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

The effect of 10,11-methylenetetradec-10-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 myristicacid. The study of each Z desaturation step revealed that the Z9-desaturase of E11–14: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. © 1994 Wiley-Liss, Inc.     

PBAN regulation of pheromone biosynthesis in female tobacco hornworm moths,Manduca sexta (L.)

Manduca sexta females that were decapitated produced no pheromone during the scotophase following decapitation, indicating that they were free of pheromone biosynthesis activating neuropeptide (PBAN). When deuterated hexadecanoic or (Z)-11-hexadecenoic acid was applied to the sex pheromone glands of decapitated or intact females of the same age, and allowed to incubate in vivo for 24 h, deuterium labeled Δ-11- and Δ-10, 12-unsaturated 16-carbon fatty acids were produced in both types of females. Injection of PBAN into intact or decapitated females 23 h after application of labeled acids had no effect on the production of unsaturated labeled fatty acids. However, deuterium labeled aldehydes were produced only in females that were injected with PBAN. Therefore, in this species, PBAN activates the process by which fatty acyl precursors in the pheromone gland are converted into the pheromonal aldehydes. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Evidence for both humoral and neural regulation of sex pheromone biosynthesis in Spodoptera littoralis

Selected tissues presumably involved in the control of sex pheromone production were analyzed by ELISA for the presence of PBAN-like immunoreactivity (PBAN-IR) in Spodoptera littoralis. The temporal distribution pattern of PBAN-IR in the hemolymph is similar to that of pheromone production in the gland. On the other hand, analysis of the retrocerebral complex, brain-subesophageal ganglion complex, and terminal abdominal ganglion (TAG) revealed similar PBAN-IR levels in both photophase and scotophase periods. Pheromonotropic activity exhibited by both hemolymph and TAG, as determined by a modified in vitro bioassay, agrees with the results of the immunochemical analyses. Severing the ventral nerve cord anterior to the TAG impaired normal sex pheromone production by second-scotophase females. These results are discussed in the context of how sex pheromone biosynthesis is regulated by PBAN in S. littoralis. © 1996 Wiley-Liss, Inc.     

Regulation of pheromone biosynthesis in the "Z strain" of the European corn borer, Ostrinia nubilalis

The regulation of pheromone biosynthesis by the neuropeptide PBAN in the Z strain of the European corn borer, Ostrinia nubilalis, was investigated using labeled intermediates. Injection of radiolabeled acetate showed PBAN did not influence the de novo synthesis of saturated fatty acids in the gland. When deuterium-labeled myristic acid was topically applied to the gland, females injected with PBAN produced more labeled pheromone than did control females, indicating that PBAN controls one of the later steps of pheromone biosynthesis. Although more myristic acid was Delta11-desaturated in the gland in the presence of PBAN, this was counterbalanced by less Delta11-desaturation of palmitic acid, indicating that desaturase activity did not change overall. This change in flux of myristic acid through to pheromone was shown to be caused by increased reduction of fatty acid pheromone precursors occurring in the presence of PBAN.     

Regulation of sex pheromone biosynthesis in two noctuid species,S. littoralis and M. brassicae,may involve both PBAN and the ventral nerve cord

