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Stimulation of sex pheromone production by head extract in Spodoptera littoralis at different times of the photoperiod.

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Archives of Insect Biochemistry and Physiology 211-220 (1988)
Stimulation of Sex Pheromone Production by
Head Extract in Spodoptera littoralis at
Different Times of the Photoperiod
Teresa Martinez and Francisco Camps
Department of Biological Organic Chemistry, Centro de Investigacidn y Desarroilo (C.S.I. C.),
Barcelona, Spain
Adult females of Spodoptera littoralis (Lepidoptera: Noctuidae) showed a
cyclic pattern of sex pheromone production, and high titers of (Z,E)-9,11tetradecadienyl acetate, the major component of its pheromone blend, were
only detected during scotophase. Maximal amounts of pheromone were
extracted approximately 2 h into second scotophase. Decapitation before the
beginning of darkness inhibited normal production of pheromone, and no
calling behavior was observed. Injection of brain-suboesophageal ganglion
(Br-SOG) homogenate at the onset of scotophase restored pheromone
production in decapitated females to the levels characteristic of second
scotophase. Pheromone biosynthesis was also stimulated in decapitated
females during photophase. The response to Br-SOG homogenate injection
was dose-dependent. The pheromonotropic activity of Br-SOC extract was
the same when females were injected during photophase or scotophase.
Key words: pheromone biosynthesis-activating neuropeptide, calling behavior, Lepidoptera
The occurrence of circadian rhythms of sexual activity in Lepidoptera
suggests that production and release of sex pheromone as well as calling
behavior are well-regulated physiological events.
Research on the control mechanism(s) of sex pheromone production in
this order has only begun recently. Although in the leafroller moth, Platynota
Acknowledgements: We thank Dr. Cemma Fabrias for the mass spectrometry analyses and
her interest in this work and Dr. Jan A. Veenstra for valuable discussions and his help in the
preparation of the manuscript. Financial support from C.A.I.C.Y.T. (grant 84/0087), C.S.I.C.
(grant 85/263), and a postdoctoral fellowship (to T.M.) from the Spanish Ministry of Education
and Science i s gratefully acknowledged.
Received June 17,1988; accepted September 12,1988.
Address reprint requests to Dr. Teresa Mart'tnez,Department of Biological Organic Chemistry
(C.S.I.C.), Jordi Cirona Salgado 18-26,08034 Barcelona, Spain.
0 1988 Alan R. Liss, Inc.
Martinez and Camps
stultana, exogenous treatment with the juvenile hormone analog ZR-512 was
found to reduce pheromone titers in virgin females [l],no involvement of
the corpora d a t a in the control of pheromone production has been reported
in two other species studied, Heliothis zeu [2] and Lyrnantria dispur [3].
Raina and Klun [2] provided the first experimental evidence that a hormonal factor from the head was involved in the control of sex pheromone
production in females of H.zeu. These researchers indicated that the active
factor was present in the brain both during photophase and scotophase, but
it was only released into the hemolymph during the scotophase to stimulate
pheromone production. Subsequently, the pheromonotropic factor was found
to be a neuropeptide originating in the subesophageal ganglion [4].Similar
pheromone biosynthesis-stimulating activity has recently been reported
in head extracts of two other moth species [5,6]. On the other hand, a neural mechanism has been partially implicated in the control of pheromone
production in L. dispur, based on experiments of ventral nerve cord transsection [3].
In the present study, the control of sex pheromone production in Spodopteru littoralis, a widespread polyphagous pest, has been addressed. We have
focused our attention on the possibility of stimulating pheromone biosynthesis during the photophase in a species that normally produces and releases its sex pheromone during darkness [7l,
S. littoralis were reared in the laboratory at 25 _+ 2°C with a 16L:8D
photoregime. Larvae were fed an artificial diet [8], and adults were provided
with a 10% aqueous sucrose solution. Pupae were sexed, and adults were
kept in separate containers. Most animals emerged within 3 h before the
onset of scotophase; these were the only ones used in the experiments.
