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Sex pheromone biosynthesis in the leafroller moth Planotortix excessana by ╬Ф10 desaturation.

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Archives of insect Biochemistry and Physiology 8:l-9 (1988)
Sex Pheromone Biosynthesis in the Leafroller
Moth Planotortrix excessana by A10
Desaturation
S.P. Foster and W.L. Roelofs
Department of Entomology, New York State Agricultural Experiment Station, Geneva
With the use of deuterium-labeled saturated fatty acids coupled with gas
chromatography-mass spectrometric analysis, biosynthesis of the sex
pheromone component (a-8-tetradecenyl acetate in the greenheaded
leafroller moth Planotortrix excessana was shown t o proceed via A10
desaturation of palmitate. The resultant (Z)-lO-hexadecenoate is two carbon
chain-shortened t o the precursor (a-8-tetradecenoate. The minor component
(a-10-tetradecenyl acetate i s biosynthesized by A10 desaturation of myristate.
This is the first confirmation of A10 desaturation in an eukaryotic system.
Key words: deuterium-labelling, 0-8-tetradecenyl acetate, 0-lo-tetradecenyl acetate,
Planofortrix excessana (greenheaded leafroller moth), delta-10 desaturation, sex
pheromone biosynthesis
INTRODUCTION
Insect sex pheromones, particularly in the Lepidoptera, have been extensively studied over the last 20 years [l], especially with regard to chemical
identification and behavioral responses. In recent years, one area of this field
that has received more attention is the biosynthesis of insect sex pheromone
chemicals. The similarity of many Lepidopteran sex pheromone chemicals to
naturally occurring fatty acids suggests that sex pheromone biosynthesis in
the Lepidoptera occurs by a similar fatty-acid synthesis.
Acknowledgments: We thank Wanda Hansen and Dr J.R. Clearwater for rearing and shipping
the insects to New York. We thank the New Zealand-United States of America Cooperative
Science Program for a grant (to SPF) aiding this cooperation. This research was supported by
National Science Foundation grant PCM-8406348.
Dr. Foster i s a visiting scientist from the Entomology Division, DSIR, Mt. Albert Research
Centre, Private Bag, Auckland, New Zealand.
Received September 9,1987; accepted March 7,1988.
Address reprint requests to Dr. S.P. Foster, Department of Entomology, New York Agricultural
Experiment Station, Geneva, N Y 14456.
0 1988 Alan R. Liss, Inc.
2
Foster and Roelofs
Early radiolabelling studies on Trichoplusia ni, the cabbage looper [2],
confirmed that the major component (Z)-7-dodecenyl acetate was biosynthesized from acetate. More-recent work has begun to determine the different
processes involved in pheromone biosynthesis. A two-carbon chain-shortening [3] enzyme and a A l l desaturase [4] have been identified in several
moths, and their general use has been invoked to explain the biosynthesis of
many known sex pheromone chemicals in moths [5,6].
Although the use of these two classes of enzymes in sex pheromone
biosynthesis is probably common in the Lepidoptera, there are also a number
of sex pheromone chemicals that are unlikely to be explained by biosynthesis
involving A l l desaturation. One such chemical is Z8-14:OAc, which among
other examples is produced by the endemic New Zealand leafroller moth
Planotortrix excessanat (Walker). Galbreath et al. [q identified the sex pheromone of a Christchurch (midcoastal South Island) population of this species
as a mixture of Z8-14:OAc and 14:OAc. Following this, Lofstedt and Roelofs
[8] analyzed the sex pheromone gland of this same population for fatty acyl
intermediates and found the likely precursors, Z8-14:Acyl and 14:Acyl, as
well as relatively large quantities of the unusual Z10-16:Acyl. They proposed
that the sex pheromone component Z8-14:OAc is biosynthesized from Z1016:Acyl, which is itself formed by A10 desaturation of palmitate.
We have now studied this system using deuterium-labelled fatty acids
coupled with GC-MS* [9,10], to show that all known pheromone components can be biosynthesized from palmitic acid. Moreover, the unsaturated
component, Z8-14:OAc, is shown to be biosynthesized by the route proposed
by Lofstedt and Roelofs [8].
METHODS AND MATERIALS
Insects and Gland Extracts
P. excessuna were reared in New Zealand from a colony originally collected
from Christchurch. They were fed on a synthetic diet incorporating dried
Acrnena srnithii (Poiret) [q.Pupae were sexed in New Zealand, and young
female pupae only were shipped to Geneva, New York, by airfreight. Upon
arrival, pupae were placed in a 16:8 1ight:dark cycle and were used 2 days
after emergence, approximately half an hour before the onset of the scotophase period. Insects that had emerged en route were used immediately
upon arrival.
