A pentapeptide of the C-terminal sequence of PBAN with pheromonotropic activity

Peptides ranging in length from 4 to 18 amino acid residues representing various sequence fragments of Helicoverpa (Heliothis)zea-pheromone biosynthesis activating neuropeptide (Hez-PBAN) were synthesized and tested for pheromonotropic activity. Biological activity resides in the C-terminus and the C-terminal pentapeptide (Phe-Ser-Pro-Arg-Leu-NH₂) represents the minimum sequence essential for induction of pheromone production. The C-terminal hexapeptide (Tyr-Phe-Ser-Pro-Arg-Leu-NH₂) had significantly higher activity at the lower doses of 100 and 10 pmol and may represent a tryptic cleavage product of PBAN.     

PBAN-induced sex pheromone biosynthesis in Heliothis peltigera: Structure,dose, and time dependent analysis

A structure-activity relationship study of Hez-PBAN was performed with respect to its pheromonotropic activity, using Heliothis peltigera as the test animal. The activity of N- and C-terminally derived sequences was examined in a time- and dose-dependent mode. Using a variety of Hez-PBAN-derived fragments at two doses (1 and 10 pmol) and at different times post-injection (5–120 min), we were able to demonstrate that peptides lacking 12 and 16 amino acids from their N-terminus are as potent as the full length PBAN, and that the C-terminally derived hexapeptide was capable of stimulating sex pheromone production to a similar extent as PBAN 1–33NH₂, when its activity was analyzed at shorter post-injection times. Within the C-terminal sequence, the amide was found to play a crucial role. In addition, it was observed that the region between amino acids 9 and 13 is important for the biological activity of the full length PBAN. The fact that the pheromonotropic activity of the hexapeptide was similar to that of the full length PBAN, under specific conditions, suggests that this sequence constitutes the biologically active site of the neuropeptide. The discovery that PBAN-derived peptides reacted in a time- and dose-dependent mode, strengthens the assumption that proteolytic enzymes interfere with the pheromonotropic activity of the PBAN-derived fragments. The ability of a variety of peptides to stimulate sex pheromone biosynthesis suggests two possible mechanisms: (1) Existence of multiple pheromonotropic mechanisms which may be mediated by multiple PBAN receptors that are activated at different kinetics; (2) Existence of only one mechanism mediated by short C-terminally derived peptides. In the first case, the C-terminally derived sequences fulfill the conformational requirement of only one class of receptors, and other regions in the PBAN molecule (e.g., 9–13) fulfill the conformational requirements of a second (or other) class of receptors. In the second case, the C-terminally derived sequence is the only conformationally important sequence, and other sequences, which were found to be essential for the biological activity, serve other non-conformational purposes (e.g., protection against proteolytic degradation). © 1995 Wiley-Liss, Inc.     

Effect of the pheromone biosynthesis activating neuropeptide on sex pheromone biosynthesis in Spodoptera littoralis isolated glands

The control of Spodoptera littoralis sex pheromone biosynthesis has been investigated with synthetic pheromone biosynthesis activating neuropeptide (PBAN) and different labeled tracers using an in vitro isolated gland system. Responsiveness of the glands to PBAN stimulation was impaired by careless tissue manipulation. The fact that PBAN is active in the isolated gland system suggests that this might be a target organ for this peptide in S. littoralis. As reported previously with Br-SOG extracts and intact females, label incorporation into the pheromone increased in glands treated with PBAN from all the precursors tested. However, the formation of labeled intermediates from d₅E11–14:Acid also occurred in glands incubated in the absence of the peptide, but the amounts of d₅Z9, E11–14:Acid were lower in PBAN treated glands than in controls. These results indicate that PBAN controls pheromone biosynthesis in S. littoralis by regulating the reduction of acyl moieties. © 1994 Wiley-Liss, Inc.     

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.     

