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(Z)-10-heptadecen-2-one and 2-tridecanone biosynthesis from [1-14C]acetate by Drosophila buzzatii
Insect Biochem. Molec. Biol. Vol.23, No. 3, pp. 375 380, 1993
0965-1748/93$6.00+ 0.00 Copyright'~) 1993PergamonPressLtd
Printed in GreatBritain.All rightsreserved
(Z)- 10-Heptadecen-2-one and 2-Tridecanone Biosynthesis From [1-14C]Acetate by Drosophila buzzatii P A U L J. SKIBA,* L A R R Y L. JACKSON*']" Received 19 May 1992; revised and accepted 21 September 1992
The in vitro incorporation of [1-14Clacetate into (Z)-10-heptadecen-2-one, the major aggregation pheromone component of D. buzzatii, was greatest in the microsomal fraction of ejaculatory bulbs from mature male D. buzzatii. [1J4CIAcetate was also incorporated into 2-tridecanone, an ejaculatory bulb component that inhibits the aggregation activity of (Z)-10-heptadecen-2-one. Radiolabeled acetate was incorporated into the 2-ketones only by the ejaculatory bulbs from mature male flies. Optimal in vitro pheromone biosynthesis by intact ejaculatory bulbs occurred at pH 6.8 in phosphate buffer. Although radiolabel was incorporated into tissue lipids at all ages tested, label was not detected in pheromone components until the insects were 4 days post-eclosion. Labeled acetate was most actively incorporated into 2-ketones by male flies of 5-6 days of age. The amount of labeled acetate present in the 2-ketones increased with incubation time, reaching a plateau at 16 h. Of the subcellular fractions, the microsomes incorporated the most radiolabel into pheromone components, with about 7% of the recovered label present in the 2-ketones. Aggregation pheromone biosynthesis Ejaculatory bulb Microsomes
Drosophila buzzatii
2-Tridecanone
INTRODUCTION
[l-14C]Acetate
(Z)- 10-Heptadecen-2-one
(Z)-10-heptadecen-2-one were transferred to the female during mating. Unexpectedly, 2-tridecanone inhibited the aggregation activity of (Z)-10-heptadecen-2-one in D. buzzatii (Schaner and Jackson, 1992). While much is known about the presence of aggregation pheromones in Drosophila, little is known about Drosophila aggregation pheromone biosynthesis. In the present study, we analyzed male D. buzzatii for the in vitro incorporation of radiolabel from [1-14C]acetate into (Z)-10-heptadecen-2-one, 2-tridecanone, and other lipids to determine the site of pheromone biosynthesis.
Mature males of the Drosophila species studied thus far produce aggregation pheromones which attract both males and females of any age (Bartelt and Jackson, 1984; Bartelt et al., 1985; Bartelt et al., 1986; Moats et al., 1987; Bartelt et al., 1989; Schaner et al., 1989a, b). The male-produced aggregation pheromones act synergistically with food odors, and are not present in newly emerged males or in virgin females of any age (Bartelt et al., 1986; Schaner et al., 1989a). In a number of species in the mulleri subgroup, the ejaculatory bulbs of mature males contain saturated and unsaturated 2-ketones. (Z)-10-Heptadecen-2-one was identified as a component of the aggregation pheromone of D. mulleri (Bartelt et al., 1989) and as the major aggregation pheromone component of D. buzzatii, D. serido, and D. martensis (Schaner and Jackson, 1992). D. buzzatii males store large quantities of 2-tridecanone (230ng/male) and (Z)-10-heptadecen-2-one (690ng/ male) in the ejaculatory bulb. The amount of (Z)-10-heptadecen-2-one in the ejaculatory bulb and on the cuticle of males increased with age. Only trace quantities of
MATERIALS AND METHODS
*Chemistry and BiochemistryDepartment, Montana State University, Bozeman, MT 59717, U.S.A. tAuthor for correspondence.
D. buzzatii (Strain 15081-1291) from the National Drosophila Species Resource Center, Bowling Green, Ohio were reared in 1 liter glass containers on yeasted Instant Drosophila Medium 4-24 (Carolina Biological Supply Company, Burlington, N.C.) at ambient laboratory temperatures (20-23°C) using a 16-8 h light,lark cycle. At less than 8 h post-eclosion, flies were separated by sex and the males placed in 3.2 cm i.d. x 10 cm vials with yeasted Instant Drosophila Medium. Sodium [1-taC]acetate (50mCi/mmol) was obtained from Research Products International, Mount Prospect, I11. A stock solution of 100 ktCi in 100/~1 of water was prepared for addition to in vitro incubations.