Pheromone extracts were made by dissecting the extruded pheromone
glands with fine forceps under a binocular microscope and placing the
+Tortricidae: Tortricinae
*Abbreviations: CI = chemical ionization; DMSO = dimethyl sulfoxide; FAMEs = fatty acid
methyl esters; CC = gas chromatograph; CC-MS = gas chromatography-mass spectrometry;
D3-14:COOH = [14,14,14-D3]myristic acid; D3-16:COOH = [16,16,16-D3]palmitic acid; 12:OAc
= dodecyl acetate; 13:OAc = tridecyl acetate; 14:OAc = tetradecyl acetate; Z8-14:OAc =
8-tetradecenyl acetate; Z10-14:OAc = (a-IO-tetradecenyl acetate; 16:OAc = hexadecyl acetate;
14Acyl = tetradecanoate; Z8-14Acyl = (a-8-tetradecenoate; Z10-14:Acyl = (a-lo-tetradecenoate; Z10-16:Acyl = (a-IO-hexadecenoate.
(a-
Sex Pheromone Biosynthesis in the Leafroller Moth
3
excised glands in approximately 10-20 pl of distilled Skelly B (petroleum
ether fraction). The glands were left to extract for approximately 16 h at
ambient temperature before analysis. Extracts for analysis of FAMEs were
made by the same method, except that glands were allowed to extract in
distilled dichloromethane for 16 h at 3°C.
The fatty acyl groups present in the gland extract were converted to the
corresponding methyl esters by base methanolysis [ll]. The dichloromethane
extract was decanted away from the glands and evaporated to apparent
dryness with a gentle stream of nitrogen. The residue was allowed to react
with 25 p1 of 0.5 M KOH in methanol for at least 30 min at ambient temperature. The products were acidified by the addition of 25 pl of 1.0 M HC1
(aqueous). The resultant FAMEs were extracted with 50 p1 of Skelly B.
The pheromone gland of the female moth was extruded and held in this
position by a closed alligator clip. The labelled acids (see below) were applied
topically as DMSO solutions (ca. 10 pg per p1) to the extruded gland. Approximately 0.2 pl of DMSO was applied to the gland by a 1.0 pl syringe. The
insect was placed in the dark with the gland extruded for about an hour, to
allow the DMSO to absorb into the gland, after which the clip was removed
and the gland allowed to return to its normal position. The insects were
placed in the dark for an additional 3 h until the glands were dissected and
extracted. In these experiments a large amount was applied to the gland, and
losses of the liquid occurred as a result of absorption into other parts of the
insect. As a result, no attempts were made to quantify the fate of the labelled
fatty acid. Extracts of 5-7 female pheromone glands were analysed by GCMS.
Chemicals
Omega labelled D3-16:COOH and D3-14:COOH were purchased from
KOR Isotopes, Cambridge, MA, and ICON Services Inc., Summit, NJ, respectively. They were both greater than 98% isotopically pure, as determined
by mass spectrometry.
Synthetic reference acetates and FAMEs were generally available from this
laboratory. FAMEs that were not available were prepared from the corresponding carboxylic acid by acid methanolysis [l2],by reaction with a methano1:benzene:sulfuric acid (30:15:1) solution at 100°C. The carboxylic acids, if
not available, were synthesized from the alcohol by reaction with a solution
of pyridinium dichromate in dimethylformamide [B]
.
Analyses
Before mass spectral analysis, extracts were analyzed by capillary gas
chromatography with a Hewlett-Packard 5880 GC with splitless injector and
flame ionization detector. A polar 50 m x 0.25 mm i.d. Silar lOC, fused silica
column (Quadrex Corporation, New Haven, CT was used. The carrier gas
was nitrogen at a linear flow velocity of l2 cm s-! The GC was programmed
80-140°C at 5°C min-' after an initial delay of 1min, then to 180°C at 2°C
min-l. These conditions allowed some resolution of synthetic standards of
isotopomers of both acetates and FAMEs. An internal reference standard of
13:OAc was used.
4
Foster and Roelofs
To follow the fate of the deuterium label a Hewlett-Packard 5985 Quadrupole mass spectrometer interfaced with a Hewlett-Packard 5840 GC (with
splitless injection) was used in the CI mode with isobutane as the reactant
gas. For increased sensitivity, the mass spectrometer was used in the selected
ion mode. The mass spectrometer was capable of monitoring up to five ion
groups (at different time periods), with up to four ions per group. Each ion
in a time group was scanned for 250 ps consecutively every second. For the
types of chemicals studied here, CI mass spectrometry with isobutane gives
the molecular ion plus one mass unit [(M+l)+] as the most intense ion of
the spectrum. Therefore, the ion corresponding to the (M+4)+ ion of the
unlabeled compound was scanned to observe incorporation of the D3-label.
