone (200300 ng/fly) was isolated from hexane extracts of the ejaculatory bulb of sexually mature male flies. Males released very little, if any, Z11-20: Ac to the food at any time. During mating there was a transfer of ca. 100 ng of ZI 120:Ac to the female's reproductive tract. The mated female fly transferred the ZI 1-20: Ac to the surrounding surfaces in just a few hours after mating. In bioassay in a wind-tunnel olfactometer, ZI 1-20:Ac was not attractive alone, but was synergistic with fermenting food or with acetone. Although D. malerkotliana has no (Z)- 1 l-octadecenyl acetate (Z 11-18 : Ac), it was as attracted to Z 11-18 : Ac as to an equal quantity of Z11-20 : Ac. D. melanogaster and D. simulans, however, responded to the ZI 1-18:Ac that they produced and did not respond to Z I 1-20: Ac. Key Words--Drosophila malerkotliana, Diptera, Drosophilidae, aggregation pheromone, (Z)- 1 l-eicosenyl acetate.
INTRODUCTION A g g r e g a t i o n p h e r o m o n e s h a v e b e e n d e m o n s t r a t e d in 11 species o f Drosophila: D. virilis ( B a r t e l t a n d J a c k s o n , 1984; B a r t e l t et al., 1985a); D. a. americana, D. a. texana, D. novamexicana, a n d D. lummei (Bartelt et al., 1986); D. borealis a n d D. littoralis ( B a r t e l t et al., 1988); D. hydei ( M o a t s et al., 1987); D. melanogaster ( B a r t e l t et al., 1985b), D. simulans ( S c h a n e r et al,, 1987); and D. mulleri ( B a r t e l t et al., 1989). In m a n y o f the c l o s e l y related s p e c i e s , the 265 0098-0331/8910100-0265$06.00/0 ~) 1989 Plenum Publishing Corporation
26 6
SCHANER ET AL.
aggregation pheromones had the same or similar chemical structures, but between some species groups there appeared to be considerable differences in the chemical structures of the aggregation pheromones. D. melanogaster and D. simulans of the melanogaster species group and melanogaster subgroup both used (Z)-I 1-octadecenyl acetate (Z11-18 : Ac, cisvaccenyl acetate, cVA) as their aggregation pheromone (Bartelt et al., 1985b; Schaner et al., 1987). No other fly-derived coattractant was necessary, but food or food-derived odors were synergistic with the Z11-18 : Ac. In D. melanogaster and D. simulans the Z11-18 : Ac was stored in the ejaculatory bulb of sexually mature male flies, transferred to females during mating, and released by mated female flies to the food media within hours after mating. D. malerkotliana was chosen for aggregation pheromone investigation because it was in the melanogaster group but in the ananassae subgroup, and we could test whether male-derived compound(s) could function as an aggregation pheromone and whether the compound(s) were the same or differed from the Z 11-18 : Ac used by members of the melanogaster subgroup.
METHODS AND MATERIALS
Flies, Extraction, Chromatography, and Bioassay. D. malerkotliana malerkotliana (strain 14024-0391.0) was obtained from the National Drosophila Species Resource Center at Bowling Green, Ohio. It was originally collected at Mysore, India. D. melanogaster (wild type, Canton S strain) and D. simulans (strain 14021-0251.0) were as reported previously (Bartelt et al., 1985b; Schaher et al., 1987). Details of rearing, extraction, chromatography, and bioassay of the flies have been described previously (Bartelt and Jackson, 1984, Bartelt et al., 1985b; Schaner et al., 1987). Briefly, flies were separated by sex at 0-6 hr old and extracted at 6-7 days of age by soaking the flies in hexane at room temperature for 24 hr. Fractionation of the hexane extracts was on open columns of silicic acid eluted with hexane, 10% ether in hexane, 50% ether in hexane, and 10% methanol in methylene chloride. The 10% ether in hexane fraction was purified further by preparative gas chromatography (GC). Bioassays were conducted in a wind-tunnel olfactometer containing ca. 1000 (0- to 2-day-old) flies that had been without food and water for approximately 2 hr. An extract, fraction, synthetic compound, or control solvent was applied to a filter paper strip inserted around the lip of the glass vial. Each bioassay test consisted of placing two differently treated vials, to be compared, into the olfactometer for 3 min. The bioassay data was transformed to the log (x + 1) scale before analysis to stabilize variance and analysis was done by the method of Yates' (1940). Identification. Identification of the pheromone was done by comparing
