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Inhibitory effect of 10 11-methyl-enetetradec-10-enoic acid on a Z9-desaturase in the sex pheromone biosynthesis of Spodoptera littoralis.

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Archives of Insect Biochemistry and Physiology 26:279-286 (1 994)
Inhibitory Effect of 10,11 -Methylenetetradec-10-Enoic Acid on a
Z9-Desaturase in the Sex Pheromone
Biosynthesis of Spodoptera littoralis
Laura Gosalbo, Gemma FabrSs, and Francisco Camps
Departament de Quimica Organica Biologica, CID-CSIC, Barcelona, Spain
The effect of 1 0 , l l -methylenetetradec-l O-enoic acid on the sex pheromone biosynthetic pathway of Spodoptera littoralis is reported. This new cyclopropene fatty
acid inhibited the biosynthesis of the main pheromone component from labeled
myristic acid. The study of each Z desaturation step revealed that the Z9-desaturase
of E l 1-14:Acid was inhibited, whereas the Z11-desaturase of 16:Acid was not
affected. The results presented in this article agree with our hypothesis that the
methylenehexadecenoic acids are beta-oxidized in the pheromone gland to the
corresponding methylenetetradecenoic acids. (0 1994 WiIey-Liss, Inc.
Key words: sex pheromone biosynthesis, desaturase, inhibition, Spodoptera iiftoralis
INTRODUCTION
In the Egyptian armyworm, Spodoptera liftoralis, the main sex pheromone
component, Z9,Ell-l4:OAc*, is synthesized from 16:Acid by chain-shortening
followed by sequential Ell-desaturation, Z9-desaturation, reduction, and acetylation (Martinez et al., 1990). Likewise, the Z11-desaturation of this 16:Acid
Acknowledgments: We thank Isabel Millan for rearing the insects used in this study, the Ministerio
de Educacion y Ciencia for a predoctoral fellowship to L.G., and CICYT for financial support (grant
ACF-92-0178).
Received June29, 1993; accepted October 8, 1993
Address reprint requests to Gemma Fabrids, Departament de Quimica Organica Biologica, CID-CSIC,
Jordi Girona, 18-26, 08034 Barcelona, Spain.
*Abbreviations used: DMSO = dimethylsulfoxide; d314:Acid = [14,14,1 4-2H3] myristic acid; dSE1114:Acid = [13,13,14,14,1 4 - 2 H ~(E)-l1
]
-tetradecenoic acid; d2g11-16:Acid = perdeuterated ( Z ) - l l hexadecenoic acid; d3116:Acid = perdeuterated palmitic acid; E l 1-14:Acid = (E)-l1-tetradecenoic
acid; FAME =fatty acid methyl ester; FID-GC = flame ionization detector gas chromatography; GC-MS
= gas chromatography coupled to mass spectrometry; MHAs = methylenehexadecenoic acids; MTAs
= methylenetetradecenoic acids; Z9,Ell-l4:Acid = (Z,E)-9,11 -tetradecadienoic acid; Z9-14:Acid =
(Z)-g-tetradecenoic acid; Z11-16:Acid = ( Z ) - l l-hexadecenoic acid; 10-MHA = 10,11 -methylenehexadec-10-enoic acid; 10- MTA = 10,ll -methylenetetradec-10-enoic acid; 12-MHA = 12,13methylenehexadec-12-enoic acid; 14:Acid = myristic acid; 16:Acid = palmitic acid; the same kind
of nomenclature has been used for both methyl esters and acetates replacing Arid by Me and OAc,
respectively.
0 1994 Wiley-Liss, Inc.
Gosalbo et al.
280
Z11-16:Acyl
Z9-14:Acyl
3
16:Acyl
Y
14Acyl
.u.
E-ll+ Ell-14Acyl
u
'-'
29,Ell-l4:Acyl j
Fig. 1. Biosynthetic pathway of S. liftoralis sex pheromone. Z-11,Zll -desaturation; E-I 1, E l l-desaturation; Z-9.Z-desaturation; -2C, chain shortening. Open arrows indicate reduction and acetylation.
followed by beta-oxidation and further reduction and acetylation gives rise to
Z9-14:OAc, a minor pheromone component (Fig. 1).
