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THE JOURNAL OF EXPERIMENTAL ZOOLOGY 274955-263 (1996)
Neuronal Control of Post-Coital Pheromone
Production in the Moth Heliothis uirescens
SONNY B. RAMASWM, YI QIU, AND YONG IHL PARK
Department of Entomology and Plant Pathology, Mississippi State
University, Mississippi State, Mississippi 39762-9775
ABSTRACT
The mechanism involved in bringing about post-coital suppression of pheromone
production, pheromonostasis, was studied in the noctuid moth Heliothis uirescens. Mating results
in a transient suppression in pheromone production, the signal for which appears to originate in
t h e testes an d other components of th e male's reproductive system. The mating-induced
pheromonostasis is due to an ascending signal via the central nervous system that appears to
inhibit the release of the pheromonotropin, pheromone biosynthesis activating neuropeptide (PBAN),
or other potential pheromonotropic substances, and is not due to a refractoriness in response of
the sex pheromone glands to PBAN in the female. A similar mechanism is operative in several
species of moths where post-coital pheromonostasis has been observed. Sperm quality is not important for pheromonostasis in H. virescens, because males with apyrene or eupyrene sperm elicit
similar pheromonostatic responses. The pheromonostatic activity of the ecdysteroid 20-OH-ecdysone appears to be the result of a direct effect on the sex pheromone glands. 0 1996 Wiley-Liss, Inc.
The temporary or permanent suppression of female receptivity and sex pheromone production
after mating in insects (Gillott and Friedel, '77;
Ramaswamy et al., '94) may be important for the
following reasons: 1)insects in copula are susceptible to predation; 2) courting males interfere with
oviposition activity; or 3) to offset the impact of
sperm precedence (Manning, '67; Pair et al., '77;
Thornhill and Alcock, '83; Svard and McNeil, '94).
Female receptivity may be suppressed by the
transfer of male factors that inhibit sex pheromone production and receptivity or trigger a
primer response, resulting in the mobilization of
endogenous pheromonostatic factors that originate
from spermatheca or neuroendocrine centers in
the female. The presence of sperm or spermatophore(s) in the mated female's reproductive
system stimulates receptors exerting a pheromonostatic effect via the central nervous system t o trigger neuronal and/or humoral signals,
resulting in complete or partial suppression of
pheromone production and receptivity o r the
male may physically obstruct the gonopore with
a mating plug (Gillott and Friedel, '77; Thornhill and Alcock, '83; Sasaki and Riddiford, '84;
Chen, '91; Kingan et al., '93; Ramaswamy et
al., '94).
Some lepidopteran males transfer factors from
their reproductive system, which causes either
transient or permanent pheromonostasis in females (Webster and Carde, '84; Raina, '89; Mbata
0 1996 WILEY-LISS, INC.
and Ramaswamy, '90). The pheromone suppressive male factor in the noctuid moth Helicouerpa
zea has been identified as a polypeptide containing 57 amino acids with a molecular weight of
6,600 and is found in the accessory sex glands of
males. Recent studies have suggested that this
factor causes pheromonostasis by blocking the action of the pheromone biosynthesis activating neuropeptide (PBAN) and also stimulates breakdown
of existing pheromone, following an ascending signal via the central nervous system to the cephalic region (Kingan et al., '93, '95). In another
tortricid moth, Argyrotaenia uelutinana, Jurenka
et al. ('93) showed that mating results in a signal
that ascends the ventral nerve cord (VNC) to the
brain t o inhibit the release of PBAN, and thus
the sex pheromone glands lacking the pheromonotropic signal do not produce any pheromone in
mated females. Yet other mechanisms appear to
cause post-mating suppression of pheromone production in females of other lepidopterans. For example, the nervous system is implicated in mated
Epiphyas postvittana (Tbrtricidae) (Foster, '93)'
while a humoral factor has been suggested t o be
responsible for such a phenomenon in another tor-
Received July 25, 1995; revision accepted November 28, 1995.
Address reprint requests t o Sonny B. Ramaswamy, Department of
Entomology and Plant Patholom, Mississippi State University, Mississippi State, MS 39762-9775.
256
S.B. RAMASWAMY ET AL.
tricid, Planotortrix octo (Foster and Roelofs, '94),
although in neither case is the actual mode of action known. In the noctuid Heliothis uirescens the
fused testes and ejaculatory ducts of the male
were shown to be the source of the post-mating
pheromone suppressive factor, and 20-OH-ecdysone has been suggested to be the putative pheromonostatic factor (Ramaswamy and Cohen, '92;
Ramaswamy et al., '94).