375
376
PAUL J. SKIBA and LARRY L. JACKSON
To obtain body segments, male flies were placed in the freezer for 10 min to facilitate handling, then placed ventral side up on a corkboard. The head, thorax, and abdomen were separated by cutting with a scalpel. Five of each body segment were transferred to a separate vial containing 50ktl of incubation buffer (0.2mM Coenzyme A, 7 m M MgC12, l m M ATP, l m M dithiothreitol, 0 . 4 m M N A D P H , 2 m M ascorbic acid, 0.25 mM sucrose in 0.1 M potassium phosphate buffer) and 0.2/~1 [c 440,000 counts per minute (cpm) of sodium [1-J4C]acetate]. The incubation buffer, with the addition of 1 mM ATP, was essentially the same as used by Vaz et al. (1988) to study hydrocarbon biosynthesis in the housefly. The addition of ATP to the incubation buffer increased the amount of label incorporated into the 2-ketones by up to 4-fold. Unless indicated otherwise, all incubations were for 16 h at pH 6.8 and 22-24°C. Abdominal cuticles and internal tissues were obtained by placing cold anesthetized males ventral side up on a corkboard with a.dissecting pin through the thorax. The abdomen was removed with a scalpel and placed in c 20/~1 of Drosophila Ringers (Ephrussi and Beadle, 1936). Under 45 x magnification, two dissecting pins were used to detach the internal tissues from the cuticle. Dissecting pins were used to separate the internal tissues into fat body, G.I. tract, testes, accessory glands, and ejaculatory bulb. Five of each tissue type were transferred to a separate vial containing 50/~1 of incubation buffer and 0.2/~1 of [l-14C]acetate. After incubation at 22-24c'C for 16 h, the tissue and incubation media were extracted for 1 h with 200#1 of hexane:isopropanol (3:2) (Hara and Radin, 1978), evaporated to apparent dryness, methylated with 0.5M K O H in methanol for 30 min at room temperature (Christie, 1973), and dried to 5 ~1 under nitrogen prior to radio-gas chromatography (radio-GC) analysis. Using this mild base methanolysis procedure, fatty acyl groups of glycerolipids react to form methyl esters but free fatty acids and the 2-ketones do not react. Methanolysis was necessary to convert radiolabeled glycerolipids to volatile methyl esters so they would not be held up on the GC column. Radio-GC analysis used a Packard 437A GC (Packard, Downer's Grove, Ill.) fitted with a 30m DB-5 Megabore (J&W Scientific, Folsom, Calif.) column and a flame ionization detector (FID). The temperature program was initial temperature 100°C, increased at 5°C/min to 150°C, after a 1 min hold at 150°C, the temperature was increased at 3°C/min to a final temperature of 250°C. The column effluent (5.4 ml/min) was split (1:100) between the FID and a Packard 894 gas proportional counter, providing both mass and radioisotope quantitation of each sample (Fig. 1). Each data point consisted of five sets of five ejaculatory bulbs per experiment. Electron impact mass spectra obtained on a V G - M M I 6 mass spectrometer using a 30m x 0.25 mm i.d. DB-5 capillary GC column for sample introduction identified the (Z)-10-heptadecen-2-one and 2-tridecanone peaks in the chromatogram and indicated that
the 2-ketone peaks from male D. buzzatii contained no other compounds (Schaner and Jackson, 1992). A control experiment was done to verify that the sodium [1-14C]acetate stock solution was not contaminated with other radioactive compounds that might appear in the analysis. A sample of 0.5~Ci of the sodium [1-t4C]acetate stock solution was extracted with 3:2 hexane:isopr0panol and evaporated to apparent dryness under nitrogen. Alkaline methanolysis was performed as described above, and the sample analyzed by radio-GC. Less than a total of 80 cpm over background were observed for the entire run by radio-GC (detection limit: 70 cpm). To determine the pH optimum for in vitro pheromone biosynthesis, ejaculatory bulbs (five sets of five bulbs each) were incubated for 16h in 50/~1 of incubation buffer of varying pH (pH 6.0-8.0) containing 0.2 ~tl of [1-~4C]acetate, extracted, derivatized, and analyzed as above. To obtain subcellular fractions, 50 ejaculatory bulbs from 5 to 6-day old male flies were homogenized in 500#1 of incubation buffer at 4°C in a Duall tissue grinder with 20 passes of the hand-driven ground glass pestle. The homogenate was centrifuged (4°C) at 1000g for 5 min (cell debris), 10,000g for 20 min (mitochondria), and 240,000g for 30 min (microsomes) (Vaz et al., 1988). The pellets were resuspended in 500/d of incubation buffer by brief sonication, and 50#1 of the suspension (five bulb equivalents) were used for each assay. The subcellular fractions were incubated with 0.2/~1 of [1-t4C]acetate for 16h, extracted, and derivatized as above prior to radio-GC analysis. RESULTS
Of the three insect body segments, only the abdomens of mature males were able to incorporate label from [1-~4C]acetate into (Z)-10-heptadecen-2-one, with 1.1% of the recovered label incorporated into the aggregation pheromone. In addition, 0.2% of the recovered label was present in 2-tridecanone. Although both the heads and thoraces incorporated label into lipids, label was not detected in the 2-ketones in these body segments. Dissection of the abdomen into tissue groups further narrowed down the site of pheromone biosynthesis in D. buzzatii. Of the abdominal tissues, only the ejaculatory bulb was capable of pheromone biosynthesis in t~itro, with 3.3% of the recovered label (0.4% of the applied label) in the ejaculatory bulb recovered in (Z)-10heptadecen-2-one and 1.0% of the recovered label (0.1% of the applied label) present in 2-tridecanone. The testes and the accessory glands, which are both closely associated with the ejaculatory bulb, did not incorporate label into the 2-ketones. The in vitro biosynthesis of (Z)-10-heptadecen-2-one and 2-tridecanone by whole ejaculatory bulbs was optimal at pH 6.8 in incubation buffer. The incorporation of [l-14C]acetate into pheromone in the ejaculatory bulb with age (Table 1) paralleled the
2-KETONE BIOSYNTHESIS
377
A
d
f 0
i. °
B
e
a
FIGURE 1. Radio-GC of D. buzzatii ejaculatory bulbs incubated with [1- 14C]acetate in pH 6.8 incubation buffer showing the mass trace (A) and the radioactive trace (B). a 2-tridecanone; b methyl laurate; c methyl myristoleate; d methyl myristate; e (Z)-10-heptadecen-2-one; f methyl palmitoleate; g, methyl palmitate; h, palmitic acid; i, methyl oleate.