In addition to the two aforementioned ions, the corresponding (M+2)+ and
(M+3)' ions were also generally scanned, primarily to observe the diminution of intensity and increase of the corresponding (M+4)+ ion when label
was detected.
The interfaced GC was equipped with a 30 m x 0.25 mm i.d Sulpelcowax
10 (Supelco Inc., Bellefonte, PA) fused silica column. Helium at 20 cm s-l
was used as carrier gas, with a temperature program of 80-200°C at 4°C
min-l after an initial delay of 3 min.
RESULTS
Gas Chromatography-Flame Ionization Detection
Analysis of synthetic mixtures of the methyl esters of D3-16:COOH and
unlabeled palmitic acid on the Silar 1OC column showed the deuterated
isotopomer eluted some 0.2 min earlier than the nondeuterated isotopomer.
This difference was insufficient to obtain baseline separation of the two
isotopomers, but mixtures of greater than approximately 10% of one component in the other could be detected. Analysis of an extract of P. excessunu
pheromone glands treated with D3-16:COOH gave leading edges on peaks,
with the retention times of Z8-14:OAc and 14:OAc (relative to internal 13:OAc
standard), indicating some incorporation of the label into these two pheromone components.
Mass Spectrometric Analyses
The results of mass spectrometric analyses of P. excessunu pheromone
glands untreated and treated with D3-16:COOH or D3-14:COOH are illustrated in Figures 1and 2.
Application of D3-16:COOH to the sex pheromone gland of P. excessunu
resulted in an increased intensity of the corresponding (M+4)+ ion of each
of the main pheromone components Z8-14:OAc (mlz = 255) and 14:OAc (ml
z = 257), relative to those ions monitored in extracts of control (untreated)
glands (see Fig. 1).An average incorporation of the D3-label into the two
pheromone components, Z8-14:OAc and 14:0Ac, of 14.7% and 12.4%, respectively, was obtained from two runs. Additionally, the corresponding
(M+4)+ ion of ZlO-l4:OAc, a minor component of the pheromone (Clearwater and Foster, unreported data) was enhanced relative to its intensity in
untreated glands.
Sex Pheromone Biosynthesis in the Leafroller Moth
CONTROL
+D3- 14:COOH
X10 ( M f 3 )
(Mf3)-
12 14 28 Z10 16
+D3-16:COOH
X10 (
12 14 Z8 Z10 16
5
M
4
3
)
L
XI0
12 14 Z8 210 NR
Fig. 1. Relative intensities of the corresponding ( M + l ) + , ( M + 3 ) + , and (M+4)+ ions of
compounds found in pheromone gland extracts from female Planotortrix excessana for
untreated glands (control), glands treated with 14,14,14-D3-myristic acid (D3-14:COOH), and
glands treated with 16,16,16-D3-palmitic acid (D3-16COOH). The control and D3-16:COOH
diagrams are the means of two CC-MS separate runs each, whereas the D3-14COOH diagram
i s from one CC-MS run. Abbreviations used: 12 = dodecyl acetate; 14 = tetradecyl acetate;
28 = (Z)-tl-tetradecenyl acetate; Z10 = (Z)-lO-tetradecenyl acetate; 16 = hexadecyl acetate; M
= molecular ion of a chemical.
In contrast, application of D3-14:COOH to the pheromone gland of P.
excessana resulted in no significant enhancement of the corresponding (M+4)+
ion of Z8-14:OAc. However, the corresponding (M+4)+ ions of 14:OAc and
Z10-14:OAc were enhanced in this treatment, relative to these ions in untreated glands. In addition, the corresponding (M+4)+ ion of l2:OAc was
enhanced, but no detectable enhancement of the (M+4)+ ion of 16:OAc was
observed.
When the resulting FAMEs were analyzed (see Fig. 2) after base methanolysis of glands treated with D3-16:COOH, incorporation of the D3-label into
the methyl esters of the putative precursors Z8-14:Acyl and 14:Acyl was
observed. Furthermore, the label was incorporated into the methyl esters of
laurate and palmitate and, most important, into Z10-16:Acyl.
The incorporation of label into the precursors Z8-14:Acyl and 14:Acyl was
much lower (1.4% and l.8%, respectively) than into the corresponding pheromone components.