Structure and activity of Bombyx PBAN

Two structurally related molecular species of pheromone biosynthesis activating neuropeptides (PBANs), PBAN-I and -II, were isolated from adult heads of the silkworm, Bombyx mori, and characterized. PBAN-I is a carboxyl-terminally amidated 33-residue peptide. Structure-activity relationship studies revealed that 1) its carboxyl-terminal pentapeptide is the smallest size showing activity, 2) the carboxyl-terminal amide is indispensable for activity, and 3) oxidation of three Met residues in PBAN-I to Met(O) (methionine sulfoxide) caused marked enhancement of activity, and the three Met(O) residues contribute equally to the enhancement of activity. Molecular design of PBAN analogs using a carboxyl-terminal hexapeptide showed that modification of the amino-terminal amino group brought about a dramatic increase in activity. This increase was presumed to be mainly due to the increased stability in hemolymph. PBANs share the common carboxyl-terminal sequence, -Phe-Xaa-Pro-Arg-Leu-NH₂, with myotropic peptides isolated from locust and cockroach. Examination of cross-activity of these two groups of peptides revealed that PBAN and its analogs exhibited myotropic activity comparable to myotropic peptides, while myotropic peptides showed extremely high pheromonotropic activity. In B. mori, PBAN activates sex pheromone (bombykol) production presumably by promoting the reduction reaction from acyl to alcohol, which is the last step in the biosynthesis of bombykol. © 1994 Wiley-Liss, Inc.     

A pheromonotropic peptide of Helicoverpa zea,with melanizing activity,interaction with PBAN,and distribution of immunoreactivity

The sequence of an 18-amino acid residue peptide was deduced from the gene encoding PBAN and other peptides with common C-termini in Helicoverpa zea. The peptide caused melanization in larvae and pheromone production in females of H. zea, and was designated pheromonotropic melanizing peptide (Hez-PMP). The peptide has a 83% sequence homology with a pheromonotropic peptide isolated from Pseudaletia separata. PMP caused melanization and mortality when injected into larvae just before molting. Whereas intense melanization was caused with a dose of 1,000 pmol, peak mortality occurred at 100 pmol, with 50% of larvae dying within 48 h after injection. Pheromonotropic activity of PMP was dose dependent. Co-injection of Hez-PMP and Hez-PBAN into a female resulted in suppression of the pheromonotropic effect of PBAN. Whole-mount immunocytochemical studies revealed PMP-like immunoreactivity in frontal ganglion, subesophageal, thoracic, and abdominal ganglia as well as the esophageal nerve.     

Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists.

A new approach for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) agonists and antagonists using the backbone cyclization and cycloscan concepts is described. Two backbone cyclic (BBC) libraries were synthesized: library I (Ser library) was based on the active C-terminal hexapeptide sequence Tyr-Phe-Ser-Pro-Arg-Leu-NH2 of PBAN1-33NH2; whereas library II (D-Phe library) was based on the sequence of the PBAN lead linear antagonist Arg-Tyr-Phe-d-Phe-Pro-Arg-Leu-NH2. In both libraries the Pro residue was replaced by the BBC building unit Nalpha-(omega-aminoalkyl) Gly having various lengths of alkyl chain. The peptides of the two libraries were tested for agonistic and antagonistic activity. Four precyclic peptides based on two of the BBC antagonists were also synthesized; their activity revealed that a negative charge at the N-terminus of the peptide abolished antagonistic activity. We also describe the use of the reagent SiCl3I for selective deprotection of the Boc group from the building unit prior to on-resin amino-end to backbone-nitrogen (AE-BN) cyclization, during solid-phase synthesis with Fmoc chemistry.     