AGGREGATION PHEROMONE IN
Drosophila
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retention times on GC, by gas chromatography-mass spectrometry (GC-MS) of the pheromone on a VG-MM16 GC-MS and by GC-MS of the dimethyl disulfide reaction product (Nichols et al., 1986). The determination of the isomer of the double-bond was done by comparison of synthetic Z 1 1 - 2 0 : A c (Sigma Chemical, St. Louis, Missouri) and the fly-derived I 1-20:Ac on a 30-m DB1 capillary GC column with a temperature program of 140-220°C with a 4-min initial hold and a rate of 4°C/min. With this method the Z and E isomers of 918 : Ac had previously been separated by a difference in the retention times of 0.11 min. The isomer of the fly-derived 11-20 : Ac was determined by a coinjection of this compound with the synthetic Z 11-20 : Ac. Production of Z11-20:Ac with Age. Since Z 11-18:Ac was found in the ejaculatory bulb in D. melanogaster (Brieger and Butterworth, 1970), the ejaculatory bulbs of D. malerkotliana were removed, extracted with hexane, and analyzed by GC. To remove ejaculatory bulbs, the males were placed in the freezer for approximately 10 min and then placed, ventral side up, on a cork board with a dissecting pin through the thorax. The genital region was teased loose with forceps and placed in a drop of Drosophila Ringer's solution (Ephrussi and Beadle, 1936). Using two dissecting pins, the ejaculatory bulb was removed from the genital region under 45 x magnification. Two sets of five bulbs for age groups from 2 hr to 8 days were placed in 1-ml conical vials that contained 500 ng nonadecane as an internal standard in 10 #1 of hexane. The bulbs were smashed with the head of a dissecting pin and the hexane extract analyzed by GC. For comparison, we also analyzed hexane extracts of whole flies (3 × 20 flies) at each age. The flies were extracted at room temperature for 24 hr, and the hexane extracts were fractionated on open silicic acid columns described above. Z 11-20 : Ac was quantified by GC relative to the internal standard, Z11-16: Ac. Transfer during Mating. The transfer of Z 11-20 : Ac from males to females and the subsequent release of the ester from the females was investigated. Virgin females (6-7 days old) were mated with virgin males of the same age. Immediately after completion of mating, the females were either extracted or placed into an empty 8-ml vial for 6 hr and then extracted. The vials were rinsed with I ml of hexane to recover any Z 11-20 : Ac that had been released. The mated males were treated in a similar fashion and, as controls, comparable sets of extracts were obtained from males and females that had not been allowed to mate. Each extract represented 10 fly equivalents and there were four replications per extract. The Z 11-20 : Ac in the extracts was quantitated by GC relative to an internal standard. Female Reproductive Tract Removal. The female reproductive tract was also removed and the extract analyzed for the presence of Z11-20 : Ac. Immediately upon completion of mating, the female was placed in the freezer for approximately 10 min. Viewed through a 10 × dissecting scope, the female was
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held ventral side up with forceps pressed laterally on the abdomen. The ovipositor was clasped with another pair of forceps and teased until the reproductive tract, without the ovaries, was pulled away from the fly. Two sets of five reproductive tracts were placed in conical vials with an intemal standard and the extract analyzed as described previously for the ejaculatory bulbs. The hexane extract of female flies minus the reproductive tracts were analyzed by GC after they were soaked in hexane for 1 hr.