Pheromone Gland Extraction
The ovipositors, containing the pheromone gland, were excised from
females at the corresponding time and extracted individually (overnight, at
4°C) with 50 p1 dichloromethane containing 20 ng of dodecyl acetate as
internal standard. Extracts were stored at -20°C until GLC* analysis.
Gas-Liquid ChromatographicAnalyses
Samples were carefully concentrated under nitrogen, and 1pl was injected
into a capillary column. GLC was performed on an instrument equipped
with a splitless injector and a flame ionization detector. The purge valve was
*Abbreviations: Br-SOG = brain-subesophageal ganglion; GLC = gas-liquid chromatography; MS = mass spectrometry; Z9,Ell-I4:Ac = (Z,E)-9,1l-tetradecadienyl acetate; Ell-14Ac =
(E)-11-tetradecenyl acetate; Z9-14Ac = (Z)-g-tetradecenyl acetate; Z11-14Ac = (Z)-11-tetradecenyl acetate.
Stimulation of Sex Pheromone Production
opened 0.5 min after injection, and hydrogen (carrier gas) flow was 1.5 ccl
min at 30°C. A SPB-5 (30 m X 0.25 mm i.d.) fused silica Supelco column was
programmed from 120°C to 220°C at 5"Clmin after an initial delay of 4 min.
A polar fused silica Supelco column SP-2340 (30 m x 0.25 mm i.d.) was also
used in some experiments, since it allows a better separation of the minor
components of the sex pheromone blend. This column was programmed
from 80°C to 220°C at 5"Clmin after an intial delay of 10 min. Authentic
standards were used to determine the retention times of the pheromone
components: (Z,E)-9,ll-tetradecadienyl acetate, (Z)-9-tetradecenyl acetate,
(E)-11-tetradecenyl acetate, and (Z)-11-tetradecenylacetate [9,10]. Some samples were also analyzed by GLC-MS to confirm their presence in the pheromone blend. Capillary GLC-MS with electron impact mode was performed
on a Hewlett-Packard 5995 system, at 70 eV, coupled with a 5997C computer
system. The columns mentioned above, at the indicated temperature programs, were used. The characteristic ions of each compound were detected.
Throughout this study pheromone titers refer to (Z,E)-9,Stetradecadienyl
acetate, the major component of S. liftoralis sex pheromone blend.
Preparation and Injection of Br-SOG Homogenate
Complexes of Br-SOG were dissected in 0.9% NaCl from male or female
adults (- 24 h old). The optic lobes were excised, and the complexes were
immediately transferred to a homogenizer stored on dry ice. Br-SOG was
homogenized just before injection in Meyers and Miller's saline, containing
188.0 mM NaCl, 20.1 mM KC1, 9.0 mM CaC12, and 1.0 mM MgS04 Ill]. Two
Br-SOG equivalents (5-pl volume) were injected into the abdomen of decapitated females briefly anesthetized with C02, using a 50-p1 Hamilton syringe.
Injections were done at two times, either 10 h into the second photophase or
close to the onset of the second scotophase. Decapitation in the latter case
was always performed at least 20 min before darkness. Females were kept in
normal rearing conditions after injection, and pheromone glands were excised and extracted 2 h later. Thus, in the first case the glands were removed
from the animals during the photophase, and in the second case glands were
removed approximately 2 h into scotophase (when peak pheromone titers
were detected in intact virgin females).
Groups of 55-60 Br-SOG complexes from - 24-h-old females were homogenized in saline to yield a final concentration of four Br-SOG in 5 pl.
Dilutions were made containing 4, 2, 1, 0.5, and 0.25 Br-SOG equivalents in
5 p1. Each dose was injected into five second photophase females that had
been previously decapitated. Controls were injected with 5 pl saline. The
experiment was performed twice.
Data presented in Table 1were analyzed by the parameter-free Wilcoxon
two-sample test.