In the few species studied thus far, there are
considerable differences in the mechanisms of
post-mating changes in receptivity and pheromonostasis, and we wanted to determine the
mechanisms involved in post-coital pheromonostasis observed in the moth H. uirescens. Therefore, a series of studies were undertaken t o
determine: 1) importance of the adult testes in
pheromonostasis; 2) the dynamics of mating in virgin and mated females; 3) how 20-OH-ecdysone,
the putative pheromonostatic factor in H. virescens
(Ramaswamy and Cohen, '92), interacts with the
pheromonotropin PBAN to regulate sex pheromone production in mated females; 4) the role of
the VNC and the mechanisms in the observed
pheromonostasis in mated females; and 5) the impact of sperm quality on post-mating pheromonostasis.
MATERIALS AND METHODS
lkst insects
H. uirescens larvae were reared on a wheat germ
diet (Bio-Sew, Frenchtown, NJ). Pupae were sexed
and males and females held in separate containers for emergence at 14:10 1ight:dark photoperiod,
26 2 2"C, 75% relative humidity (RH). Under these
conditions, most insects emerge during the first
half of the scotophase. Moth emergence was
checked every 15 min, and those emerging between the 2nd and 4th h after lights off were
grouped and used in the tests. Adult females were
housed in 3.75 1 glass jars in a chamber at 26 2
2"C, 75% RH, and fed 5% sucrose solution. All surgical procedures and behavioral observations during scotophase were facilitated by use of flashlights
or microscopic lamps with red filters (approximately 2 lux) with a cutoff of 590 nm wavelength,
at which moth behavior is unaffected.
sisted of 21 mM KCl, 12 mM NaCl, 3 mM CaC12,
18 mM MgC12,85 mM trehalose, and 5 mM PIPES
buffer, and was brought t o pH 6.6 using KOH
(Jurenka et al., '91).
Gas chromatography
The sex pheromone glands (SPGs) were excised
during scotophase under diffused red light or under fluorescent light during photophase. The gland
was dipped for approximately 5 min in 25 pl redistilled CS, containing 100 ng Z-ll-hexadecenyl
acetate (Z11-16:AC) as internal standard. The extract was evaporated t o about 2 p1 under a gentle
stream of N2 and injected on a 30 m Alltech EconoCap Carbowax column (0.25 pm film thickness,
0.32 mm ID) in a Varian 3700 gas chromatograph
with a flame ionization detector (GC-FID).Helium
was used as the carrier gas (column head pressure 12 psi) and makeup gas (30 ml/min). The GC
was programmed for an initial temperature of
110°C for 2 min raised at the rate of 10"C/min t o
220°C and held for 5 min. The major component
of the sex pheromone of H. uirescens, Z-ll-hexadecenal (Zll-l6:ALD), was integrated and quantified by comparing its peak area with that of the
internal standard, Z11-16:AC, and is reported as
nanograms of Z-ll-l6:ALD/female.
Castration
Data presented for Helicouerpa zea suggested
that the testes are not necessary for post-mating
pheromonostasis (Raina, '89). However, methanolic extracts of HeZiothis uirescens testes injected
into conspecific virgin females elicited pheromonostasis comparable to that observed in mated
females (Ramaswamy et al., '94). Therefore, we
wanted t o determine if adult testes are necessary
for pheromonostasis in mated females of H.
uirescens. Adult males were subjected t o castration immediately after wing expansion, approximately 60-90 min after emergence. Males were
anesthetized under a continuous stream of COB
and the 5/6 dorsolateral abdominal segments
cleared of scales by vacuum aspiration. After
swabbing with 95% ethanol, a small incision was
made and after the addition of a few crystals of
phenylthiourea, the fused testes were removed by
severing at the junction of the seminal vesicles
(Callahan, '58), using sterile iridectomy scissors.
Chemicals
The wound was sealed with molten, low temperaSynthetic Hez-PBAN was purchased from Pen- ture wax. Control males were handled similarly
insula Labs (Belmont, CA). The other reagents except the reproductive system was left intact.
were purchased from Sigma (St. Louis, MO). The Males were allowed t o recuperate for approxiWeever's saline used throughout the study con- mately 24 h after surgery and paired with females
PHEROMONE PRODUCTION IN MATED HELIOTHIS
257
for mating. Over 70% of the operated individuals
survived for several days, and they mated with
virgin females readily Gonadectomized, sham-operated control, and unoperated control males were
allowed to copulate with 48-h-old virgin females.