In order to understand better the mechanism of regulation of pheromone production in moth species, we performed ELISA analyses to detect and follow pheromone biosynthesis activating neuropeptide-like immunoreactivity (PBAN-IR) in different tissues of the two noctuidae species, Spodoptera littoralis and Mamestra brassicae. Male S. littoralis and both male and female M. brassicae brain-subesophageal ganglion (Br-SEG), corpora cardiaca-corpora allata complex, and terminal abdominal ganglion extracts showed the presence of PBAN-IR during both the photophase and the scotophase. However, PBAN-IR was found only in scotophase in female hemolymph. Analysis of extracts of Br-SEG, terminal abdominal ganglion, and hemolymph after HPLC fractionation showed that the most immunoreactive fraction in all the extracts exhibited the same retention time as Hez-PBAN, suggesting that similar PBAN-like material is present in all these tissues. In vivo studies demonstrated that severing the ventral nerve cord in M. brassicae anterior to the terminal abdominal ganglion impaired normal sex pheromone production by third-scotophase females, as was previously shown in S. littoralis. Additionally, PBAN-IR levels were lower in hemolymph samples obtained at the peak of pheromone production in both S. littoralis and M. brassicae females that had the ventral nerve cord severed compared with sham operated animals. These results, along with earlier reported data, indicate that control of pheromone production in both species may involve both PBAN (or PBAN-like peptides) and the ventral nerve cord and support the hypothesis that a neural input from the ventral nerve cord triggers the release of the pheromonotropic peptide(s) into the hemolymph, which then acts directly on the pheromone gland to stimulate pheromone biosynthesis. Arch. Insect Biochem. Physiol. 37:295–304, 1998. © 1998 Wiley-Liss, Inc.

1 We thank Germán Lázaro for insect rearing.

     

Evidence for two-step regulation of pheromone biosynthesis by the pheromone biosynthesis-activating neuropeptide in the moth Heliothis virescens

The control of pheromone biosynthesis by the neuropeptide PBAN was investigated in the moth Heliothis virescens. When decapitated females were injected with [2-(14)C] acetate, females co-injected with PBAN produced significantly greater quantities of radiolabeled fatty acids in their pheromone gland than females co-injected with saline. This indicates that PBAN controls an enzyme involved in the synthesis of fatty acids, probably acetyl CoA carboxylase. Decapitated females injected with PBAN showed a rapid increase in native pheromone, and a slower increase in the pheromone precursor, (Z)-11-hexadecenoate. Total native palmitate and stearate (both pheromone intermediates) showed a significant decrease after PBAN injection, before their titers were later restored to initial levels. In contrast, the acyl-CoA thioesters of these two saturated fatty acids increased during the period when their total titers decreased. When a mixture of labeled palmitic and heptadecanoic (an acid that cannot be converted to pheromone) acids was applied to the gland, PBAN-injected females produced greater quantities of labeled pheromone and precursor than did saline-injected ones. The two acids showed similar time-course patterns, with no difference in total titers of each of the respective acids between saline- and PBAN-injected females. When labeled heptadecanoic acid was applied to the gland alone, there was no difference in titers of either total heptadecanoate or of heptadecanoyl-CoA between PBAN- and saline-injected females, suggesting that PBAN does not directly control the storage or liberation of fatty acids in the gland, at least for this fatty acid. Overall, these data indicate that PBAN also controls a later step involved in pheromone biosynthesis, perhaps the reduction of acyl-CoA moieties. The control by PBAN of two enzymes, near the beginning and end of the pheromone biosynthetic process, would seem to allow for more efficient utilization of fatty acids and pheromone than control of only one enzyme.     

Correlation between glycerolipids and pheromone aldehydes in the sex pheromone gland of female tobacco hornworm moths,Manduca sexta (L.)

Analysis by TLC and HPLC revealed that the triacylglycerols comprise the most abundant lipid class in the sex pheromone glands of Manduca sexta females. Also, conjugated olefinic acyl analogs of the major pheromone aldehydes occur principally in the triacylglycerols. The amount of triacylglycerols with conjugated diene acyl moieties significantly decreased when the period of pheromone production was extended by 7 h beyond the normal period of pheromone production by 3 injections of pheromone biosynthesis activating neuropeptide (PBAN) at 3 h intervals. This decrease indicates that the triacylglycerols stored in the gland may serve as major sources of pheromone precursors in the biosynthesis of the sex pheromone aldehydes. Furthermore, analysis of pheromone aldehydes and triacylglycerols in the gland from moths treated with PBAN showed that the proportions of the triacylglycerols with conjugated diene moieties were closely correlated with the proportions of aldehydes found in the same gland. This correlation suggests that the proportions of fatty acids bound to certain triacylglycerols regulates the proportions of aldehydes in biosynthesis of the pheromone blend in M. sexta. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Molecular cloning of pheromone biosynthesis activating neuropeptide in silkworm, Bombyx mori