increase in the amount of pheromone stored in the bulb (Schaner and Jackson, 1992). Although the ejaculatory bulb incorporated significant amounts of label into lipids at all ages tested, pheromone was not detectably labeled until 4 days post-eclosion. Label was most actively incorporated into pheromone from 5 to 6 days of age, peaking at 1162 cpm at 6 days. Between 7 and 9 days, incorporation into the pheromone remained at a nearly
constant level of c 500 cpm. Likewise, label was most actively incorporated into 2-tridecanone from 5 to 6 days of age, peaking at 625 cpm at 6 days. Between 8 and 9 days, incorporation remained at a nearly constant level of c 250 cpm. Changes in the in vitro incorporation of [1-~4C]acetate with incubation time were examined with incubations conducted from 0 to 24 h. In experiments of short
378
P A U L J. SKIBA and L A R R Y L. J A C K S O N T A B L E 1. Age dependent incorporation of radiolabel from [IJ4C]acetate into Z-10-hepta and 2-tri by ejaculatory bulbs of male D. buzzatii Age (days as adult)
Total recovered labelt (cpm _+ SD:~ in thousands)
l
44+
2 3 4 5 6 7 8 9
56_+4 40__+ 9 41 _+ 6 41 + 7 54+ 1 45 + 2 42_+ 2 47 +_ 2
4
Label in Z-10-hepta (cpm _+ SD)
Label in 2-tri (cpm _+ SD)
*
*
* * 323 + 104 686 __+97 1162_+ 188 594 + 65 484 +_ 61 449 + 68
* * * 488 _+ 59 6 2 5 + 155 433 _+ 128 278 + 79 226 + 47
*Below detectable limits ( < 60cpm). tc 440 x 103 cpm applied. :~n = five sets of five males each.
duration ( < 8 h), the overall incorporation of label into pheromone and other lipids was low (Table 2). Following a lag period, label incorporation into pheromone reached a maximum at 16 h. Since no change in the total label or pheromone label was seen from 16 to 24h of incubation, all subsequent incubations were carried out for 16h. [1-'4C]Acetate was incorporated into 2-ketones to some extent in all of the subcellular fractions (Table 3). The microsomal fraction, however, most actively incorporated label into the pheromone components, with 4.9% of the recovered label present in (Z)-10-heptadecen-2-one and 2.4% present in 2-tridecanone. The remainder of the label was recovered as methyl laurate (1%), methyl myristate (2%), methyl palmitate (33%), palmitic acid (2%), methyl palmitoleate (26%), palmitoleic acid (2%), methyl stearate (1%), methyl oleate (18%), and oleic acid (1%). The biosynthesis of 2-ketones by microsomes was optimal at pH 6.8 in incubation buffer (Fig. 2). DISCUSSION Although the ejaculatory bulb has been shown to be the site of aggregation pheromone storage in a number of Drosophila, the site of pheromone biosynthesis remained unknown. These in vitro studies support the hypothesis that the ejaculatory bulb is
the site of aggregation pheromone biosynthesis in D. buzzatii.
Of the three body segments, only the abdomen from mature males incorporated labeled acetate into the pheromone. Although both the head and thorax incorporated label into lipids, no label was present in the 2-ketones. The only abdominal tissue capable of in vitro incorporation of label from [l-~4C]acetate into (Z)-10heptadecen-2-one and 2-tridecanone in D. buzzatii was the ejaculatory bulb, strongly suggesting that aggregation pheromone biosynthesis is confined to the ejaculatory bulb. The in vitro production of 2-ketones by intact ejaculatory bulbs was maximal at pH 6.8 in potassium phosphate buffer. The pH profiles for the in vitro production of both (Z)-I 0-heptadecen-2-one and 2-tridecanone were similar. The amounts of 2-ketones produced during a 16 h incubation were comparable to the amounts produced by live flies in the same time period. In addition to the 2-ketones, label was also incorporated into fatty acids. The use of alkaline methylation enabled the amount of label present in both the free and esterified fatty acids to be determined. However, the incorporation of label into a particular fatty acyl moiety could not be correlated with the incorporation of label into either of the 2-ketones. The incorporation of [l- 14C]acetate into pheromone in the ejaculatory bulb with age paralleled the increase in the amount of pheromone stored in the bulb. 2-Ketones
T A B L E 2. Time dependent incorporation of radiolabel from [1-~4C]acetate into Z-10-hepta and 2-tri by ejaculatory bulbs of 6-day old male D. buzzatii Incubation time (h)
Total recovered labelt (cpm + SD:~ in thousands)
1
12+1
2 4 8 12 16 24
18+6 27 __+5 40 _+ 4 49 _+ 5 55 _+ 10 53 + 5
*Below detectable limits ( < 60cpm). tc 440 x 103cpm applied. :~n = Five sets of five males each.
Label in Z-10-hepta (cpm + SD)
Label in 2-tri (cpm + SD)
*
*
* 264 _+ 48 672 + 69 796 + 54 925 _+ 87 887 + 96
* * 152 _+ 51 450 _+ 22 509 _+ 99 503 ___86
2-KETONE BIOSYNTHESIS
379
TABLE 3. Incorporation of [l-~4C]acetate into Z-10-hepta and 2-tri by the subcellular fractions of D. buzzatii ejaculatory bulbs Total recovered label* (cpm + SDt in thousands)
Subcellular fraction Cellular debris Mitochondria Microsomes Soluble
36 ___3 41 + 9 33 + 2 34 + 5
Label in Z-10-hepta (cpm + SD) 312 _ 756 _ 1604 + 340 +
271 190 354 308
Label in 2-tri (cpm + SD) 174 + 96 351 __+73 782 + 1I0 197 _+59
*c 440 x 103 cpm applied. ?n = Five sets of five males each.