Martinez and Camps
TABLE 1. Stimulation of Pheromone Biosynthesis in Decapitated Females of S. littoralis by
Br-SOG HomogenateP
Experiment Donor
ng Z9,Ell-l4:Ac/female,
Intact females
4.0 f 1.4 (4) 20.3 1.5 (7)
6.9 & 1.5 (4) 33.6 f 3.3 (8)
9.8 f 0.7 (5) 43.0 f 5.5 (5) 24.3 f 2.5 (5)
31.6 f 5.8 (5)
2.8 f 0.6 (4)
29.2 f 3.4(5)
% + SEM (n)
3.9 f 0.8 (5) 26.7 f 2.4 (7)
4.2 + 1.5 (4) 39.4 f 2.1 (8)
2.7 f 0.3 (5) 26.4 & 1.1 (6)
2.5 + 0.3 (6) 18.0 f 1.3(6)
2.6 f 0.5 (5) 17.4 f 1.5(5) 6.4 f 0.9(5) 18.1 rt 2.7(5) 15.7 f 2.2(5)
20.4 3.2 (6)
22.6 + 2.7(6)
3.8 0.5 (6)
7.3 f 0.8 (6)
24.7 3.8 (6)
20.3 f 0.8 (6)
22.0 f 1.7(6)
*Second-photophase or scotophase females were used, except in experiments 8 and 9, in
which animals were 1day older. Pheromone titers of Br-SOG-injected females are in all cases
significantly different from those of saline-injected controls ( P < 0.01). Differences between
pheromone titers obtained after stimulation with Br-SOG homogenates from males and
females are not statistically significant (experiments 4 and 10; P > 0.20), nor was a significant
difference found between titers of females stimulated during photophase and scotophase
(experiments 9 and 10; P > 0.20).
In order to be able to examine the stimulatory activity of Br-SOG homogenate on pheromone production, we first determined times of maximum and
minimum pheromone titers in intact females. Analyses were performed
during the first 5 days of adult life, and pheromone glands were removed
and extracted the last hour of photophase and 2 h into scotophase. High
pheromone titers were detected only during scotophase and peaked in the
second scotophase (X = 28.6 ng per female). Comparatively low amounts
were found 1h before the onset of darkness (X = 4.7 ng per female) (Fig. 1).
In order to confirm the approximate time of maximum pheromone content
in the gland, hourly determinations were made during the first part of second
scotophase. A rapid increase followed during the first hour of darkness, and
at 2-3 h after lights-off the highest amounts of (Z,E)-9,1l-tetradecadienyl
acetate were extracted (X = 26.8 ng per female). From the third hour of
scotophase pheromone titers began to decrease (Fig. 2).
Three groups of experiments (summarized in Table 1) were performed
with decapitated S. littoralis females to determine the pheromonotropic activity of Br-SOG homogenates from males and females. Stimulation of pheromone production was initially attempted at the time pheromone titer normally
peaks in intact females. Injection of Br-SOG homogenate was close to the
beginning of the second scotophase, and pheromone glands were excised 2
Stimulation of Sex Pheromone Production
Female age ( h )
Fig. 1. Sex pheromone titers (expressed as ng of (Z,E)-9,11-tetradecadienyl acetate) in
S. littoralis virgin females as a function of age. Pheromone glands were removed in the last
hour of photophase and 2 h into scotophase. Stippled bars refer to scotophase (n = 10-15)
and open ones to photophase (n = 8). Data represent means f SEM. Arrow indicates
approximate time of emergence (2 h before darkness).
h later. Decapitation before darkness inhibited the increase in pheromone
titer characteristic of second-scotophase females. However, injection of two
Br-SOG equivalents restored pheromone production in decapitated females.
The average extractable amounts of (Z,E)-9, ll-tetradecadienyl acetate were
similar (sometimes even higher) to those of intact second-scotophase females
(Table 1, experiments 1-4). In one of these experiments, Br-SOG homogenates from male and female donors were tested on the same group of females
(emerged simultaneously) in order to compare the pheromonotropic activity
of Br-SOG in both sexes. No significant difference was found between
pheromone titers obtained after stimulation with the two types of homogenate (Table 1, experiment 4; P > 0.20); a significant difference between BrSOG homogenates from males and females was also not found in a later
experiment (Table 1, experiment 10; P > 0.20).