Pheromone biosynthesis activating neuropeptide (PBAN) is a suboesophageal ganglion secretory polypeptide of insect, which activates the pheromone gland to produce sex pheromone biosynthesis in female silkworm, Bombyx mori. A Bombyx genomic library was screened by the method of plaque hybridization using the 32P-labeled BomDH cDNA as a probe. The genomic sequence encoding PBAN has been cloned and its structure is analyzed. The PBAN gene comprises two exons interspersed by a single intron 697 bp in length. Preceding the PBAN amino acid sequence is a 32-amino acid sequence containing two FXPRL amide peptides, which are α-SGNP (Ile-Ile-Phe-Thr-Pro-Lys-Leu) and β-SGNP (Ser-Val-Ala-Asn-Pro-Arg-Thr-His-Glu-Ser-Leu-Glu-Phe-Ile-Pro-Arg-Leu), which is followed by a Gly-Arg processing site. Immediately, after the PBAN amino acid sequence is a Gly-Arg processing site and a FXPRL amide peptide γ-SGNP (Thr-Met-Ser-Phe-Ser-Pro-Arg-Leu). It is suggested that besides PBAN, 7-, 8-, and 17-residue amidated peptides wer     

Pheromonotropic stimulation of moth pheromone gland cultures in vitro

The direct neurohormonal control of pheromone biosynthesis by pheromone biosynthesis activating neuropeptide (PBAN) was demonstrated in Helicoverpa (Heliothis) spp. using pheromone gland cultures in vitro. Pheromone gland activation involved the de novo production of the main pheromone component (Z)-11-hexadecenal as revealed by radio-TLC, radio-HPLC, and radio-GC. Activation was found to be a specific response attributed to pheromone gland cultures alone. Specificity of pheromonotropic activation was demonstrated to be limited to nervous tissue extracts. A sensitive and specific radioimmunoassay was developed using [³H]-PBAN, and the spatial and temporal distribution of PBAN-immunore-activity was studied. PBAN-immunoreactivity in brain complexes was found throughout the photoperiod and in all ages. From the distribution of PBAN-immunoreactivity it appears that PBAN release is affected by photoperiod. Pheromone gland cultures were found to be competent to pheromone production irrespective of age and photoperiod. Therefore, the neuroendocrine control of pheromone production operates at the level of neuropeptide synthesis and/or release and not at the level of the target tissue itself. The involvement of cyclic-AMP as a second messenger system was demonstrated. Brain extracts and PBAN were shown to stimulate dose- and time-dependent changes in intracellular cyclic-AMP levels. The role of cyclic-AMP in this mechanism was further verified by the ability of cyclic-AMP mimetics to mimic the pheromonotropic effect of brain extracts and PBAN. However, dose-response studies using PBAN and a hexapeptide C-terminal fragment of PBAN suggested that PBAN induces a two mechanism response, one occurring at low PBAN concentrations (high affinity receptor) and another at higher PBAN concentrations (low affinity receptor). Further evidence indicating a dual receptor system was obtained with the observation that the active phorbol ester (phorbol-12-myristate 13-acetate), the diacyl-glycerol analog (1,2-dioleolyl-sn-glycerol), and the intracellular calcium ionophore (ionomycin) mimicked the physiological action of PBAN and that lithium chloride had a pheromonostatic effect. The results indicate that pheromone glands also possess receptors that are linked to inositol phosphate hydolysis. © 1994 Wiley-Liss, Inc.     