Previously (Arsequell et al., 1989; Gosalbo et al., 1992)we demonstrated that
the production of selected components of S. littoralis sex phermone can be
inhibited by several C-16 cyclopropene fatty acids with the ring at positions 10,
11, or 12. This effect is brought about by inactivation of two desaturases:
Z11-desaturase of 16:Acid and ZPdesaturase of Ell-14:Acid (Gosalbo et al.,
1992). On the basis of previously reported structure-activity relationships (Fogerty et al., 1972), the effect of the above MHAs on the latter enzyme was
unexpected. As a possible explanation, we suggested that these compounds
might be beta-oxidized to the corresponding C-14 cyclopropene acids, which
would be the actual inhibitors. Although this beta-oxidation in pheromone
glands has not been so far experimentally proven, the ability of selected MTAs
to inhibit the Z9 desaturase of Ell-14:Acid would indirectly support this
assumption. In this paper we report on the biological activity of one of these
compounds, namely ZO-MTA, on the sex pheromone biosynthetic pathway of
S. littoralis.
MATERIALS AND METHODS
Insects
Insects were maintained at 25 f 1°C with a 1ight:dark cycle of 16 h:8 h. Larvae
were reared on an artificial diet (Poitut et al., 1972).Pupae were sexed and adults
kept in separate containers. Adult females were separated daily before the onset
of the scotophase. Only virgin females that emerged within 5 h before lights-off
were used throughout this study.
Chemicals
DMSO was obtained from Sigma (St. Louis, MO), and Bom-PBAN was
obtained from Peninsula Laboratories (Belmont, CA). d31 16:Acid was purchased from Fluorochem Ltd. (Derbyshire, UK) and d314:Acid from IC Chemikalien (Munich, Germany). dsEll-14:Acid and 10-MTA were prepared in our
laboratory as described previously (Martinez et al., 1990; Gosalbo et al., 1993).
inhibition of a Z9-Desaturase
281
Treatments
Effect of 10-MTA on Z11-desaturation of d3116:Acid. Insects were briefly
anesthetized and immobilized 3 0 4 0 min before the onset of their second
scotophase and their pheromone glands topically treated with 0.1 pl of DMSO
(controls) or 0.1 pl of DMSO containing 0.1 pg of inhibitor. After a 30 rnin
incubation, 4 pg of d3116:Acid was topically applied to the glands, and the
insects, still immobilized, were placed back in the rearing chamber and the
glands excised 2 h later and processed for FAME analysis as indicated below.
Effect of 10-MTA on ZPdesturation of ds-Ell-14 Acid. These experiments
were performed as described above, using a dose of 1 pg of dsEll-14:Acid and
serial dilutions containing 0.001-1 pg of inhibitor.
Effect of PBAN on pheromone production. All the experiments that involved PBAN stimulation of pheromone production were performed 5 h before
lights-off. Females were anesthetized by brief cooling and injected with 4 p1 of
a PBAN solution in Meyers and Miller’s saline (Meyers and Miller, 1969)
containing the doses indicated. To study the effect of PBAN on the incorporation
of d3 14:Acid into pheromone, the glands were topically treated with 1 pg of
d314:Acid in DMSO (0.1 pl), and the insects were released 15 min later and
injected with 4 p1 of either saline or saline containing PBAN (12.5 pmols/female). The pheromone glands were excised 2 h after injection, and the amounts
of both natural and labeled pheromone were determined as described below.
Effect of 10-MTA on incorporation of d314:Acid into pheromone and intermediates. In these experiments, 5 h before the onset of the scotophase, the
glands were treated with DMSO (0.1 pl) with or without 10- MTA (0.1 pg) and,
after a 30 min incubation, with 1pg of d314:Acid. Insects were freed 15min after
the last topical application and injected with 4 p1 of a PBAN solution (12.5
pmol/female). The pheromone glands were excised 2 h later and extracted and
analyzed for pheromone and intermediate contents as described below.
Tissue Extraction and Analytical Methods
The Z11-desaturation of d3116:Acid was monitored by FID-GC using the
equipment and conditions previously reported (Arsequell et al., 1989; Gosalbo
et al., 1992). Individual pheromone glands were extracted with CHC13:MeOH
2:l (ST, overnight), and the d29Zll-l6:Me/d3116:Me ratios were calculated
after usual base methanolysis of the lipidic extracts.
The ZPdesaturation of d5El l-14:Acid was determined by GC-MS analysis of
methanolyzed extracts with the same equipment and conditions described in a
previous paper (Gosalbo et al., 1992). Groups of three glands were extracted
and methanolyzed under standard conditions, and the ratios between ions 243
and 245 (molecular ions for dsZ9,Ell-l4:Me and d5El1-l4:MeTrespectively)
were determined.