In the next set of experiments a possible stimulatory effect of Br-SOG on
pheromone production was investigated during photophase, when only low
amounts of pheromone were detected in S. ZittomZis females. Injection of two
Br-SOG equivalents into second-photophase decapitated females stimulated
pheromone production. Mean pheromone titers were similar (or even higher)
to those of intact second-scotophase females (Table 1, experiments 5-8).
Martinez and Camps
Second scotophase ( h1
Fig. 2. Sex pheromone titers (expressed as ng of (Z,E)-9,11-tetradecadienyl acetate) of virgin
females during the second scotophase (first 5 hours). Determinations were made hourly from
the beginning of darkness (0 h). Each point represents mean & SEM (n = 10).
These results suggested that the active substance was as effective in photophase as in scotophase. In order to confirm this notion, stimulation with a
single Br-SOG extract was attempted in the same group of females, during
photophase and the following scotophase. This experiment was performed
once with Br-SOG from male donors and twice with Br-SOG from females.
No significant difference was found between pheromone titers of females
stimulated during photophase and those stimulated during scotophase (Table 1,experiments 9 and 10; P > 0.20).
Stimulation of pheromone production during photophase was found to
be dependent on the dose of Br-SOG homogenate injected (Fig. 3).
The relative amounts of (Z,E)-9,1l-tetradecadienylacetate and minor components of the sex pheromone blend of S. littoralis were determined in intact
second-scotophase females (2 h into scotophase) and compared with those
in decapitated females injected with Br-SOG homogenate. Females stimulated at the onset of scotophase showed a pheromone blend with normal
proportions of its components. However, when stimulation was performed
during photophase, the ratio of (Z)-9-tetradecenyl acetate to (Z,E)-9,11-tetradecadienyl acetate was found to be higher than in intact second-scotophase
females (Table 2).
It is worth mentioning that after decapitation females exhibited repetitive
abdominal movements with extrusion and retraction of the ovipositor; in
Stimulation of Sex Pheromone Production
5 -
Br-SOG equivalents
Fig. 3. Effect of Br-SOG homogenate injection into decapitated females on pheromone titers
(expressed as ng of (Z,E)-9,11-tetradecadienyl actetate). Injections and pheromone gland
excisions were performed during the second photophase. Each point represents mean
SEM (n = 10). S, saline-injected females.
TABLE 2. Relative Amounts (YO)of Pheromone Components in S. littoralis Females*
Br-SOG homogenate-injected females
5.6 f 0.3
10.4 0.8
13.0 k 0.8
70.9 f 1.0
5.3 k 0.4
9.5 f 0.5
12.7 k 1.3
72.4 f 0.9
5.0 k 0.5
10.3 f 0.8
17.9 k 2.0
66.9 f 1.4
*Data represent means f SEM (n = 9).
ahtact second-scotophase females.
some females this behavior was accompanied by oviposition. However, animals were never observed to expose their pheromone gland or display a
"calling posture." Thus, although injection of Br-SOG homogenate induced
pheromone production in decapitated females, those animals were not observed to call.
Like other moth species, S . Zitturdis virgin females show a cyclic pattern of
sex pheromone production. Only during the scotophase, when the females
display a calling behavior, were high pheromone titers detected. Maximal
amounts of pheromone (25-30 ng) were extracted during the second and
Martinez and Camps
third scotophases ( l-2-day-old females), and gland pheromone content
peaked around 2 h into scotophase.
In a recent study, Dunkelblum et al. [7] published some data on pheromone titers in S. littovalis females, indicating that 6-8 ng of (Z,E)-9,ll-tetradecadienyl acetate were the largest amounts extracted from l-3-day-old
females. Although we can not rule out the possibility of strain differences as
a partial explanation for the disparity between their results and ours, the
extraction procedure (15 min in hexane) used by Dunkelblum et al. [7l is
probably the main reason for the above differences. We found an average of
8 ng per female when following their procedure. However, if the same glands
were subsequently extracted in dichloromethane (overnight, at 4"C), an
average of 16.5 ng of additional pheromone per female was obtained.