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     

Potent activity of a PK/PBAN analog with an (E)-alkene,trans-Pro mimic identifies the Pro orientation and core conformation during interaction with HevPBANR-C receptor

The pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family plays a multifunctional role in an array of important physiological processes in insects, including regulation of sex pheromone biosynthesis in moths. A cyclic PK/PBAN analog (cyclo[NTSFTPRL]) retains significant activity on the pheromonotropic HevPBANR receptor from the tobacco budworm Heliothis virescens expressed in CHO-K1 cells. Previous studies indicate that this rigid, cyclic analog adopts a type I β-turn with a transPro over residues TPRL within the core PK/PBAN region. An analog containing an (E)-alkene, trans-Pro mimetic motif was synthesized, and upon evaluation on the HevPBANR receptor found to have an EC₅₀ value that is not statistically different from a parent C-terminal PK/PBAN hexapeptide sequence. The results, in aggregate, provide strong evidence for the orientation of Pro and the core conformation of PK/PBAN neuropeptides during interaction with the expressed PBAN receptor. The work further identifies a novel scaffold with which to design mimetic PBAN analogs as potential leads in the development of environmentally favorable pest management agents capable of disrupting PK/PBAN-regulated pheromone signaling systems.     

Induction of cuticular melanization in Spodoptera littoralis larvae by PBAN/MRCH: Development of a quantitative bioassay and structure function analysis

PBAN (also termed melanization and reddish coloration hormone, MRCH) is a cerebral factor known to regulate sex pheromone biosynthesis and cuticular melanization in moths. In the present study we developed a quantitative method (based on computerized image analysis of cuticles) to determine the effect of Helicoverpa zea PBAN (Hez-PBAN) on cuticular melanization and to study the structure-activity relationship of the neuropeptide in Spodoptera littoralis larvae. The results indicate that Hez-PBAN stimulates cuticular melanization in an interspecific manner, and that the minimal dose evoking formation of melanins is between 3–10 pmol/larva. Higher doses of Hez-PBAN did not stimulate melanization any further. Examination of the structure-activity relationship of Hez-PBAN revealed that the first eight N-terminal amino acids are not essential for the melanotropic activity and that the activity resides in the C-terminal region. Within this region the C-terminal amide was found to play a very important role. © 1996 Wiley-Liss, Inc.     

Effect of synthetic pban and derived peptides on sex pheromone biosynthesis in Heliothis peltigera (Lepidoptera: Noctuidae)

The activity of synthetic Heliothis zea PBAN (Hez-PBAN) and four shorter peptides on the sex pheromone biosynthesis in Heliothis peltigera was investigated in order to characterize their biological potency, and to determine the structure-activity relationship. Hez-PBAN (PBAN 1–33) is very potent and stimulates sex pheromone biosynthesis at the picomolar range both in photophase and scotophase. Removal of eight amino acids from the N-terminal region of the peptide Hez-PBAN had only a minor effect on the biological activity. A shorter fragment of Hez-PBAN, lacking 18 amino acids from the N-terminus, was less active. Two short peptides, consisting of eight and six amino acids, derived from the C-terminal region of Hez-PBAN had very little biological activity. In addition, it was found that PBAN 1–33 undergoes oxidation during storage. The oxidation of the peptide resulted in a loss of its biological activity, which could be restored by reduction with N-methylmercaptoacetamide. Unlike PBAN 1–33, PBAN 9–33 did not lose activity as a function of time, and its activity was fully preserved after prolonged storage. The results indicate that PBAN 1–33 and PBAN 9–33 have similar activities, and that the sequence containing the eight N-terminal amino acids is not essential for the biological activity of Hez-PBAN on the biosynthesis of H. peltigera sex pheromone.     

Development of a highly sensitive ELISA for the determination of PBAN and its application to the analysis of hemolymph in Spodoptera littoralis

