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 ~. 350 a E 4~o $ i 250- Pheromone Titer Timeincopula a' T 350 300 = 3 fi 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. 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