were first detected in the e j a c u l a t o r y b u l b o f D. buzzatii at 3 d a y s post-eclosion, a n d the a m o u n t o f 2-ketones s t o r e d in the e j a c u l a t o r y b u l b increased m o s t r a p i d l y between 4 a n d 6 d a y s o f age before reaching a p l a t e a u at 8 d a y s ( S c h a n e r a n d J a c k s o n , 1992). O u r e x p e r i m e n t s indicate t h a t r a d i o l a b e l was first i n c o r p o r a t e d into (Z)10-heptadecen-2-one at 4 d a y s post-eclosion, was m o s t actively i n c o r p o r a t e d between 5 a n d 6 d a y s o f age, a n d then t a p e r e d off. Similarly, label was first detected in 2 - t r i d e c a n o n e at 5 d a y s o f age, a n d was m o s t actively i n c o r p o r a t e d between 5 a n d 6 d a y s before t a p e r i n g off. As expected, there was a lag p e r i o d after label first a p p e a r e d in lipids before label b e g a n to a p p e a r in the p h e r o m o n e c o m p o n e n t s . This is consistent with the h y p o t h e s i s t h a t a labeled p r e c u r s o r p o o l needs to be established before the p h e r o m o n e can be p r o d u c e d . A l t e r n a t i v e l y , the a m o u n t o f label present in the 2-ketones c o u l d have been b e l o w detection ( < 70 cpm). A l t h o u g h label a p p e a r e d in lipids in the earliest incub a t i o n (1 h), the 2-ketones were n o t labeled until 4 h into the i n c u b a t i o n . Thereafter, the a m o u n t o f label incorp o r a t e d into the 2-ketones increased steadily until 16 h, with no increase in the a m o u n t o f label i n c o r p o r a t e d between 16 a n d 24 h. O f the subcellular fractions, the m i c r o s o m a l fraction i n c o r p o r a t e d the greatest a m o u n t o f label into the p h e r o m o n e c o m p o n e n t s . L a b e l e d 2-ketones were also p r o d u c e d by the m i t o c h o n d r i a l fraction from e j a c u l a t o r y bulbs. These results are consistent with p r e v i o u s studies o f p h e r o m o n e biosynthesis in o t h e r insects. B o t h the m i c r o s o m a l a n d m i t o c h o n d r i a l fractions f r o m houseflies c o n v e r t e d (Z)-9-tricosene to the e p o x i d e 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 , with the micro-
200
160
.,9 "~
&~ c
~
~ c
,2o 80
0 5.5
60
6.5
7.0
Z5
8.0
pH
FIGURE 2. pH Dependent biosynthesis of (Z)-10-heptadecen-2-one (O) and 2-tridecanone ( 0 ) from [lt4C]acetate by the microsomal fraction of D. buzzatii ejaculatory bulbs. Values are mean + standard deviation; n = five sets of five males each.
somes having the highest activity ( A h m a d et al., 1987). Similarly, subcellular f r a c t i o n a t i o n o f the p h e r o m o n e g l a n d from the spruce b u d w o r m indicated that the p h e r o m o n e biosynthetic activities involved in c o n v e r t i n g ( Z ) - l l - t e t r a d e c a n o i c acid into (Z)-I 1-tetradecyl acetate are located in the m i c r o s o m e s ( M o r s e a n d Meighen, 1986). In s u m m a r y , these in vitro e x p e r i m e n t s d e m o n s t r a t e d that a g g r e g a t i o n p h e r o m o n e biosynthesis in D. buzzatii is confined to the e j a c u l a t o r y bulb. In a d d i t i o n , only m a t u r e male flies (greater than 4 d a y s o f age) were c a p a b l e o f 2-ketone biosynthesis. T h e a m o u n t s o f 2ketones p r o d u c e d d u r i n g a 16 h i n c u b a t i o n were c o m p a r a b l e to the a m o u n t s p r o d u c e d by live males in the same time period. Similar to previous insect studies, the m i c r o s o m a l fraction from D. buzzatii e j a c u l a t o r y bulbs exhibited the greatest biosynthetic activity. Investig a t i o n s continue to o p t i m i z e the i n c u b a t i o n buffer with necessary c o f a c t o r s at a p p r o p r i a t e c o n c e n t r a t i o n s for m a x i m a l 2-ketone biosynthesis. The o t h e r subcellular fractions are being investigated to d e t e r m i n e whether they c o n t a i n m i c r o s o m e s with 2-ketone biosynthesis activity or w h e t h e r there are c o m p o n e n t s o f 2-ketone biosynthesis in these subcellular fractions.
REFERENCES
Ahmad S., Kirkland K. E. and Blomquist G. J. (1987) Evidence for a sex pheromone metabolizing cytochrome P-450 mono-oxygenase in the housefly. Archs Insect Biochem. Physiol. 6, 121-140. Bartelt R. J. and Jackson L. L. (1984) Hydrocarbon component of the Drosophila virilis (Diptera: Drosophilidae) aggregation pheromone: (Z)-10-heneicosene. Ann. ent. soc. Am. 77, 364-371. Bartelt R. J., Schaner A. M. and Jackson L. L. (1985) 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 pheromones in five taxa of the Drosophila virilis species group. Physiol. Ent. 11, 367 376. Bartelt R. J., Schaner A. M. and Jackson L. L (1989) Aggregation pheromone components in Drosophila mulleri: a chiral ester and an unsaturated ketone. J. chem. Ecol. 15, 399-412. Christie W. W. (1973) Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of Lipids. Pergamon Press, Oxford. Ephrussi B. and Beadle G. W. (1936) A technique for transplantation in Drosophila. Am. Nat. 70, 218~25. Hara A. and Radin N. S. (1978) Lipid extraction of tissues with a low-toxicity solvent. Analyt. Biochem. 90, 420-426. 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 (Diptera: Drosophilidae). J. chem. Ecol. 13, 451-462.
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PAUL J. SKIBA and LARRY L. JACKSON
Morse D. and Meighen E. A. (1986) Pheromone biosynthesis and the role of functional groups in pheromone specificity. J. chem. Ecol. 12, 335 351. Schaner A. M. and Jackson L. L. (1992) (Z)-10-Heptadecen-2-one and other 2-ketones in the aggregation pheromone blend of Drosophila martensis, D. buzzatii, and D. serido. J. chem. Ecol. 18, 53 64. Schaner A. M., Benner A. M., Leu R. D. and Jackson L. L. (1989a) Aggregation pheromone of Drosophila mauritiana, Drosophila yakuba, and Drosophila rajasakari. J. chem. Ecol. 15, 1249 1257. Schaner A. M., Jackson L. L., Graham K. J. and Leu R. D. (1989b) (Z)-ll-Eicosenyl acetate, an aggregation pheromone
component in Drosophila malerkotliana. J. chem. Ecol. 15, 265-273. Vaz A. H., Blomquist G. J. and Reitz R. C. (1988) Characterization of the fatty acyl elongation reactions involved in hydrocarbon biosynthesis in the housefly, Musca domestica L. Insect Biochem. 18, 177--184.