RESULTS AND DISCUSSION
Demonstration, Purification, and Identification of Pheromone. Initial attempts to demonstrate an aggregation pheromone in D. malerkotliana were patterned after previous experiments with D. melanogaster (Bartelt et al., 1985b) and D. simulans (Schaner et al., 1987). The hexane extract of mature males, with a food synergist, was clearly active in bioassay, and the hexane extract of mature females was, with food, also about twice as attractive as the food (Table 1, A). The male fly hexane fraction alone was not attractive. Although food was an effective coattractant, we felt that using a synergist that was less attractive might clarify pheromonal effects and make pheromone identification more rapid. Acetone was the preferred coattractant, which lead to the identification of the aggregation pheromone in D. melanogaster (Bartelt et al., 1985b). Compounds such as acetone and ethyl acetate are known to be produced by molds and yeasts (Fogleman, 1982) and could well influence the behavior of the flies under natural conditions. When acetone was used as a coattractant in D. malerkotliana (Table 1, B), the male fly hexane extract plus acetone was about 10 times more attractive than the female fly hexane extract plus acetone. Acetone alone was not attractive. Both sexes of flies respond nearly equally to the hexane extracts of mature male flies. Since the attractive nature of the male fly extracts was much greater than that of female fly extracts, this paper will concentrate on the male fly-derived pheromone. After fractionation of the hexane extract of male flies on silicic acid, only the 10% ether-hexane fraction was active. The remaining fractions from the extract of male flies were not different in activity from the control solvent. By capillary GC, the male 10% ether-hexane fraction was predominantly one component. GC-MS of the major peak in the male 10% ether-hexane fraction indicated that the component was an acetate ester of a monounsaturated alcohol with 20 carbons. GC-MS of the dimethyl disulfidetreated fraction revealed that the double bond in the 20-carbon alcohol of the ester was at 11. Synthetic (Z)-I 1-eicosenyl acetate (abbreviated Z 11-20 : Ac) compared identically with the fly-derived component in GC and GC-MS. GC comparison of the fly-derived compound and the synthetic Z 11-20 : Ac identified the fly-derived 11-20 : Ac as the Z isomer. Z 11-20 : Ac was present in the
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269
TABLE 1. FOUR SERIES OF BIOASSAY EXPERIMENTS WITH EXTRACTS OF O.
materkotliana TO ISOLATE AND CHARACTERIZE AGGREGATION PHEROMONE. Mean bioassay catchh
Treatment~ A:
Synergism of food" by hexane extracts of flies Fermenting food Female fly hexane extract + food Male fly hexane extract + food Male fly hexane extract B: Comparison of hexane extracts from mate and female flies with acetone as a coattractant Male fly hexane extract + acetonea Female fly hexane extract + acetone Acetone control C: Test for equivalence of synthetic Z 11-20 : Ac with extracts, fractions and purified compounds Synthetic Z11-20: Ac + acetone Male-derived Z 11-20 : Ac + acetone 10% ether in bexane fraction + acetone Male fly hexane extract + acetone Acetone control D: Synergism between Z I 1-20 : Ac and fermenting food Synthetic Z 11-20 : Ac + food Fermenting food Z 11-20 : Ac Hexane control
(N = 18) 12.3d 26.8c 56.5b 0.7a (N = 8) 13.0b 1.2a 0.4a (N = 16) 11.0c 6.1 b 9.5bc 6.1b 1.0a (N = 24) 73.3c 5.3b 1.2a 0.7a
"All fly-derived fractions and their synthetic counterparts were used at 1 fly equivalent per test (220 ng of Z 11-20 : Ac). ~'In any experiment, means followed by the same letter were not significantly different at the 5% level (LSD). 'Fermenting food is Formula 4-24 Instant Drosophila Medium (Carolina Biological Supply, Burlington, North Carolina) to which active yeast has been added at least 24 hr previous to testing. aAcetone, 10 #1, was added to the filter paper with the extracts.
h e x a n e e x t r a c t o f m a t u r e m a l e flies at a c o n c e n t r a t i o n o f ca. 2 0 0 rig/fly, a n d there was no indication of the presence of Z 11-18:Ac
in t h e e x t r a c t . T h i s is
t h e first r e p o r t o f Z 1 1 - 2 0 : A c as an a g g r e g a t i o n p h e r o m o n e in Drosophila. The male hexane extract, 10% ether-hexane fraction, synthetic Zll20 : A c , a n d m a l e - d e r i v e d Z 1 1 - 2 0 : A c w e r e c o m p a r e d b y b i o a s s a y (all at 2 2 0 ng Z 1 1 - 2 0 : A c ; T a b l e 1, C ) . A l l w e r e s i g n i f i c a n t l y g r e a t e r t h a n t h e c o n t r o l ( P
I-> I.-- 300
> 200 ..J W I00
20
200 ZII-20:AC
2000 (ng)
20000
FiG. 1. Activity of D. malerkotliana towards different doses of Z 11-20 : Ac. Relative activities are 100% for the 200-rig dose of Z l l - 2 0 : A c ( - 1 fly eq) and 0% for the controls (N = 8). In each case, 10 /zl of acetone was used as a coattractant. Spacing along the x axis is logarithmic.
SCHANER ET AL.