DISCUSSION
Our results with deuterium-labeled saturated fatty acids show that the sex
pheromone components Z8-14:OAc and 14:OAc of P. excessma are both
6
Foster and Roelofs
+D3- 16:COOH
CONTROL
P+3)
,A
X10
14
28-14
210-14
210-16
(M+3)
d
x10
14
28-14
210-14
210-16
Fig. 2. Relative intensities of the corresponding (M + I ) + ,(M+3)+,and (M +4)+ ions of fatty
acid methyl esters from base-methanolysed pheromone gland extracts from female Planotortrix excessam for untreated glands (control) and glands treated with 16,16,16-D3-palmiticacid
(D3-16:COOH). The control diagram is from one run, whereas t h e D3-16COOH diagram is
the mean of two separate runs. Abbreviations used: 14 = methyl myristate; 28-14 = methyl
(3-8-tetradecenoate; 210-14 = methyl (3-IO-tetradecenoate; 210-16 = methyl (a-IO-hexadecenoate; M = molecular ion of a chemical.
biosynthesized from palmitic acid. In contrast, only 14:OAc can be biosynthesized directly from myristic acid. Another sex pheromone component of
P. excessunu, ZlO-l4:OAc, was also biosynthesized from myristic acid, as was
the saturated l2:OAc also found in the gland [S]. However, no label from
myristic acid was detected in the other saturated acetate found in the gland,
16:0Ac, (Foster, unreported data), suggesting that it cannot be biosynthesized
from myristic acid (at least to the detectable level of ca. 0.5% incorporation).
The incorporation of label from palmitic acid into the fatty-acyl precursors
of these chemicals is consistent with the results described here. The label is
incorporated into both putative precursors and additionally into the unsaturated hexadecenoate, Z10-16:Acyl. Our interpretation of these results (Fig. 3)
is the same as that of Lofstedt and Roelofs [S] and invokes the use of both
desaturation and two-carbon chain-shortening.
Our evidence for biosynthesis of ZS-14:OAc from palmitic acid is consistent with the first step being A10 desaturation of palmitate to produce Z1016:Acyl, which is chain-shortened by two carbons to the putative precursor
ZS-14:Acyl. This precursor in turn is reduced and acetylated to the pheromone component.
The alternative route of AS desaturation to produce ZS-14:Acyl directly
from myristate is highly unlikely because of the nonincorporation of label
from myristic acid into the pheromone component. Further evidence for a
Sex Pheromone Biosynthesis in the Leafroller Moth
HMMEWOATE
DELTA-10 DESANRAllON
/
//
----__
7
II
0
BnA-OXIDAllON
------+
'WR
ETPJDEWOATE
1
\
II
0
o
1
R
BEIA-OXIDATION
1
1
1
DELTA-10 OES4NRAllON
I1
R
R
(2)- 10-TEIWDECPIOATE
(Z)--&TEIRADECENOATE
1,
REOUCTIDN AN0
ACEMATlON
1
(Z)-O-TEIWDECPM
ACElATE
0
0
I1
I
REDUCTION AND
ACEMAllON
(Z)-lO-TEIWDECWrL
0
It
ACETATE
Fig. 3. Proposed pathways for biosynthesis of the sex-pheromone components, (a-8-tetradecenyl acetate (a-10-tetradecenyl acetate, and tetradecyl acetate in the leafroller moth
Planotortrix excessana.
A10 desaturase in the gland is provided by the presence of small amounts of
Z10-14:OAc and its precursor, Z10-14:Acyl, which are probably biosynthesized directly from myristate (see Fig. 3).
The chain shortening of Z10-16:Acyl to Z8-14:Acyl and of palmitate to
myristate and laurate probably occurs by &oxidation of the carboxyl group,
as reported for the chain shortening of palmitate to myristate in Argyvofaeniu
citrana sex pheromone biosynthesis [3]. In this chain-shortening process the
position of unsaturation shifts two carbons closer to the carboxyl group,
leaving the carbon atom in the omega position unchanged.
We have no evidence for chain elongation of added palmitic or myristic
acids in the gland, although it is possible that it occurs to a small extent, as
in A. velutinana [9], but remains undetected.
An interesting observation from these results is that the label was incorporated in relatively greater quantity into the pheromone components than
into the fatty acyl precursors. This phenomenon is also apparent in the
results from a similar study on A. velutinana [9]. Although free fatty acids
have not been found in pheromone glands of moths [14], this observation
implies that they are preferentially converted to a lipid that is more readily
converted to the pheromone components than other lipids containing the
fatty acyl precursors of the pheromone present in the gland. This would tend
to support the work of Bjostad and Roelofs [9], who failed to obtain incorporation of label from labelled triacylglycerols into the pheromone components
8
Foster and Roelofs
of A . velutinana. Although the triacylglycerols contain the greatest amount of
the pheromone precursors in this insect, this result led Bjostad and Roelofs
[9] to speculate that the triacylglycerols act as a “dumping ground’’ for the
excess acyl moieties not used in pheromone biosynthesis.