Acknowledgements--This research was supported by NSF grant DBC-8509976. Contribution No. J-2790 from the Montana Agricultural Experiment Station.
0965-1748/93$6.00+ 0.00 Copyright'~) 1993PergamonPressLtd
Printed in GreatBritain.All rightsreserved
(Z)- 10-Heptadecen-2-one and 2-Tridecanone Biosynthesis From [1-14C]Acetate by Drosophila buzzatii P A U L J. SKIBA,* L A R R Y L. JACKSON*']" Received 19 May 1992; revised and accepted 21 September 1992
The in vitro incorporation of [1-14Clacetate into (Z)-10-heptadecen-2-one, the major aggregation pheromone component of D. buzzatii, was greatest in the microsomal fraction of ejaculatory bulbs from mature male D. buzzatii. [1J4CIAcetate was also incorporated into 2-tridecanone, an ejaculatory bulb component that inhibits the aggregation activity of (Z)-10-heptadecen-2-one. Radiolabeled acetate was incorporated into the 2-ketones only by the ejaculatory bulbs from mature male flies. Optimal in vitro pheromone biosynthesis by intact ejaculatory bulbs occurred at pH 6.8 in phosphate buffer. Although radiolabel was incorporated into tissue lipids at all ages tested, label was not detected in pheromone components until the insects were 4 days post-eclosion. Labeled acetate was most actively incorporated into 2-ketones by male flies of 5-6 days of age. The amount of labeled acetate present in the 2-ketones increased with incubation time, reaching a plateau at 16 h. Of the subcellular fractions, the microsomes incorporated the most radiolabel into pheromone components, with about 7% of the recovered label present in the 2-ketones. Aggregation pheromone biosynthesis Ejaculatory bulb Microsomes
Drosophila buzzatii
2-Tridecanone
INTRODUCTION
[l-14C]Acetate
(Z)- 10-Heptadecen-2-one
(Z)-10-heptadecen-2-one were transferred to the female during mating. Unexpectedly, 2-tridecanone inhibited the aggregation activity of (Z)-10-heptadecen-2-one in D. buzzatii (Schaner and Jackson, 1992). While much is known about the presence of aggregation pheromones in Drosophila, little is known about Drosophila aggregation pheromone biosynthesis. In the present study, we analyzed male D. buzzatii for the in vitro incorporation of radiolabel from [1-14C]acetate into (Z)-10-heptadecen-2-one, 2-tridecanone, and other lipids to determine the site of pheromone biosynthesis.
Mature males of the Drosophila species studied thus far produce aggregation pheromones which attract both males and females of any age (Bartelt and Jackson, 1984; Bartelt et al., 1985; Bartelt et al., 1986; Moats et al., 1987; Bartelt et al., 1989; Schaner et al., 1989a, b). The male-produced aggregation pheromones act synergistically with food odors, and are not present in newly emerged males or in virgin females of any age (Bartelt et al., 1986; Schaner et al., 1989a). In a number of species in the mulleri subgroup, the ejaculatory bulbs of mature males contain saturated and unsaturated 2-ketones. (Z)-10-Heptadecen-2-one was identified as a component of the aggregation pheromone of D. mulleri (Bartelt et al., 1989) and as the major aggregation pheromone component of D. buzzatii, D. serido, and D. martensis (Schaner and Jackson, 1992). D. buzzatii males store large quantities of 2-tridecanone (230ng/male) and (Z)-10-heptadecen-2-one (690ng/ male) in the ejaculatory bulb. The amount of (Z)-10-heptadecen-2-one in the ejaculatory bulb and on the cuticle of males increased with age. Only trace quantities of
MATERIALS AND METHODS
*Chemistry and BiochemistryDepartment, Montana State University, Bozeman, MT 59717, U.S.A. tAuthor for correspondence.
D. buzzatii (Strain 15081-1291) from the National Drosophila Species Resource Center, Bowling Green, Ohio were reared in 1 liter glass containers on yeasted Instant Drosophila Medium 4-24 (Carolina Biological Supply Company, Burlington, N.C.) at ambient laboratory temperatures (20-23°C) using a 16-8 h light,lark cycle. At less than 8 h post-eclosion, flies were separated by sex and the males placed in 3.2 cm i.d. x 10 cm vials with yeasted Instant Drosophila Medium. Sodium [1-taC]acetate (50mCi/mmol) was obtained from Research Products International, Mount Prospect, I11. A stock solution of 100 ktCi in 100/~1 of water was prepared for addition to in vitro incubations.