Coupling and uncoupling times and duration of
mating were determined. One hour after uncoupling, female SPGs were excised and processed
for quantification of pheromone titer. ?b determine
the success of the surgical procedures, an autopsy
was performed at the end of the tests.
ethanol or 2% ethanol alone. This dose was chosen because it suppressed pheromone production
in virgin females at a level comparable t o that in
mated H . virescens females (Ramaswamy and
Cohen, '92). Pheromone titers of 20-OH-ecdysoneinjected and control females were quantified 1 h
after injection. An autopsy was performed at the
end of the tests t o determine the success of the
surgical procedures.
VNC transection
served in mated females results from the lack of
a pheromonotropic signal or is due to the inability of the SPG to respond to such a signal, isolated abdomen and isolated gland culture assays
were used (Jurenka et al., '91; Ramaswamy et al.,
'95). For the isolated abdomen assay, the abdomen was excised at the junction of the 5th abdominal segment of mated females (3 h after
uncoupling) or similarly aged virgin females and
incubated for 1h with 20 p1 Weever's saline containing 1 pmol synthetic Hez-PBAN or saline
alone (control). The cut ends of the abdomens were
placed on 20 p1 drops of culture medium on sterile 9 cm plastic Petri dish bottoms, covered, and
placed on a bench at ambient laboratory temperature (26 2 2'C) and light. After incubation, the
SPGs were excised, extracted, and subjected to GC
analysis.
To prepare isolated SPGs for incubation, the ovipositors from mated females were excised 3 h after uncoupling and placed on a 20 pl drop of
Weever's saline. The ovipositor tip was cut and
then the ovipositor was cut lengthwise along the
dorsal surface. Remnants of the oviduct, hindgut,
fat bodies, and any other adhering tissues were
removed. The remaining mostly pheromone gland
tissue was rinsed, gently swabbed on a Kimwipes,
and placed epidermis side down on a 10 pl drop
of Weever's saline containing 2 pmol synthetic
Hez-PBAN or saline alone (control) in sterile 9 cm
plastic Petri dish bottoms. After incubation for 1h,
the glands were removed, wiped dry on a Kimwipes,
and extracted for GC analysis. Pheromone glands
from comparably aged intact virgin females (i.e.,
aged to coincide with the end of the incubation of
the isolated glands from mated females) were extracted also to determine pheromone titers.
To test the role of the VNC in mating and post-
Isolated abdomen and isolated SPG assays
To determine whether the pheromonostasis ob-
mating pheromone production, virgin females
were operated on during the first photophase (<8
h old) after emergence. The test insects were anesthetized with C02 and the VNC was cut anterior
t o the terminal abdominal ganglion (TAG) by inserting a pair of iridectomy scissors through the
intersegmental membrane parallel t o the body
surface and cutting. Sham-operated control insects
were treated similarly, except that the cut was
made in the abdominal intersegmental area lateral t o the VNC. These operated individuals were
allowed t o recuperate for approximately 40 h and
were compared with similarly aged, untreated control insects. Females of the 3 treatments were
paired with 2-day-old normal, virgin males in
groups of 5 pairs in 3.75 1 glass jars lined with
moist paper toweling in the bottoms and provided
with 5 x 15 cm strips of vertically hanging paper
toweling as perches. Small (30 ml) plastic cups
containing cotton soaked in 5% sucrose served as
food sources. 'ItYenty-five females from each treatment were observed once every 15 min under a
red flashlight for mating during the third scotophase. Mating pairs were placed in individual 500
ml plastic cups lined with moist Kimwipes and
provisioned with 5% sucrose. Coupled moths were
observed once every 15 min to determine the time
of uncoupling. Mating frequency distributions during scotophase and length of time in copula were
quantified. SPGs of VNC-transected, sham-operated, and unoperated control females were excised
1 h after uncoupling, extracted, and subjected t o
GC analysis.