For pheromone titer determinations, individual glands were extracted with
40 pl of hexane containing 10 ng of 12:OAc as internal standard (1 h, room
temperature), and the samples were analyzed by FTD-GC as reported previously (Martinez and Camps, 1988).
The incorporation of d314:Acid into pheromone and intermediates was determined in groups of three pheromone glands, which were first extracted with
282
Cosalbo et al.
100 pl of hexane containing 60 ng of 12:OAc as internal standard (1 h, room
temperature). After pheromone extraction, the tissues were transferred and
soaked in 200 pl of CHC13:MeOH 2:l (SOC, overnight), and lipids thus extracted
were methanolyzed under usual conditions, adding 100 ng of 12:Me to the
samples before workup for quantification. The analyses were carried out by
GC-MS, using a Fisons gas chromatograph (8000 series) coupled to a Fisons MD800 mass selective detector. The system was equipped with a Hewlett Packard
HP-1 capillary column (30 m x 0.20 mm), which was programmed either from
60-300°C at 8"C/min (pheromone analysis) or from 80-220°C at 5"C/min and
then to 280°C at 10"C/min (FAME analysis). The selected ions monitored in
pheromone analysis were 168 (M.+-60for 12:OAc),252 (M.+for natural Z9,Ell14:OAc), and 255 (M.+for d39,Ell- 14:OAc).In analysis of intermediates, the
selected ions were 238 (M.7 for natural Z9,Ell-l4:Me), 241 (M.+for d a 9 , E l l 14:Me),240(M.+fornatural E11-14:Me),243 (M-+for d3Ell-l4:Me), and 245 (M.+
for d314:Me). To quantify the amounts of d39,Ell-14:OAc1 an aliquot of each
sample was also analyzed by FID-GC under the conditions previously reported
(Martinez and Camps, 1988). The actual amount of d3Z9,Ell-l4:OAc was
calculated as [255/(252 + 255)] multiplied by the total amount obtained in the
FID-GC analysis. To assess the effect of 10-MTA on the Z9-desaturase of
Ell-l4:Acid, the ratios between ions 241 and 243 were determined. Likewise,
the effect of 10-MTA on the Ell-desaturase of 14:Acid was estimated from the
ratios between ions 243 and 245.
Statistics
Data were analyzed by the unpaired two-tail t test, after log (x + 1)transformation of the data when variances were unequal.
RESULTS
The effect of 10-MTAon the different desaturases involved in the biosynthetic
pathway of S. Iitforalis sex pheromone was studied using several tracers and
experimental conditions. In a first set of experiments, the effect of 10-MTA on
Z11-desaturation of 16:Acid was investigated with d3116:Acid as labeled precursor, following the same procedure previously reported (Arsequell et al.,
1989; Gosalbo et al., 1992).No significant difference was found in the amounts
of d29Z11-16:Acid produced by controls and insects treated with 0.1 pg of
10-MTA, as concluded from the ratios between the corresponding perdeuterated methyl esters in the FID-GC analyses (Fig. 2).
In a second group of experiments, the effect of 10-MTA on the ZPdesaturation of Ell-14:Acid was studied using dsEll-14:Acid as tracer. The inhibitory
activity of 10-MTA on this step was found to be dose-dependent (Fig. 31, and
statistically significant effects were observed with all the doses tested, except
for 1 ng.
Finally, the effect of 10-MTA on sex pheromone production was studied with
d314:Acid as tracer. In order to stimulate its conversion into d3Z9,E11-14:OAc
during the photophase, PBAN was also injected into these females. In a first set
of experiments, we found that PBAN stimulated an increase in pheromone titer
in a dose-dependent manner when injected into whole females at mid-photo-
Inhibition of a Z9-Desaturase
283
DMSO 10-MTA
Fig. 2. Effect of 10-MTA on Z11 -desaturation of djil6:Acid. Ratios between dmZ11-16:Me and
d3116:Me, obtained from the GC traces, are given in the ordinate. Data are expressed as mean k SEM
of 8-1 0 individual replicates. The difference between control and treatment is not statistically
significant ( P > 0.1).
phase (Fig. 4A). After analysis of the dose-response curve, a dose of 12.5
pmols/female was chosen for further treatments. As found for natural pheromone, amounts of d39,Ell-l4:OAc formed from d314:Acid were higher in
females injected with 12.5 pmols of PBAN than in controls (Fig. 4B). However,
the females that had been treated with 10-MTA produced significantly lower
amounts of d$9,Ell-l4:0Ac from d314:Acid than controls after PBAN stimulation, as concluded from both FID-GC and GC-MS analyses (Fig. 5A). The analyses
of FAME profiles revealed that the ratios between ions 243 and 245 and those
between ions 241 and 243 were significantlylower in treatments than in controls
(Fig. 58). Thus, both the Ell-desaturase of l4:Acid and the Z9-desaWase of
Ell-14:Acid were apparently inhibited by 10-MTA in these experiments.