Termination of sex pheromone production in mated females of the silkworm moth

A mating duration of more than 6 h was necessary to permanently terminate the production of the sex pheromone (bombykol) in the silkworm moth, Bombyx mori L. (Lepidoptera: Bombycidae), although the female formed a bursa copulatrix including a spermatophore and laid fertilized eggs even after mating for only 0.5 h. The 6-h mated female again produced bombykol if given an injection of synthetic pheromonotropic neuropeptide (PBAN), which is known to activate pheromone biosynthesis in a virgin female. Extracts of brain-suboesophageal ganglion (SG) complexes, which were removed from 6- and 24-h mated females, showed strong pheromonotropic activities. These results indicated that the pheromone gland of the mated female maintained its ability to biosynthesize bombykol; however, it could not produce pheromone due to a suppression of PBAN secretion from the SG. Furthermore, bombykol titers did not decrease after mating in females with a transected ventral nerve cord, even after the injection of a spermatophore extract, suggesting that the suppression of PBAN secretion was mediated by a neural signal and not by a substance in the spermatophore. The mated females accumulated (10E, 12Z)-10,12-hexadecadienoic acid, a precursor of bombykol biosynthesis, in their pheromone glands as did decapitated females. © 1996 Wiley-Liss, Inc.     

Stimulation of sex pheromone production by proteinaceous extracts of the bursa copulatrix in the redbanded leafroller moth

Pheromone biosynthesis in the redbanded leafroller moth, Argyrotaenia velutinana, was stimulated by homogenates of the bursa copulatrix. Although pheromonotropic activity was also extractable from the ovary, the activity of pheromone biosynthesis activating neuropeptide (PBAN) or bursa extracts was not impaired in isolated abdomens by removal of the ovary. Response to the bursa extracts was dependent on the dose administered and the time of incubation. Amounts of pheromone present in adult females of different ages appeared to be correlated with the extractable amount of pheromonotropic activity from their bursa copulatrix. Decapitation did not result in the suppression of burse factor production. Homogenates of the bursa elicited similar effects in both isolated gland and isolated abdomen incubations, but the brain neuropeptide, PBAN, was less active in the former than in the latter. Bursa extracts stimulated pheromone production in isolated abdomen incubations deprived of the bursa copulatrix, but PBAN did not. Loss of activity of bursa homogenates after treatment with either pronase E or carboxypeptidase Y indicated that the pheromonotropic factor is a proteinaceous substance. The mechanism through which pheromone production is regulated in redbanded leafroller moths is discussed. © 1992 Wiley-Liss, Inc.     

Hormonal control of female sex pheromone biosynthesis in the redbanded leafroller moth,Argyrotaenia velutinana

Pheromone biosynthesis in female redbanded leafroller moths (RBLR) is under control of a neuropeptide produced in the brain. A bioassay consisting of isolated abdomens was developed to test the mode of action of the pheromone biosynthesis activating neuropetide (PBAN). Pheromone titer and incorporation of radiolabeled acetate into pheromone could be monitored with this bioassay. Synthetic PBAN with sequences identical to PBAN isolated from Heliothis zea and Bombyx mori were active in inducing synthesis of pheromone in RBLR. Removal of the ventral nerve cord in isolated abdomens did not inhibit the action of PBAN. Small amounts of PBAN-like activity was found in hemolymph collected from normal females but not from decapitated females. Severing the VNC in vivo in normal females did not lower the pheromone titer. These data indicate that PBAN is released into the hemolymph and then travels to its site of action. A two-fold increase in both pheromone titer and radiolabeled acetate incorporation upon incubation with PBAN was shown with isolated pheromone glands. However, the differences between control and PBAN-induced values were smaller than those obtained with the isolated abdomen culture bioassay where a seven-fold increase was observed. A decrease in pheromone titer was seen upon the in vivo removal of the corpus bursae from normal females. Removal of the corpus bursae in the isolated abdomen cultures also abolished the activity of PBAN. However, cutting the cervix bursae and leaving the corpus bursae in the abdomen culture increased both titer and radiolabeled acetate incorporation into pheromone without the presence of PBAN. An aqueous extract made from the corpus bursae of 5-day-old females was also active by itself in inducing pheromone biosynthesis in the isolated abdomen cultures. Experiments performed using newly emerged females confirmed that the corpus bursae extracts will induce pheromone biosynthesis. These results indicate that both PBAN and the corpus bursae are involved in controlling pheromone biosynthesis in RBLR.     