To test the involvement of the VNC in the
pheromonostatic activity of 20-OH-ecdysone7virgin females <8 h old were subjected t o VNC
transection and sham operation as above during Pheromonotropic activity of head extracts of
mated and virgin females
scotophase. VNC-transected and sham-operated
sibling virgin females were injected 40 h after surTo test if the suppression in post-mating pherogery with 1 pg of 20-OH-ecdysone in 5 pl of 2% mone titer is due t o cessation of the production of
258
S.B. RAMASWAMY ET AL.
a pheromonotropic factor by the brain-subesophageal ganglion-corpora cardiaca (brain-SEG-CC)
complex, heads of females 1 h after uncoupling
and heads of comparably aged virgin females were
excised and homogenized in a glass-glass homogenizer in 1 mM HC1 in 50% methanol. The extracts were sonicated with an Artek Systems
Dismembrator fine probe sonicator for 15 sec at a
setting of 60 and then centrifuged at 12,000 rpm
for 15 min at 4°C.The supernatants were dried
in a Savant Speed-Vac concentrator. The residues
were resuspended in Weever's saline at the rate
of 2 female equivalents (FEY10 pl.Abdomens from
decapitated, 24-h-old virgin females were incubated in the head extracts for 1h, then the glands
were excised and extracted for quantification of
pheromone titer.
Sperm quality
'lb determine whether sperm quality is important in the post-mating suppression of pheromone
production, 24-h-old virgin H. virescens females
[designated wild type (WT)] were coupled with virgin, WT males o r H. subflexa x H. uirescens backcross males (designated BC) (Laster, '72). The BC
insects used here have been back-crossed and in
culture for >250 generations (M. Laster, USDAA R S , Stoneville, MS, personal communication).
They were raised on a wheat germ diet in multicellular rearing trays (King and Hartley, '85) at
the Southern Field Crop Insect Management
Laboratory, USDA-ARS, Stoneville, MS, and were
shipped by courier express. Upon receipt, male BC
pupae were entrained t o conditions used for the
WT males and females (see above) for at least 48
h during the pupal stage t o ensure that the pupae had been entrained (Roush and Schneider, '85)
to our laboratory conditions. The BC males exhibited an emergence profile similar to that observed
for our WT males, with peak adult emergence restricted predominantly to the period between 2
and 4 h after lights off. Virgin BC and WT males
were paired with 24-h-old virgin WT females in
groups of 5 pairs as before, and observations on
coupling and uncoupling were as before. Mating
frequency, coupling and uncoupling times, and
mating duration were determined. SPGs were excised 1h post-uncoupling and extracted for pheromone quantification as above.
ference (LSD) test was used to separate means.
In some experiments involving comparisons of
pairs of treatments, t-tests were used.
RESULTS AND DISCUSSION
Importance of testes in post-mating
pheromonostasis
The post-mating depletion of pheromone observed in H. virescens females mated to normal
or sham-operated males is not evident in those
mated to castrated males (Fig. 1).Pheromone titer in the latter group is similar t o that of comparably aged virgin females. However, castration did
not impair the mating ability as normal males and
castrated males remained in copula for 208.8 2
32.8 and 210.0 2 32.4 min (mean 2 SE), respectively Eggs from females mated to normal males
showed 100% hatchability, while those mated t o
castrated males laid comparable numbers of eggs
of which a small percentage (45%) hatched. This
was a surprising finding since we expected castrated males not to fertilize any eggs in females.
It is possible that the seminal vesicles were not
completely excised in some males during castration,
and these store sperm, even in newly emerged adult
males (Callahan, '58; Chapman, '82).
The lack of post-mating suppression in females
mated to castrated males is, on the one hand, not
surprising as methanolic or phosphate buffered
saline (PBS) extracts of testes injected into normal, virgin females elicited a post-injection suppression in pheromone titer that was comparable
t o the post-mating suppression in pheromone titer seen in H. virescens (Ramaswamy et al., '94).
250
-a
-5
I
a
a
T - r
c:
5
150
0
fi
4ib:
100
50
G
0
Virgin
C-mated
S-mated
N-mated
Fig. 1. Sex pheromone titers in virgin H. virescens females
and those mated t o castrated (C-mated), sham-operated (SStatistical analysis
mated), or normal (N-mated)males. Bars represent the mean
Data were transformed using the formula Y = f SE (n = 8-16 for each treatment). Different letters indicate
log(1 + X) and subjected to analysis of variance significantly different groups as determined by ANOVA fol(ANOVA). Fisher's protected least significant dif- lowed by Fisher's protected LSD test ( P < 0.05).