272
increase in Z 11-20 : Ac. Over the next few days there is a gradual increase to a plateau o f about 300 ng/fly (Figure 2). The assays o f ejaculatory bulb contents are somewhat higher than the 24-hr hexane extracts o f whole flies. The same trend in Z 11-20 : Ac production is observed from the whole fly and ejaculatory bulb extracts. Although the whole fly extracts are much easier to perform, they do not provide a reliable quantitative estimation. Virgin females possess no Z I 1-20 : Ac. However, males transferred over half o f the Z l 1-20 : Ac they possessed to the female during mating. Immediately after mating about 85% o f the male-transferred Z 1 1 - 2 0 : A c is in the female's reproductive tract. Within 6 hr after mating, the female transferred approximately one third o f the Z I 1-20 : Ac into an empty vial. Males, virgin or mated, emitted no Z I 1 - 2 0 : A c into an empty vial that they occupied for 6 hr. Therefore, as in D. melanogaster and D. simulans (Bartelt et al., 1985b; Schaner et al., 1987), it is the mated female that emits the pheromone into the environment although it is male-produced. In summary, sexually mature D. malerkotliana males produce Z 11-20: Ac,
A
~- 3 0 0 E ¢-
c~ 2 0 0
/
(5 cxl !
N
/
~ I00 0 I-Z
0 0
I
I
i
I
I
I
I
I
I
0
I
2
3
4
5
6
7
8
AGE OF ADULT FLIES (days) FIG. 2. Amounts of ZI 1-20 : Ac extracted from virgin males D. malerkotliana of various ages. Solid circles indicate the Z 11-20 : Ac from a 24-hr hexane soak of the whole fly, and open circles indicate the Z I 1-20 : Ac within the hexane extract of ejaculatory bulbs. The bars indicate standard deviation.
AGGREGATION PHEROMONE IN Drosophila
273
w h i c h is s t o r e d in the e j a c u l a t o r y b u l b , t r a n s f e r r e d to the r e p r o d u c t i v e tract o f f e m a l e s d u r i n g m a t i n g , a n d r e l e a s e d f r o m the f e m a l e s to the s u r r o u n d i n g surfaces a f t e r m a t i n g . A l o n g w i t h food o d o r s , Z 1 1 - 2 0 : A c acts as an a g g r e g a t i o n p h e r o m o n e to attract D. m a l e r k o t l i a n a o f e i t h e r sex to food a n d p o t e n t i a l m a t i n g sites. Acknowledgments--The research was supported by NSF grant DCB-8509976. The paper is contribution No. J-2044 in the Montana Agricultural Experiment Station Scientific 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. 77:364-37 I. BARTELT, R.J., JACKSON, L.L., and SCHANER, A.M. 1985a. Ester components of the aggregation pheromone of Drosophila virilis (Diptera: Drosophila). J. Chem. Ecol. 11 : 1197-1208. BARTELT, R.J., SCHANER,A.M., and JACKSON, L.L. 1985b. cis-Vaccenyl acetate as an aggregation pheromone in Drosophila melanogaster. J. Chem. Ecol. 11:1747-1756. BARTELT, R.J., SCHANER, A.M., and JACKSON, L.L. 1986. Aggregation pheromone in five taxa of the Drosophila virilis species group. Physiol. Entomol. I 1:367-376. 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. BARTELT, R.J., SCHANER, A.M., and JACKSON, L.L. 1989. Aggregation pheromone compounds in Drosophila mulleri: A chirat ester and an unsaturated ketone. J. Chem. Ecol. 15:399-412. BRtEGER. G., and BU'rTERWORTH,G.M. 1970. Drosophila melanogaster: Identity of male lipid in reproductive system. Science 167:1262. EPHRUSSJ, B., and BEADLE, G.W. 1936. A technique of transplantation in Drosophila. Am. Nat. 70:218-225. FOGLEMAN,J.C. 1982. The role of volatiles in the ecology of catophilic Drosophila, pp. 191-206, in J.S.F. Barker and W.T. Starmer (eds.). Ecological Genetics and Evolution: The CactusYeast Drosophila Model System. Academic Press, Sydney, Australia. MOATS, R.A., BAaTEL'r, 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. NICHOLS, P.D., GUCKERT, J.B., and WroTE, D.C. 1986. Determination of monounsaturated fatty acid double-bond position and geometry for microbial monocultures and complex consortic by capillary GC-MS of their dimethyl disulphide adducts. J. Microbiol. Methods 5:49-55. SCHANER, A.M., BARTELT, R.J., and JACKSON, L.L. t987. Z-I 1-Octadecenyl acetate is an aggregation pheromone in Drosophila simulans. J. Chem. Ecol. 13:1777-1786. YATES, F. 1940. The recovery of interblock information in balanced incomplete block designs. Amz. Eugen. 10:317-325.