A highly sensitive enzyme linked immunosorbent assay (ELISA) for the determination of the pheromone biosynthesis activating neuropeptide (PBAN) has been developed. Six antisera have been obtained that recognize the carboxyl terminal side of this peptide. Two immunogens have been rationally designed and synthesized in order to direct antibody specificity, using as haptens PBAN or PBAN(20-33) with a Cys residue attached to their amino-terminal side. The Cys thiol group has been used to covalently bind the peptide to keyhole limpet hemocyanin (KLH) by using N-succinimidyl-4-(maleidimidomethyl) cyclohexane carboxylate (SMCC) as a convenient heterobifunctional cross-linker. Several usable competitive immunoassays have been obtained by synthesizing eight different coating antigens and screening the sera against all of them. The best assay was obtained with antibody 4 using Cys-Hez-PBAN(20-33) coupled to bovine serum albumin (BSA) through the Lys groups by using the homobifunctional cross-linker dimethylpimelidate dihydrochloride (DMP) as the coating antigen. The optimized assay allows to detect PBAN at concentrations as low as 1 fmol/well (l₅₀ = 2.5 fmol/well). An extraction procedure for the hemolymph has been developed that allows to perform PBAN measurements in this tissue even after a tenfold dilution. In these conditions matrix effect is negligible. Preliminary results on the presence of PBAN like immunoreactivity (PBAN-IR) in the hemolymph of Spodoptera littoralis females are reported.© 1995 Wiley-Liss, Inc.     

Comparison of the effects of pyrokinins and related peptides identified from arthropods on pupariation behaviour in flesh fly (Sarcophaga bullata) larvae (Diptera: Sarcophagidae)

Peptides from the pyrokinin/PBAN family and some structurally related compounds identified in various arthropods were tested for acceleration of puparial contraction in flesh fly larvae. Modifications of behavioural patterns of pupariation were further studied for the active compounds using a behavioural analysis based on the recording of changes in tension of the cuticle. Nine peptides belonging to the pyrokinin/PBAN family (Lem-PK, Pea-PK-5, Lom-PK II, Hez-PBAN, Bom-DH-I), identified in five different insect species, two pyrokinin peptides derived from the genome of Drosophila melanogaster (capa-3, and hugin), and two pyrokinins identified from the white shrimp Penaeus vannamei were very active in the pupariation assay, with threshold doses within the range of 0.1-5.0 pmol larva(-1). High activity was also detected for a related peptide ETH1 from Drosophila. All of these peptides share a C-terminal PRLamide, which is essential and sufficient for the activity. Interestingly, two other structurally related peptides from Drosophila--ETH2 and capa-1--which feature conservative changes (Ile and Val, respectively) at the C-terminal Leu position, were inactive within a physiological range of concentrations. It is clear that the receptor mediating the acceleration of puparial contraction behaviour is sensitive to the introduction of greater steric bulk at the C-terminal Leu position. The peptides that accelerated pupariation showed very similar patterns of muscular and cuticular activity.     

The pheromone biosynthesis activating neuropeptide (PBAN) receptor of Heliothis virescens: identification, functional expression, and structure-activity relationships of ligand analogs

Pheromone biosynthesis activating neuropeptide (PBAN) promotes synthesis and release of sex pheromones in moths. We have identified and functionally expressed a PBAN receptor from Heliothis virescens (HevPBANR) and elucidated structure-activity relationships of PBAN analogs. Screening of a larval CNS cDNA library revealed three putative receptor subtypes and nucleotide sequence comparisons suggest that they are produced through alternative splicing at the 3'-end. RT-PCR amplified preferentially HevPBANR-C from female pheromone glands. CHO cells expressing HevPBANR-C are highly sensitive to PBAN and related analogs, especially those sharing the C-terminal pentapeptide core, FXPRLamide (X=T, S or V). Alanine replacements in the C-terminal hexapeptide (YFTPRLamide) revealed the relative importance of each residue in the active core as follows: R5>L6>F2>P4>T3>Y1. This study provides a framework for the rational design of PBANR-specific agonists and/or antagonists that could be exploited for disruption of reproductive function in agriculturally important insect pests.     

Backbone cyclic peptide antagonists, derived from the insect pheromone biosynthesis activating neuropeptide, inhibit sex pheromone biosynthesis in moths.