375
376
PAUL J. SKIBA and LARRY L. JACKSON
To obtain body segments, male flies were placed in the freezer for 10 min to facilitate handling, then placed ventral side up on a corkboard. The head, thorax, and abdomen were separated by cutting with a scalpel. Five of each body segment were transferred to a separate vial containing 50ktl of incubation buffer (0.2mM Coenzyme A, 7 m M MgC12, l m M ATP, l m M dithiothreitol, 0 . 4 m M N A D P H , 2 m M ascorbic acid, 0.25 mM sucrose in 0.1 M potassium phosphate buffer) and 0.2/~1 [c 440,000 counts per minute (cpm) of sodium [1-J4C]acetate]. The incubation buffer, with the addition of 1 mM ATP, was essentially the same as used by Vaz et al. (1988) to study hydrocarbon biosynthesis in the housefly. The addition of ATP to the incubation buffer increased the amount of label incorporated into the 2-ketones by up to 4-fold. Unless indicated otherwise, all incubations were for 16 h at pH 6.8 and 22-24°C. Abdominal cuticles and internal tissues were obtained by placing cold anesthetized males ventral side up on a corkboard with a.dissecting pin through the thorax. The abdomen was removed with a scalpel and placed in c 20/~1 of Drosophila Ringers (Ephrussi and Beadle, 1936). Under 45 x magnification, two dissecting pins were used to detach the internal tissues from the cuticle. Dissecting pins were used to separate the internal tissues into fat body, G.I. tract, testes, accessory glands, and ejaculatory bulb. Five of each tissue type were transferred to a separate vial containing 50/~1 of incubation buffer and 0.2/~1 of [l-14C]acetate. After incubation at 22-24c'C for 16 h, the tissue and incubation media were extracted for 1 h with 200#1 of hexane:isopropanol (3:2) (Hara and Radin, 1978), evaporated to apparent dryness, methylated with 0.5M K O H in methanol for 30 min at room temperature (Christie, 1973), and dried to 5 ~1 under nitrogen prior to radio-gas chromatography (radio-GC) analysis. Using this mild base methanolysis procedure, fatty acyl groups of glycerolipids react to form methyl esters but free fatty acids and the 2-ketones do not react. Methanolysis was necessary to convert radiolabeled glycerolipids to volatile methyl esters so they would not be held up on the GC column. Radio-GC analysis used a Packard 437A GC (Packard, Downer's Grove, Ill.) fitted with a 30m DB-5 Megabore (J&W Scientific, Folsom, Calif.) column and a flame ionization detector (FID). The temperature program was initial temperature 100°C, increased at 5°C/min to 150°C, after a 1 min hold at 150°C, the temperature was increased at 3°C/min to a final temperature of 250°C. The column effluent (5.4 ml/min) was split (1:100) between the FID and a Packard 894 gas proportional counter, providing both mass and radioisotope quantitation of each sample (Fig. 1). Each data point consisted of five sets of five ejaculatory bulbs per experiment. Electron impact mass spectra obtained on a V G - M M I 6 mass spectrometer using a 30m x 0.25 mm i.d. DB-5 capillary GC column for sample introduction identified the (Z)-10-heptadecen-2-one and 2-tridecanone peaks in the chromatogram and indicated that
the 2-ketone peaks from male D. buzzatii contained no other compounds (Schaner and Jackson, 1992). A control experiment was done to verify that the sodium [1-14C]acetate stock solution was not contaminated with other radioactive compounds that might appear in the analysis. A sample of 0.5~Ci of the sodium [1-t4C]acetate stock solution was extracted with 3:2 hexane:isopr0panol and evaporated to apparent dryness under nitrogen. Alkaline methanolysis was performed as described above, and the sample analyzed by radio-GC. Less than a total of 80 cpm over background were observed for the entire run by radio-GC (detection limit: 70 cpm). To determine the pH optimum for in vitro pheromone biosynthesis, ejaculatory bulbs (five sets of five bulbs each) were incubated for 16h in 50/~1 of incubation buffer of varying pH (pH 6.0-8.0) containing 0.2 ~tl of [1-~4C]acetate, extracted, derivatized, and analyzed as above. To obtain subcellular fractions, 50 ejaculatory bulbs from 5 to 6-day old male flies were homogenized in 500#1 of incubation buffer at 4°C in a Duall tissue grinder with 20 passes of the hand-driven ground glass pestle. The homogenate was centrifuged (4°C) at 1000g for 5 min (cell debris), 10,000g for 20 min (mitochondria), and 240,000g for 30 min (microsomes) (Vaz et al., 1988). The pellets were resuspended in 500/d of incubation buffer by brief sonication, and 50#1 of the suspension (five bulb equivalents) were used for each assay. The subcellular fractions were incubated with 0.2/~1 of [1-t4C]acetate for 16h, extracted, and derivatized as above prior to radio-GC analysis. RESULTS
Of the three insect body segments, only the abdomens of mature males were able to incorporate label from [1-~4C]acetate into (Z)-10-heptadecen-2-one, with 1.1% of the recovered label incorporated into the aggregation pheromone. In addition, 0.2% of the recovered label was present in 2-tridecanone. Although both the heads and thoraces incorporated label into lipids, label was not detected in the 2-ketones in these body segments. Dissection of the abdomen into tissue groups further narrowed down the site of pheromone biosynthesis in D. buzzatii. Of the abdominal tissues, only the ejaculatory bulb was capable of pheromone biosynthesis in t~itro, with 3.3% of the recovered label (0.4% of the applied label) in the ejaculatory bulb recovered in (Z)-10heptadecen-2-one and 1.0% of the recovered label (0.1% of the applied label) present in 2-tridecanone. The testes and the accessory glands, which are both closely associated with the ejaculatory bulb, did not incorporate label into the 2-ketones. The in vitro biosynthesis of (Z)-10-heptadecen-2-one and 2-tridecanone by whole ejaculatory bulbs was optimal at pH 6.8 in incubation buffer. The incorporation of [l-14C]acetate into pheromone in the ejaculatory bulb with age (Table 1) paralleled the
2-KETONE BIOSYNTHESIS
377
A
d
f 0
i. °
B
e
a
FIGURE 1. Radio-GC of D. buzzatii ejaculatory bulbs incubated with [1- 14C]acetate in pH 6.8 incubation buffer showing the mass trace (A) and the radioactive trace (B). a 2-tridecanone; b methyl laurate; c methyl myristoleate; d methyl myristate; e (Z)-10-heptadecen-2-one; f methyl palmitoleate; g, methyl palmitate; h, palmitic acid; i, methyl oleate.
increase in the amount of pheromone stored in the bulb (Schaner and Jackson, 1992). Although the ejaculatory bulb incorporated significant amounts of label into lipids at all ages tested, pheromone was not detectably labeled until 4 days post-eclosion. Label was most actively incorporated into pheromone from 5 to 6 days of age, peaking at 1162 cpm at 6 days. Between 7 and 9 days, incorporation into the pheromone remained at a nearly
constant level of c 500 cpm. Likewise, label was most actively incorporated into 2-tridecanone from 5 to 6 days of age, peaking at 625 cpm at 6 days. Between 8 and 9 days, incorporation remained at a nearly constant level of c 250 cpm. Changes in the in vitro incorporation of [1-~4C]acetate with incubation time were examined with incubations conducted from 0 to 24 h. In experiments of short
378
P A U L J. SKIBA and L A R R Y L. J A C K S O N T A B L E 1. Age dependent incorporation of radiolabel from [IJ4C]acetate into Z-10-hepta and 2-tri by ejaculatory bulbs of male D. buzzatii Age (days as adult)
Total recovered labelt (cpm _+ SD:~ in thousands)
l
44+
2 3 4 5 6 7 8 9
56_+4 40__+ 9 41 _+ 6 41 + 7 54+ 1 45 + 2 42_+ 2 47 +_ 2
4
Label in Z-10-hepta (cpm _+ SD)
Label in 2-tri (cpm _+ SD)
*
*
* * 323 + 104 686 __+97 1162_+ 188 594 + 65 484 +_ 61 449 + 68
* * * 488 _+ 59 6 2 5 + 155 433 _+ 128 278 + 79 226 + 47
*Below detectable limits ( < 60cpm). tc 440 x 103 cpm applied. :~n = five sets of five males each.