Tissue localization and partial characterization of pheromone biosynthesis activating neuropeptide inAchaea janata

Female sex pheromone production in certain moth species have been shown to be regulated by a cephalic endocrine peptidic factor: pheromone biosynthesis activating neuropeptide (PBAN), having 33 amino acid residues. Antisera against syntheticHeliothis zea-PBAN were developed. Using these polyclonals, immunoreactivity was mapped in the nervous system ofAchaea janata. Three distinct groups of immunopositive secretory neurons were identified in the suboesophageal ganglion; and immunoreactivity was observed in the corpora cardiaca, thoracic and in the abdominal ganglia. From about 6000 brain sub-oesophageal ganglion complexes, the neuropeptide was isolated; and purified sequentially by Sep-pak and reversed phase high performance liquid chromatographic methods. Identity of purified PBAN fraction was confirmed with polyclonal antibody by immunoblotting. Molecular mass of the isolated peptide was determined by matrix-assisted laser desorption/ionization mass spectrometry, and was found to be 3900 Da, same as that of knownH. zea-PBAN. Radiochemical bioassay confirmed the pheromonotropic effect of the isolated neuropeptide in this insect     

CONTROL OF SEX PHEROMONE BIOSYNTHETIC PATHWAY BY PBAN IN ASIAN CORN BORER OSTRINIA FURNACALlS*

Abstract Sex pheromone titer in Ostrinia furnacalis was significantly decreased to a very low level by decapitation, but it could be restored by injection of head extract prepared from both male and female moths or synthetic pheromone biosynthesis activating neuropepide (PBAN). This fact indicates that pheromone production is under the control of a PBAN-like factor. The sex pheromone biosynthetic pathway of O. furnacalis originates with the biosynthesis of palmitic acid and followed by A14 desaturation, chain shortening, reduction and acetylation to form the pheromone components, (Z) and (E)-12-tetradecenyl acetate. In order to determine which step in the pathway is controlled by PBAN, the incorporation of different labeled precursors into the pheromone and its intermediate were studied. Our results suggest that PBAN controls pheromone biosynthesis in O. furnacalis by mainly regulating an early step from acetate to palmitic acid.     

Regulation of pheromone production in virgin and mated females of two tortricid moths

Sex pheromone titers in females of two tortricid moths, Epiphyas postvittana and Planotortrix octo, did not significantly vary between the scotophase and photophase. Pheromone production in these two species is controlled by a factor located in the head of the respective females, probably the pheromone biosynthesis-activating neuropeptide (PBAN). Unlike that reported for the related tortricid, Argyrotaenia velutinana, the bursa copulatrix in female E. postvittana and P. octo does not appear to contain a factor that stimulates pheromone production. After mating, female E. postvittana permanently shut down pheromone production. In contrast, pheromone titer in mated P. octo females is reduced to a level approximately half that of similar-age virgins. While the abdominal nervous system is involved in the inactivation of pheromone production in mated E. postvittana females and probably acts to stop release of PBAN from the corpora cardiaca, the abdominal nervous system is not involved in effecting the decreased pheromone titers of mated P. octo females. It is possible that in the latter species, a humoral factor(s) is responsible for effecting the decreased pheromone titers, possibly through affecting the release of PBAN from the corpora cardiaca. Bioassaying head extracts allowed changes in PBAN titer in female E. postvittana to be inferred. PBAN titers remain roughly constant in virgins but increase after mating. This suggests that PBAN is biosynthesized throughout the life of an adult virgin female at approximately the same rate as it is released. Furthermore, it appears that the decline in pheromone titer observed in older E. postvittana females is probably due to a decline in competency of the gland to produce pheromone rather than to a decrease in PBAN titer in older females. © 1994 Wiley-Liss, Inc.     