259
PHEROMONE PRODUCTION IN MATED HELZOTHIS
However, in a closely related species, Helicouerpa
zea, Raina ('89) had shown that testes were unimportant in post-mating suppression of pheromone titers. One possible explanation for the
discrepancy between the findings of Raina ('89)
and those reported here is that we castrated males
during the adult stage, while Raina ('89) castrated
males during the larval stage. Alternatively, it may
be attributed t o species differences. The pheromonostatic activity of testicular extracts in Heliothis uirescens (Ramaswamy et al., '94) and the
inability of castrated males t o cause post-mating
suppression in pheromone (Fig. 1) suggest that
the testes are indeed a source of a putative
pheromonostatic factor in this species.
In a few species of moths such as Manduca sexta
(Sphingidae) (Sasaki and Riddiford, '84), Lymantria dispar (Lymantriidae) (Giebultowicz et al.,
,911, and E. postuittana (Foster, '931, post-coital
suppression of pheromone production apparently
requires the presence of sperm. It is possible that
in H. uirescens non-testicular pheromonostatic factors and sperdtesticular factors together elicit
post-coital pheromonostasis.
lights off and mating was completed in most individuals by 4 h after lights off (Fig. 3). The only
apparently negative effect of VNC transection was
a reduction in the duration of remaining in copula
(Fig. 2). While all three groups of females showed
similar profiles of distribution of mating during
scotophase, a few VNC-transected females appeared to mate later in the scotophase (Fig. 3).
The ability of VNC-transected females t o mate
as successfully as normal females further questions the validity of the claim for the involvement
of VNC in pheromone production in heliothine
moths. Teal et al. ('89) and Christensen et al. ('91)
had suggested that the VNC is necessary for
pheromone production and release in Helicoverpa
zea and Heliothis uirescens. Data from the current studies on the successful mating of VNCtransected H . virescens further corroborate the
conclusions of Rafaeli et al. ('go), Jurenka et al.
('91), Rafaeli ('94), and Ramaswamy et al. ('95)
concerning the lack of involvement of VNC in
pheromone production in heliothines.
Normal and sham-operated females mated t o
normal, virgin males exhibited similar levels of
post-coital reduction in pheromone production at
Role of VNC in mating and post-mating
1 h after uncoupling (Fig. 4). However, VNCpherornonostasis
transected females, which mated successfully with
Virgin H. virescens females with transected similar temporal mating frequencies, exhibited no
VNCs mate as readily as do sham-operated and post-coital suppression in pheromone production
normal females (Fig. 2). During the observation (Fig. 4). One may conclude from these latter data
period of the third scotophase after emergence, that the mating-induced suppression in phero11, 16, and 16 females, respectivelx of the 25 each mone production is transmitted as an ascending
normal, sham-operated, and VNC-transected fe- signal via the VNC t o some structure anterior to
males mated. Females initiated mating 1 h after the terminal abdominal ganglion, as transection
of the VNC was done anterior to the terminal ab-
Normal
Sham Opcratcd
VNC-Transecled
n
Virgin
Normal
Sham
VNC- Transected
0.0
Fig. 2. Duration of copulation of normal, sham-operated,
and VNC-transected H. uirescens females mated t o normal
males. Bars represent the mean 2 SE (n = 11-16 for each
treatment). Different letters indicate significantly different
groups as determined by ANOVA followed by Fisher's protected LSD test (P < 0.05).
Hours into Scotophase
Fig. 3. Distribution of mating pairs during scotophase of
normal, sham-operated, and VNC-transected H. virescens females mated to normal males. N = 11-16 for each treatment.
S.B. RAMASWAMY ET AL.
260
200
-$5
350
T
I
a
150
a
l
-4
l-
2
4*
2
4
T
T
30
G
3
3
100
-5
r(
37
R
d
T
a
a
300
250
Y
'
G
3
a
50
I+
0
Virgin
Normal
Sham
VNC- Transected
Fig. 4. Sex pheromone titers in normal, sham-operated,
and VNC-transected H. virescens females mated to normal
males. Bars represent the mean 2 SE (n = 11-16 for each
treatment). Different letters indicate significantly different
groups as determined by ANOVA followed by Fisher's protected LSD test ( P < 0.05).
dominal ganglion. Similar ascending neuronal signals have been suggested to bring about post-coital
pheromonostasis in A. uelutinana (Jurenka et al.,
'931, E. postvittana (Foster,'931, and Helicoverpa zea
(Kingan et al., '95). Ascending neuronal signals
are necessary also to bring about a switch from
virgin to mated female behaviors in M . sexta
(Sasaki and Riddiford, '84; Stringer et al., '85). In
contrast, in the tortricid moth I! octo, a humoral
mechanism for post-coital pheromonostasis has
been suggested (Foster and Roelofs, '94). These
findings suggest that different mechanisms, including neuronal and humoral, have evolved to
bring about post-mating pheromonostasis in the
different species of moths.