duration ( < 8 h), the overall incorporation of label into pheromone and other lipids was low (Table 2). Following a lag period, label incorporation into pheromone reached a maximum at 16 h. Since no change in the total label or pheromone label was seen from 16 to 24h of incubation, all subsequent incubations were carried out for 16h. [1-'4C]Acetate was incorporated into 2-ketones to some extent in all of the subcellular fractions (Table 3). The microsomal fraction, however, most actively incorporated label into the pheromone components, with 4.9% of the recovered label present in (Z)-10-heptadecen-2-one and 2.4% present in 2-tridecanone. The remainder of the label was recovered as methyl laurate (1%), methyl myristate (2%), methyl palmitate (33%), palmitic acid (2%), methyl palmitoleate (26%), palmitoleic acid (2%), methyl stearate (1%), methyl oleate (18%), and oleic acid (1%). The biosynthesis of 2-ketones by microsomes was optimal at pH 6.8 in incubation buffer (Fig. 2). DISCUSSION Although the ejaculatory bulb has been shown to be the site of aggregation pheromone storage in a number of Drosophila, the site of pheromone biosynthesis remained unknown. These in vitro studies support the hypothesis that the ejaculatory bulb is
the site of aggregation pheromone biosynthesis in D. buzzatii.
Of the three body segments, only the abdomen from mature males incorporated labeled acetate into the pheromone. Although both the head and thorax incorporated label into lipids, no label was present in the 2-ketones. The only abdominal tissue capable of in vitro incorporation of label from [l-~4C]acetate into (Z)-10heptadecen-2-one and 2-tridecanone in D. buzzatii was the ejaculatory bulb, strongly suggesting that aggregation pheromone biosynthesis is confined to the ejaculatory bulb. The in vitro production of 2-ketones by intact ejaculatory bulbs was maximal at pH 6.8 in potassium phosphate buffer. The pH profiles for the in vitro production of both (Z)-I 0-heptadecen-2-one and 2-tridecanone were similar. The amounts of 2-ketones produced during a 16 h incubation were comparable to the amounts produced by live flies in the same time period. In addition to the 2-ketones, label was also incorporated into fatty acids. The use of alkaline methylation enabled the amount of label present in both the free and esterified fatty acids to be determined. However, the incorporation of label into a particular fatty acyl moiety could not be correlated with the incorporation of label into either of the 2-ketones. The incorporation of [l- 14C]acetate into pheromone in the ejaculatory bulb with age paralleled the increase in the amount of pheromone stored in the bulb. 2-Ketones
T A B L E 2. Time dependent incorporation of radiolabel from [1-~4C]acetate into Z-10-hepta and 2-tri by ejaculatory bulbs of 6-day old male D. buzzatii Incubation time (h)
Total recovered labelt (cpm + SD:~ in thousands)
1
12+1
2 4 8 12 16 24
18+6 27 __+5 40 _+ 4 49 _+ 5 55 _+ 10 53 + 5
*Below detectable limits ( < 60cpm). tc 440 x 103cpm applied. :~n = Five sets of five males each.
Label in Z-10-hepta (cpm + SD)
Label in 2-tri (cpm + SD)
*
*
* 264 _+ 48 672 + 69 796 + 54 925 _+ 87 887 + 96
* * 152 _+ 51 450 _+ 22 509 _+ 99 503 ___86
2-KETONE BIOSYNTHESIS
379
TABLE 3. Incorporation of [l-~4C]acetate into Z-10-hepta and 2-tri by the subcellular fractions of D. buzzatii ejaculatory bulbs Total recovered label* (cpm + SDt in thousands)
Subcellular fraction Cellular debris Mitochondria Microsomes Soluble
36 ___3 41 + 9 33 + 2 34 + 5
Label in Z-10-hepta (cpm + SD) 312 _ 756 _ 1604 + 340 +
271 190 354 308
Label in 2-tri (cpm + SD) 174 + 96 351 __+73 782 + 1I0 197 _+59
*c 440 x 103 cpm applied. ?n = Five sets of five males each.