Neuroendocrine control of pheromone production in moths

In many moth species regulation of pheromone production has been attributed to the timely release of a pheromone biosynthesis activating neuropeptide (PBAN). The gene encoding PBAN has been sequenced in two moth species. Immunochemical studies as well asin situ hybridization and Northern analysis of PBAN encoding mRNA have localized the neuroendocrine cells responsible for the production of PBAN and have traced the neuronal network of PBAN immunoreactivity. Release into the bloodstream has been demonstrated, the target tissue delineated, and the signal transduction pathway and its modulation analyzed. This paper reviews the current status of research concerning the neuroendocrine control of pheromone production in Lepidopterans and presents some recent developments concerning the receptors involved in the pheromonotropic activity. In this study, we report on the use of a biologically active photoaffinity-biotin-labeled derivative of PBAN N-[N-(4-azido-tetrafluorobenzoyl-biocytinyloxyl-succinimide) and show the presence of a protein (estimated molecular weight of 50 kDa) which specifically binds to PBAN in membrane preparations of pheromone glands. Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No.2279-E, 1997 series     

RNA interference of pheromone biosynthesis-activating neuropeptide receptor suppresses mating behavior by inhibiting sex pheromone production in Plutella xylostella (L.)

Sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN) in many lepidopteran species. We cloned a PBAN receptor (Plx-PBANr) gene from the female pheromone gland of the diamondback moth, Plutella xylostella (L.). Plx-PBANr encodes 338 amino acids and has conserved structural motifs implicating in promoting G protein coupling and tyrosine-based sorting signaling along with seven transmembrane domains, indicating a typical G protein-coupled receptor. The expression of Plx-PBANr was found only in the pheromone gland of female adults among examined tissues and developmental stages. Heterologous expression in human uterus cervical cancer cells revealed that Plx-PBANr induced significant calcium elevation when challenged with Plx-PBAN. Female P. xylostella injected with double-stranded RNA specific to Plx-PBANr showed suppression of the receptor gene expression and exhibited significant reduction in pheromone biosynthesis, which resulted in loss of male attractiveness. Taken together, the identified PBAN receptor is functional in PBAN signaling via calcium secondary messenger, which leads to activation of pheromone biosynthesis and male attraction.     

Effects of decapitation and PBAN injection on amounts of triacylglycerols in the sex pheromone gland of Manduca sexta (L.)

The correlation between triacylglycerols containing conjugated diene fatty acyl moieties and pheromone aldehydes in the sex pheromone glands of females of Manduca sexta was investigated. Females decapitated 15 h after adult emergence neither called nor produced pheromone during the natural period of pheromone production on the subsequent two nights. However, these females could be stimulated to produce sex pheromone for prolonged periods by repeated injection of synthetic pheromone biosynthesis activating neuropeptide (PBAN). Gas chromatographic analysis of methanolysis products of lipids extracted from the pheromone glands of decapitated and intact females showed no differences in the amounts of fatty acyl precursors of pheromone. High performance liquid chromatographic analysis of the triacylglycerols containing conjugated diene analogues of the pheromone components (diene TG), obtained 24 and 48 h after decapitation, showed that the total amounts of these components were not affected by decapitation. The amounts of all diene TG peaks declined significantly when decapitated females were stimulated to produce pheromone during a 7 h period by repeated injection of PBAN at 3 h intervals but recovered when pheromone production subsided. These results indicate that PBAN induces liberation of pheromone precursors from the triacylglycerols during pheromone biosynthesis but does not induce replenishment of this storage pool. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America