DISCUSSION
Our investigations on the development of desaturase inhibitors (Arsequell et
al., 1989; Gosalbo et al., 1992) have led us to a new cyclopropene fatty acid,
Fig. 3 . Effect of different doses of 10-MTA on Z9-desaturation of dsEl1- 14:Acid. Determinations
were performed as indicated in Materials and Methods. Abscissa: The amount of inhibitor administered. Ordinate: Ratios between the abundances of ions 243 (dsZ9,Ell-l4:Me) and 245 (dsEl114:Me) from the GC-MS analyses. Each point represents mean i SEM of eight determinations with
groups of three glands. Asterisks indicate statistical significance: * P < 0.05; **P i0.0005.
"':
cd
C
30 -
20 -
bd
fb
10 -
01 :a,
0
i
,
,
,
10
20
,
,
.
30
,
40
. ,
,
XI
dose (pmols)
B
T
1.0
0.5
0.0
Fig. 4. A: Dose-dependence of PBAN-stimulation of sex pheromone production in S. littoralis.
Abscissa: The amount of PBAN injected. Ordinate: Amounts of Z9,E11-14:OAc calculated from the
GC traces. Data are mean f SEM of six individual females, except for 50 pmols, with which only four
replicates were obtained. Statistical significance among means is indicated by different letters beside
each bar ( P < 0.05). B: PBAN effect on production of d3Z9,Ell-l4:OAc from d314:Acid. Bars represent
mean SE of six groups of three females. Differences between saline and PBAN-injected females are
statistically significant at P i 0.02.
+
H DMSO
E3 10-MTA
Fig. 5. A: Effect of 10-MTA on production of d3Z9,Ell-l4:OAc from dj14:Acid. Amounts of
d3Z9,Ell-l4:0Ac were obtained from both FID-GC and GC-MS analyses. B: Effect of 10-MTA on
the Z9-desaturase of E l 1-14:Acid. Ratios between ions 241 and 243 were obtained from the GC-MS
analyses. C: Effect of 10-MTA on the E l 1-desaturase of 14:Acid. Ratios between ions 243 and 245
were obtained from the CC-MS analyses. In all figures, data are means SE of nine groups of three
females/group. Differences between controls and treatments are statistically significant at P < 0.05 in
all cases. The same glands were used for the analysis of both pheromone and intermediates.
+
Inhibition of a Z9-Desaturase
285
10-MTA, which interferes with the Z9-desaturase of E l 1-14:Acid in the biosynthesis of the sex pheromone of S. littoralis.
In previous articles (Arsequell et al., 1989; Gosalbo et al., 1992) we reported
the ability of selected MHAs to inhibit both the desaturation of 16:Acid to
Z11-16:Acid and that of Ell-14:Acid to Z9,Ell-l4:Acid. Although the former
effect was expected, the latter was somewhat surprising, since, except for
10-MHA, these compounds did not have one of the critical structural requirements to inhibit the Z9-desaturase enzyme (Fogerty et al., 1972), namely neither
C-9 nor C-10, which are the positions desaturated in E l l - 14:Acid, included in
the ring. This requirement would be present in the C-14 cyclopropene fatty
acids that would result from beta-oxidation of these MHAs. Thus, we anticipated that the MHAs might get chain-shortened in the pheromone gland to the
corresponding MTAs, which would be the actual inhibitors of the Z9-desahrase
enzyme. Although final confirmation of this assumption awaits the detection
of MTAs in pheromone glands incubated with MHAs, the results presented in
this article support this possibility. Thus, 10-MTA, which would result from
chain-shortening of 12-MHA, inhibited the ZPdesaturase of E l I-14:Acid in
similar extent to that previously described for 12-MHA (Gosalbo et al., 1992)
under similar experimental conditions. However, it is noteworthy that the
activities exhibited by any of the MHAs assayed or by 10-MTA on the ZPdesaturase of Ell-l4:Acid are lower than those of the MHAs on the Z11-desaturase
of 16:Acid (ArsequeI1 et al., 1989; Gosalbo et al., 1992). On the other hand,
10-MTA had no effect on the Z11-desaturation of 16:Acid.This lack of activity,
as compared to the effectiveness of the C-16 analog 10-MHA (Gosalbo et al.,
1992), suggests that the length of the alkyl substituent at C-12 is relevant for this
inhibitory activity.