Our results are obtained from the entire lipid content of the gland; therefore, the isotopomer content of the fatty acyl moieties in all of the lipid
classes is averaged. If one lipid class (or chemical) is preferentially converted
to the pheromone, we may see incorporation of label in this class to the same
extent as observed in the pheromone components.
This investigation is the first demonstration of A10 desaturase activity in
an insect sex pheromone system and, to our knowledge, in any eukaryotic
system [15,16]. This unique A10 desaturase activity may be considerably more
widespread in moth sex pheromone biosynthesis. Although many femaleproduced moth pheromones may be explained by the action of a A l l desaturase, a number of species, especially in the Olethreutinae subfamily of the
Tortricidae, use even-numbered A8 and A10 unsaturated 12- and 14-carbon
compounds [5]. These chemicals may be formed directly by A10 desaturation
of a saturated intermediate, or less directly in combination with two-carbon
@-oxidationchain shortening. We hope to further characterize this desaturase
activity and make comparisons with the A9 and A l l desaturases found in
other systems [17,16].
LITERATURE CITED
1. Tamaki Y: Sex pheromones. In: Comprehensive Insect Physiology, Biochemistry and
Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, New York, Vol9, pp 145-191
(1985).
2. Jones IF, Berger RS: Incorporation of [l-’4C]acetate into cis-7-dodecen-1-01 acetate, a sex
pheromone in the cabbage looper (Trichoplusia ni). Envir Ent 7, 666 (1978).
3. Wolf WA, Roelofs WL: A chain-shortening reaction in orange tortrix moth sex pheromone
biosynthesis. Insect Biochem 23, 375 (1983).
4. Bjostad LB, Roelofs WL: Sex pheromone biosynthesis in Trichoplusia ni: Key steps involve
delta-11 desaturation and chain shortening. Science 220, 1387 (1983).
5. Roelofs WL, Brown RL: Pheromones and evolutionary relationships of Tortricidae. Annu
Rev Ecol Syst 23, 395 (1982).
6. Roelofs WL, Bjostad LB: Biosynthesis of Lepidopteran pheromones. Bioorg Chem 12, 279
(1984).
7. Galbreath RA, Benn MH, Young H, Holt VA: Sex pheromone components in the New
Zealand leafroller Planoforfrix excessana (Lepidoptera: Tortricidae). Z Naturforsch 40c, 266
(1985).
8. Lofstedt C, Roelofs WL: Sex pheromone precursors in two primitive New Zealand Tortricid moth species. Insect Biochem 25, 729 (1985).
9. Bjostad LB, Roelofs WL: Sex pheromone biosynthesis in the red-banded leafroller moth,
studied by mass-labeling with stable isotopes and analysis with mass spectrometry. J
Chem Ecol 12, 431 (1986).
10. Lofstedt C, Elmfors A, Sjogren M, Wijk E: Confirmation of sex pheromone biosynthesis
from (16-D3) palmitic acid in the turnip moth using capillary gas chromatography. Experientia 42, 1059 (1986).
11. Litchfield C: Analysis of Triglycerides. Academic Press, New York, p 32 (1972).
12. Mangold HK: Aliphatic Lipids. In: Thin Layer Chromatography: A Laboratory Handbook.
Stahl E, ed. Springer, New York, pp 363-424 (1969).
13. Corey EJ, Schmidt G: Useful procedures for the oxidation of alcohols involving pyridinium
dichromate in aprotic media. Tetrahedron Lett 5, 399 (1979).
Sex Pheromone Biosynthesis in the Leafroller Moth
9
14. Bjostad LB, Wolf WA, Roelofs WL: Total lipid analysis of the sex pheromone gland of the
redbanded leafroller moth, Argyrofueniu velufinunu, with reference to pheromone biosynthesis. Insect Biochem 11, 73 (1981).
15. James AT: The specificity of mammalian desaturases. Adv Exp Med Biol83, 51 (1977).
16. Wolf WA, Roelofs WL: Properties of the All-desaturase enzyme used in cabbage looper
moth sex pheromone biosynthesis. Arch Insect Biochem Physiol3, 45 (1986).
17. Wang DL, Dillwith JW, Ryan RO, Blomquist GJ, Reitz RC: Characterization of the acylCoA desaturase in the housefly, Muscu dornesficuL. Insect Biochem 12, 545 (1982).
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