Mechanisms involved in 20-OH-ecdysoneinduced pheromonostasis
20-OH-ecdysone elicited significant suppression
in pheromone production in both sham-operated
and VNC-transected females (Fig. 5); however,
suppression by 20-OH-ecdysone was only approximately 40% compared with the >70% suppression
due to mating. These data suggest that the injected ecdysteroid brings about its pheromonstatic
activity in a manner different from the normal
mating-induced pheromonostasis, without the involvement of any ascending neuronal signals. It
remains t o be seen if the ecydsteroid has a direct
effect on the SPGs, either eliciting the breakdown
of pheromone produced, as suggested for the action of the pheromonostatin in H. zea (Kingan et
al., '95), or if it inhibits the pheromonotropic effect of PBAN.
b
200
b
150
100
50
0
EtOH
ECD
Sham-Operated
EtOH
ECD
VNC-Transected
Fig. 5. Sex pheromone titers in virgin sham-operated and
VNC-transected H. virescens females injected with 2% ethanol (EtOH) or 1 pg 20-OH-ecdysone (ECD) in 2% ethanol.
Bars represent the mean 2 SE (n = 12 for each treatment).
Different letters indicate significantly different groups as determined by ANOVA followed by Fisher's protected LSD test
( P < 0.05).
Responses of SPGs from mated females in
abdomen and isolated gland culture
Both isolated abdomens (Fig. 6) and isolated
SPGs (Fig. 7) from mated females produced significantly more pheromone when incubated with
PBAN than with saline alone. These results suggest that the occurrence of post-coital pheromonostasis in Heliothis virescens is the result of
the lack of PBAN or other pheromonotropins
rather than due t o the gland's refractoriness t o
the pheromonotropic signal. A similar mechanism
of post-coital pheromonostasis has been reported
for the totricid A. uelutinana (Jurenka et al., '93).
2M
rrl
I Abdomen Culture
&
52ooj
a
-T-
PBAN
Saline
1 pmol
Fig. 6. Sex pheromone titers in glands of mated female
H. virescens abdomens isolated and incubated for 1 h with 1
pmol PBAN in 20 pl Weever's saline or saline alone. Bars
represent the mean 2 SE (n = 14 for each treatment). Different letters indicate significantly different groups as determined by paired t-test ( P = 0.001).
261
PHEROMONE PRODUCTION IN MATED HELIQTHZS
the brain-SEG-CC complex of A. velutinana, a tortricid species (Jurenka et al., '93). Neuronal and
humoral inhibition of release of pheromonotropin
a
from the brain-SEG-CC complex have been inT
a
ferred also for the tortricids E. postvittana and I?
octo, respectively (Foster, '93; Foster and Roelofs,
'94). In the few species of moths in which the
mechanisms of post-coital pheromonostasis have
been examined closely, the data suggest that insects belonging to different families exhibit simiVirgin P E A N Saline
lar mechanisms of post-coital pheromonostasis. It
2 pmol
is possible that such a phenomenon may be
Fig. 7. Sex pher rnone titers in mated female SPGs iso- widespread in the Lepidoptera. However, how
lated and incubated for 1 h with 2 pmol PBAN in 10 pl this neuronal signal inhibits the release of the
Weever's saline or saline alone and titer in comparably aged
virgin female glands. Bars represent the mean 2 SE (n = 10 pheromonotropin( s) is unknown for any species of
for each treatment). Different letters indicate significantly Lepidoptera and remains t o be studied.
Gland Culture
1L
different groups as determined by ANOVA followed by Fisher's
protected LSD test (P< 0.05) for pheromone titer.