The effect of 10-MTA on pheromone production was studied using d314:Acid
as tracer. In previous papers (Arsequell et al., 1989; Gosalbo et al., 1992),
treatments with both inhibitor and precursor were performed shortly before the
onset of the scotophase, and pheromone gland excision was carried out 2 h into
the dark period, when maximum pheromone titers are reached (Martinez and
Camps, 1988). However, since we found that insect handling close to the
scotophase caused a threefold reduction in pheromone production and that
pheromone biosynthesis could be stimulated in the photophase by injection of
brain-subesophageal ganglion extracts (Martinez et al., 19901, we decided to
carry out these inhibition experiments in the photophase, using synthetic PBAN
to stimulate pheromone production. In a first series of experiments, we confirmed that synthetic Born-PBAN was able to cause an increase in pheromone
titer in S. littoralis. Likewise, we found that, as previously observed for different
labeled precursors and with brain-subesophageal ganglion extracts (Martinez
et al., 19901, females treated with dsl4:Acid and further injected with PBAN
produced higher amounts of labeled pheromone than saline-injected animals.
In these experiments, amounts of natural Z9,Ell- 14:OAc were also higher in
PBAN than in saline-injected insects (mean k SE: 23.4 k 3.5 and 6.8 k 2.1,
respectively; n = 6; P < 0.002). In the inhibition studies, insects that had been
treated with 10-MTA produced significantly less d3Z9,Ell-l4:OAc from
d314:Acid than controls after PBAN stimulation. The analysis of intermediates
confirmed that inhibition of the Z9-desaturase of Ell-14:Acid was involved in
286
Gosalbo et al.
the observed reduction in labeled pheromone production. In these experiments,
inhibition of the Ell-desaturase of 14:Acid did apparently occur as well. However, we must not disregard the possibility that inhibition of this E l l-desaturase
may not be caused by 10-MTA itself, but by the dsEll-14:Acid that accumulates
as a result of inhibition of the Z9-desaturase.
In summary, we have prepared a new cyclopropene fatty acid, structurally
related to myristic acid, which inhibits the Z9-desaturation of Ell-14:Acid to
the corresponding diene system, but it does not affect the Z11-desaturation of
16:Acid. Further studies about the mode of action of these inhibitors on the
different desaturases involved in the biosynthetic pathway of S. littoralis sex
pheromone are underway in our laboratory.
LITERATURE CITED
Arsequell G, Fabrias G, Camps F (1989): Inhibition of a delta-11 desaturase in Spodoptera littoralis
by 12,13-methylenehexadec-12-enoic
acid. Insect Biochem 19:623427.
Fogerty AC, Johnson AR, Pearson JA (1972): Ring position in cyclopropene fatty acids and stearic
acid desaturation in hen liver. Lipids 7:335-338.
Gosalbo L, Fabrias G, Arsequell G, Camps F (1992): Inhibition of sex pheromone biosynthesis in
Spodopteru littoralis by cyclopropene fatty acids. Insect Biochem Mol Biol22:687490.
Gosalbo L, Barrot M, Fabrias G, Arsequell G, Camps F (1993):Synthesisof deuterated cyclopropene
fatty esters structurally related to palmitic and myristic acids. Lipids 28:11251130.
Martinez T, Camps F (1988):Stimulation of sex pheromone production by head extract in Spodopteru
litfordis at different times of the photoperiod. Arch Insect Biochem Physiol9:211-220.
Martinez T, Fabrias G, Camps F (1990):Sex pheromone biosynthetic pathway in Spodoptera littoralis
and its activation by a neurohormone. J Biol Chem 2651381-1387.
Meyers J, Miller T (1969): Starvation-induced activity in cockroach Malpighian tubules. Ann
Entomol SOCAm 62:725.
Poitut S, Bues R, Le Rumeur C (1972): Elevage sur milieu artificiel simple de deux noctuelles
parasites du cotton Earias insulana et Spodoptera littoralis. Ent Exp Appl20:341-350.
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