were first detected in the e j a c u l a t o r y b u l b o f D. buzzatii at 3 d a y s post-eclosion, a n d the a m o u n t o f 2-ketones s t o r e d in the e j a c u l a t o r y b u l b increased m o s t r a p i d l y between 4 a n d 6 d a y s o f age before reaching a p l a t e a u at 8 d a y s ( S c h a n e r a n d J a c k s o n , 1992). O u r e x p e r i m e n t s indicate t h a t r a d i o l a b e l was first i n c o r p o r a t e d into (Z)10-heptadecen-2-one at 4 d a y s post-eclosion, was m o s t actively i n c o r p o r a t e d between 5 a n d 6 d a y s o f age, a n d then t a p e r e d off. Similarly, label was first detected in 2 - t r i d e c a n o n e at 5 d a y s o f age, a n d was m o s t actively i n c o r p o r a t e d between 5 a n d 6 d a y s before t a p e r i n g off. As expected, there was a lag p e r i o d after label first a p p e a r e d in lipids before label b e g a n to a p p e a r in the p h e r o m o n e c o m p o n e n t s . This is consistent with the h y p o t h e s i s t h a t a labeled p r e c u r s o r p o o l needs to be established before the p h e r o m o n e can be p r o d u c e d . A l t e r n a t i v e l y , the a m o u n t o f label present in the 2-ketones c o u l d have been b e l o w detection ( < 70 cpm). A l t h o u g h label a p p e a r e d in lipids in the earliest incub a t i o n (1 h), the 2-ketones were n o t labeled until 4 h into the i n c u b a t i o n . Thereafter, the a m o u n t o f label incorp o r a t e d into the 2-ketones increased steadily until 16 h, with no increase in the a m o u n t o f label i n c o r p o r a t e d between 16 a n d 24 h. O f the subcellular fractions, the m i c r o s o m a l fraction i n c o r p o r a t e d the greatest a m o u n t o f label into the p h e r o m o n e c o m p o n e n t s . L a b e l e d 2-ketones were also p r o d u c e d by the m i t o c h o n d r i a l fraction from e j a c u l a t o r y bulbs. These results are consistent with p r e v i o u s studies o f p h e r o m o n e biosynthesis in o t h e r insects. B o t h the m i c r o s o m a l a n d m i t o c h o n d r i a l fractions f r o m houseflies c o n v e r t e d (Z)-9-tricosene to the e p o x i d e 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 , with the micro-
200
160
.,9 "~
&~ c
~
~ c
,2o 80
0 5.5
60
6.5
7.0
Z5
8.0
pH
FIGURE 2. pH Dependent biosynthesis of (Z)-10-heptadecen-2-one (O) and 2-tridecanone ( 0 ) from [lt4C]acetate by the microsomal fraction of D. buzzatii ejaculatory bulbs. Values are mean + standard deviation; n = five sets of five males each.
somes having the highest activity ( A h m a d et al., 1987). Similarly, subcellular f r a c t i o n a t i o n o f the p h e r o m o n e g l a n d from the spruce b u d w o r m indicated that the p h e r o m o n e biosynthetic activities involved in c o n v e r t i n g ( Z ) - l l - t e t r a d e c a n o i c acid into (Z)-I 1-tetradecyl acetate are located in the m i c r o s o m e s ( M o r s e a n d Meighen, 1986). In s u m m a r y , these in vitro e x p e r i m e n t s d e m o n s t r a t e d that a g g r e g a t i o n p h e r o m o n e biosynthesis in D. buzzatii is confined to the e j a c u l a t o r y bulb. In a d d i t i o n , only m a t u r e male flies (greater than 4 d a y s o f age) were c a p a b l e o f 2-ketone biosynthesis. T h e a m o u n t s o f 2ketones p r o d u c e d d u r i n g a 16 h i n c u b a t i o n were c o m p a r a b l e to the a m o u n t s p r o d u c e d by live males in the same time period. Similar to previous insect studies, the m i c r o s o m a l fraction from D. buzzatii e j a c u l a t o r y bulbs exhibited the greatest biosynthetic activity. Investig a t i o n s continue to o p t i m i z e the i n c u b a t i o n buffer with necessary c o f a c t o r s at a p p r o p r i a t e c o n c e n t r a t i o n s for m a x i m a l 2-ketone biosynthesis. The o t h e r subcellular fractions are being investigated to d e t e r m i n e whether they c o n t a i n m i c r o s o m e s with 2-ketone biosynthesis activity or w h e t h e r there are c o m p o n e n t s o f 2-ketone biosynthesis in these subcellular fractions.
REFERENCES
Ahmad S., Kirkland K. E. and Blomquist G. J. (1987) Evidence for a sex pheromone metabolizing cytochrome P-450 mono-oxygenase in the housefly. Archs Insect Biochem. Physiol. 6, 121-140. Bartelt R. J. and Jackson L. L. (1984) Hydrocarbon component of the Drosophila virilis (Diptera: Drosophilidae) aggregation pheromone: (Z)-10-heneicosene. Ann. ent. soc. Am. 77, 364-371. Bartelt R. J., Schaner A. M. and Jackson L. L. (1985) 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 pheromones in five taxa of the Drosophila virilis species group. Physiol. Ent. 11, 367 376. Bartelt R. J., Schaner A. M. and Jackson L. L (1989) Aggregation pheromone components in Drosophila mulleri: a chiral ester and an unsaturated ketone. J. chem. Ecol. 15, 399-412. Christie W. W. (1973) Lipid Analysis: Isolation, Separation, Identification and Structural Analysis of Lipids. Pergamon Press, Oxford. Ephrussi B. and Beadle G. W. (1936) A technique for transplantation in Drosophila. Am. Nat. 70, 218~25. Hara A. and Radin N. S. (1978) Lipid extraction of tissues with a low-toxicity solvent. Analyt. Biochem. 90, 420-426. 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 (Diptera: Drosophilidae). J. chem. Ecol. 13, 451-462.
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PAUL J. SKIBA and LARRY L. JACKSON
Morse D. and Meighen E. A. (1986) Pheromone biosynthesis and the role of functional groups in pheromone specificity. J. chem. Ecol. 12, 335 351. Schaner A. M. and Jackson L. L. (1992) (Z)-10-Heptadecen-2-one and other 2-ketones in the aggregation pheromone blend of Drosophila martensis, D. buzzatii, and D. serido. J. chem. Ecol. 18, 53 64. Schaner A. M., Benner A. M., Leu R. D. and Jackson L. L. (1989a) Aggregation pheromone of Drosophila mauritiana, Drosophila yakuba, and Drosophila rajasakari. J. chem. Ecol. 15, 1249 1257. Schaner A. M., Jackson L. L., Graham K. J. and Leu R. D. (1989b) (Z)-ll-Eicosenyl acetate, an aggregation pheromone
component in Drosophila malerkotliana. J. chem. Ecol. 15, 265-273. Vaz A. H., Blomquist G. J. and Reitz R. C. (1988) Characterization of the fatty acyl elongation reactions involved in hydrocarbon biosynthesis in the housefly, Musca domestica L. Insect Biochem. 18, 177--184.
Acknowledgements--This research was supported by NSF grant DBC-8509976. Contribution No. J-2790 from the Montana Agricultural Experiment Station.