Pheromonotropic activity of mated and
virgin female cephalic extracts
Sperm quality and pheromonostasis
In several species of moths, including M . sexta,
L. dispal; and E. velutinana, the presence of sperm
or spermatophore has been implicated in the
switch from virgin female to mated female behavHead extracts from both virgin and mated fe- ior (Sasaki and Riddiford, '84; Giebultowicz et al.,
males elicited pheromonotropic activity, compa- '91; Foster, '93). In the current study, while BC
rable t o 0.5 pmol synthetic Hez-PBAN in an males remained in copula significantly longer than
isolated abdomen assay (Fig. 8). These findings did WT males (Fig. 9), both types of males elicsuggest that the brain-SEG-CC complex in mated ited similar levels of suppression of post-coital
females is indeed competent at producing the pheromone titers in WT females. These findings
pheromonotropin(s),but that their release appears of the post-mating pheromone suppressive activt o be inhibited by the ascending neuronal signal. ity when females were mated t o BC or WT males
Post-coital pheromonostasis is also the result of are similar t o the report of Raina and Stadelinhibition of release of the pheromonotropin from bacher ('go), who found approximately 90%reduction in pheromone production by females mated
t o WT or BC males. The BC males of the H.
a
I
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350
a
E
4~o
$
i
250-
Pheromone Titer
Timeincopula
a'
T
350
300
=
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g
E
Saline
PBAN
0.5 pmol
Virgin
Mated
Head Extract
Fig. 8. Sex pheromone titers in pheromone glands of decapitated, virgin female H. vireseens abdomens incubated for
1 h with 0.5 pmol PBAN in 20 pl Weever's saline or saline
alone or head extracts of mated or virgin females. Bars represent the mean 5 SE (n = 10 for each treatment). Different
letters indicate significantly different groups as determined
by ANOVAfollowed by Fisher's protected LSD test (P < 0.05).
200-
a
Virgin
W T x WT
WTxEC
Crosses
Fig. 9. Duration of copulation and sex pheromone titers
in virgin H. virescens females and those mated to WT and
BC males. Bars represent the mean f SE (n = 20 for each
treatment). Duration of copulation and sex pheromone titers
were analyzed separately. Different letters indicate significantly different groups as determined by ANOVA followed by
Fisher's protected LSD test ( P < 0.05) for pheromone titer
and by paired t-test (P < 0.05) for duration of copulation.
262
S.B. RAMASWAMY ET AL.
subflexa x H. uirescens crosses produce anucleate
apyrene sperm and WT males of H. virescens produce nucleated eupyrene sperm (Laster, ’72;
LaChance, ’84), suggesting that sperm quality is
unimportant for pheromonstasis. BC males also
are as responsive to pheromone from WT females
in the field as are WT males (Ramaswamy et al.,
’85). These findings of the effectiveness of mate
location and pheromonostasis by BC males could
allay fears of the ineffectiveness of BC males in
field situations for suppression of populations of
H. uirescens (M. Laster, USDA-ARS, Stoneville,
MS, personal communication).
In conclusion, post-coital pheromonostasis in H .
uirescens is the result of a n ascending neuronal
signal that inhibits release of pheromonotropin(s)
by the brain-SEG-CC complex and is not due to
an effect of mating inducing a refractoriness in
response of SPGs t o pheromonotropic signals.
Both the testes and other non-testicular factors
appear to be necessary for pheromonostasis;
however, sperm quality is not important for
pheromonostasis in H . uirescens, because males
with apyrene o r eupyrene sperm elicit similar
pheromonostatic responses. The pheromonostatic activity of the ecdysteroid 20-OH-ecdysone is not mediated via t h e VNC t o t h e
brain-SEG-CC complex, but is likely the result
of a direct effect on the SPGs. The mechanism
of pheromonostasis that occurs in H. uirescens
surprisingly is different from that observed in
a close relative, Helicouerpa zea, in which
pheromonostasis results from inhibition of the
pheromonotropic activity of PBAN (Kingan et
al., ’93, ’95), but rather is similar to mechanisms
of pheromonostasis observed in unrelated tortricid moths (Jurenka et al., ’93). The involvement of ascending neuronal signal inhibiting
release of a pheromonotropin(s) as the underlying mechanism of post-coital pheromonostasis appears t o be common t o several species
of moths belonging to two disparate families.
Whether such a mechanism is broadly applicable t o the other lepidopterans remains to
be seen.
ACKNOWLEDGMENTS
Drs. G. Baker, R. Luttrell, E. Nebeker, and
two anonymous reviewers are thanked for comments. Research was conducted a s part of
project MIS-6536. Approved for publication as
Journal Article No. 5-8811,Mississippi Agricultural and Forestry Experiment Station, Mississippi State University.
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