On the other hand, studies on pheromone titer in different moth species
show that individual variation in the quantity of gland-extractable sex pheromone is frequently observed [l2,13]. We have also found some variability in
the responsiveness to stimulation with Br-SOG homogenate. For example,
when comparing the average pheromone titers obtained in the experiments
presented in Table 1with mean titers in the dose-response experiment, the
latter values are relatively low. However, in this type of experiment, we
cannot rule out the possibility that some enzyme degradation of the active
factor has occurred during the serial dilutions of the homogenate. A rapid
loss of biological activity in crude Br-SOG preparations has been reported in
Heliothis [4].
The fact that pheromone production and sexual activity in S. littoralis
appear to be restricted to a certain time of the photoperiodic cycle indicates
that precise control mechanisms must be implicated. Decapitation of S.
littoralis virgin females before the onset of the second scotophase was found
to inhibit normal production of pheromone. Only reduced titers were detected in those females 2 h after lights-off. Calling behavior was also inhibited
after decapitation, although females showed repetitive abdominal movements with extrusion of the ovipositor. These findings are in agreement with
the observations reported by Tang et al. [3], who indicated that females of L.
dispar did not call normally after transsection of the ventral nerve cord. In the
moths P. stultana [l] and Chilo suppressalis [5], calling was also inhibited by
decapitation. However, while some decapitated S. littoralis females were
observed to oviposit, in P. stultana oviposition did not occur [l].
Injection of Br-SOG homogenate induced pheromone production in decapitated females, but no evidence of calling was ever observed in those
animals. These results indicate that calling behavior in S. littoralis females is
not controlled by the same mechanism as pheromone production. An intact
nervous connection of the brain and/or the subesophageal ganglion with the
terminal abdominal ganglion appears to be essential for calling behavior in
Hyalophora cecropia [14] and Manduca sexta [15].
Raina and Klun [2] reported that a factor from the head is involved in the
control of sex pheromone production in H. zea. These researchers demonstrated that injection of crude Br-SOG extracts into neck-ligated females
caused a significant increase in pheromone titers when the animals were
injected during the scotophase. At that time, the stimulatory activity was
Stimulation of Sex Pheromone Production
also found in female hemolymph. If, as their data strongly suggest, a neurohormone regulates sex pheromone biosynthesis, then one may expect that it
can also stimulate pheromone biosynthesis during the photophase. However, Raina et al. [4] and Raina and Menn [16] have recently indicated that
pheromone production in H. zed was not stimulated when Br-SOG homogenate was injected into ligated females during photophase, and they have
suggested the existence of some additional control mechanism.
The results reported here demonstrate that pheromone production in S.
littoralis females can also be stimulated during photophase. Similar responsiveness in decapitated females is achieved after injection of Br-SOG homogenate during photophase and scotophase, as evidenced by the amounts of
pheromone extracted at both times. Preliminary results indicate that stimulation of pheromone production during the light period can also be achieved
in another moth species, Thaumetopoea pityocampa (Notodontidae), and thus
suggest that S. littoralis is not an exception in this respect. A normal pheromone blend was produced in S. littoralis females after injection of T. pityocumpa Br-SOG homogenate (unpublished observations). These findings are
in agreement with the indication that the pheromone biosynthesis-activating
neuropeptide does not regulate pheromone specificity [16].
Average pheromone amounts found in decapitated control females during
scotophase were usually a little higher than those of controls extracted in
photophase (Table 1, experiments 9 and 10; P < 0.01). Since decapitations
were always performed during photophase, these findings suggest that in S.
liftoralis the active factor may start to be released into the hemolymph a little
earlier than the lights-off signal. On the other hand, the reduced pheromone
titers found in these decapitated females, when compared with the amounts
extracted from intact second-scotophase animals, would be in agreement
with the hypothesis that a continuous presence of the active factor is required
to maintain pheromone production [2,5].
The results reported in the present study support the existence of a
hormonal control of pheromone production in S. littomlis, although they do
not rule out the possibility that a neural mechanism can mediate the response. Furthermore, the ability to stimulate pheromone biosynthesis in S.
littoralis females during the photophase suggests that the necessary cellular
machinery is available and can probably be activated at any time of the
photoperiodic cycle in this species.
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2. Raina AK, Klun JA: Brain factor control of sex pheromone production in the female of the
corn earworm moth. Science 225, 531 (1984).
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