We describe an application of the backbone cyclization and cycloscan concept for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) antagonists capable of inhibiting sex pheromone biosynthesis in Heliothis peltigera female moths. Two backbone cyclic (BBC) sub-libraries were designed and synthesized. The structure of the first sub-library ([Arg27]PBAN27-33NH2, termed the Ser sub-library) was based on the active C-terminal hexapeptide sequence (Tyr-Phe-Ser-Pro-Arg-Leu-NH2) of PBAN1-33NH2, which was found to comprise its active core. The second sub-library ([Arg27, D-Phe30]PBAN27-33NH2, termed the D-Phe sub-library) was based on the sequence of the lead antagonist Arg-Tyr-Phe-(D)Phe-Pro-Arg-Leu-NH2. In both sub-libraries the Pro residue was replaced by an Nalpha(omega-amino-alkyl)Gly building unit having various lengths of the alkyl chain. All the cyclic peptides in each sub-library had the same primary sequence and the same location of the ring. The members of each library differed from each other by the bridge size and bridge chemistry. Screening of the two libraries for pheromonotropic antagonists resulted in the disclosure of four compounds that fully inhibited sex pheromone biosynthesis at 1 nmol and were devoid of agonistic activity. All antagonistic peptides originated from the D-Phe sub-library. Substitution of the D-Phe30 amino acid with a Ser resulted in a loss of antagonistic activity. Agonistic activities were exhibited by peptides from both sub-libraries.     

Studies on the status of tyrosyl residues in phospholipases A2 fromNaja naja atra andNaja nigricollis snake venoms

Two phospholipases A₂ (PLA₂) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA₂ were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca ²⁺; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca²⁺. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA₂ could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity.Tyrosine-63-modifiedN. naja atra PLA₂ exhibited the same Ca²⁺-induced difference spectra as that of native PLA₂, indicating that the Ca²⁺-binding ability of Tyr-63-modifiedN. naja atra PLA₂ was not impaired. However, Tyr-3-modified PLA₂ and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca²⁺, revealing that the Ca²⁺-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca²⁺ binding. AtpH 8.0, both native PLA₂ enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca²⁺, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved.     

Temporal distribution of PBAN‐like immunoreactivity in the hemolymph of Mamestra brassicae females in relation to sex pheromone production and calling behavior

A direct enzyme‐linked immunosorbent assay has been developed and applied to the analysis of PBAN immunoreactivity in female hemolymph of the cabbage armyworm, Mamestra brassicae. PBAN‐IR determinations have been carried out with third scotophase insects at different times of the photoperiod. The rhythm of calling and the pattern of pheromone production by third scotophase females at different times of the photoperiod have also been determined. PBAN‐IR and calling are well correlated. However, whereas pheromone titers decrease, both PBAN‐IR levels and percentage of calling females remain high in the last hours of the scotophase. These results are discussed in the context of the regulation of sex pheromone biosynthesis in M. brassicae. Arch. Insect Biochem. Physiol. 40:80–87, 1999. © 1999 Wiley‐Liss, Inc.     

Effect of the insecticide nerametrine EK-15 on the activity of soil microorganisms

The effect of the insecticide Nerametrine EK-15 (containing an active supercypermetrine component) on nitrification, nitrogen fixation, CO₂ production and cellulase activity of soil microorganisms was investigated. Four soil types were sampled from various localities. Supercypermetrine at 31 pmol/kg soil affected remarkably the metabolic activity of all soil samples tested by producing CO₂ after a 1-d exposure. After a 14-d exposure no difference in the metabolic activity related to CO₂ production was noticed in the case of garden soil where the insecticide at 31 pmol/kg soil and the unaffected control were used. As far as other samples are concerned, the supercypermetrine concentration amounting to 31 pmol/kg soil explicitly inhibited the metabolic activity of soil microorganisms. On the other hand, concentrations of 0.61 and 6.1 pmol/kg soil stimulated the metabolic activity of soil in the locality of Senica. The soil samples enriched with nutrients (organic nitrogen in urea) manifested an evident inhibition at 31 pmol/kg soil. The nitrification activity of all soil types was interrupted at 61 pmol/kg soil. Supercypermetrine 0.12 pmol/L stopped completely nitrogen fixation withA. chroococcum and that corresponding to 0.3 pmol/L stopped aerobic cellulase decomposition.     

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.