Ada Rafaeli

Identification of a G protein-coupled receptor for pheromone biosynthesis activating neuropeptide from pheromone glands of the moth Helicoverpa zea

Pheromone biosynthesis-activating neuropeptide (PBAN), a peptide produced by the subesophageal ganglion, is used by a variety of moths to regulate pheromone production. PBAN acts directly on pheromone gland cells by using calcium and cAMP as second messengers. We have identified a gene encoding a G protein-coupled receptor (GPCR) from pheromone glands of the female moth Helicoverpa zea. The gene was identified based on sequence identity to a group of GPCRs from Drosophila that are homologous to neuromedin U receptors in vertebrates. The full-length PBAN receptor was subsequently cloned, expressed in Sf9 insect cells, and shown to mobilize calcium in response to PBAN. This response was dose-dependent (EC50 = 25 nM) with a maximum response at 300 nM and a minimal observable response at 10 nM. Four additional peptides produced by the PBAN-encoding gene were also tested for activity, and it was determined that three had similar activity to PBAN and the other was slightly less active. Peptides belonging to the same family as PBAN, namely pyrokinins, as well as the vertebrate neuromedin U peptide also induced a calcium response. We have identified a GPCR for the PBAN/pyrokinin family of peptides with a known function of stimulating pheromone biosynthesis in female moths. It is related to several receptors from insects (Drosophila and Anopheles) and to neuromedin U and ghrelin receptors from vertebrates.

Pheromone biosynthesis activating neuropeptide (PBAN): Regulatory role and mode of action

This review focuses on the endocrine regulation of reproductive behavior in moth species with particular emphasis on Helicoverpa spp. Reproductive behavior in most adult moths is dependent on the release of a unique blend of sex pheromones by the females to attract conspecific males. Mating, on the other hand, results in a loss of sexual receptivity due to the transfer of secretions from the male accessory glands, which renders females unattractive to ensuing mates. Synchronization of sexual behavior is attained by the timely release of Pheromone-Biosynthesis-Activating Neuropeptide (PBAN), a member of the PBAN/Pyrokinin neuropeptide family, characterized by a common amino acid sequence FXPRLamide motif in the C-terminus. PBAN is released into the hemolymph of females during the scotophase and is drastically reduced after mating, contributing to the loss in female receptivity. Pheromone production is age-dependent and Juvenile Hormone is involved in its regulation. PBAN activates pheromone production through its binding to a PBAN-Receptor (PBAN-R) and subsequent up-regulation of key enzymes in the biosynthetic pathway. The PBAN-R gene was identified as a member of the G-protein coupled receptor family (GPCRs), classified with the vertebrate subfamily of neuromedin U receptors. Using both biochemical and in silico mutagenesis studies, putative binding sites are predicted. Differential expression studies reveal its localization in pheromone glands, neural tissues and the male aedeagus. In the latter tissue, no activity and/or receptor-binding can be detected in response to PBAN. These results raise many questions concerning the evolutionary role of the PBAN/Pyrokinin receptors belonging to the GPCR family.

PBAN regulation of pheromone biosynthesis in female moths

Publisher Summary This chapter discusses the pheromone biosynthesis activating neuropeptide (PBAN) regulation of pheromone biosynthesis in female moths. The neuropeptide regulates pheromone production in most of the moths. It belongs to the pyrokinin/PBAN family of peptides based on functional cross-reactivity with the active sequence FXPRL amide at the C-terminal end. The gene encoding for PBAN encodes for several pyrokinin/PBAN-like peptides in addition to PBAN. Studies on the site of PBAN action indicate that pheromone gland cells have a receptor for PBAN that starts the signal cascade of events to trigger the pheromone biosynthetic pathway. Normal activation of pheromone production may involve several interdependent mechanisms including both neural and humoral modulators. Addressing molecular mechanisms involved in pheromone production led to the elucidation of receptor proteins involved in both the stimulation and feedback inhibition of pheromone biosynthesis. In addition, such molecular studies reveal the identity and structure of the key enzymes involved in eliciting the biosynthetic pathway.

Drosophila melanogaster sex peptide stimulates juvenile hormone synthesis and depresses sex pheromone production in Helicoverpa armigera

Previous studies demonstrate that virgin female adult Helicoverpa armigera (Lepidoptera: Noctuidae) moths exhibit calling behaviour and produce sex pheromone in scotophase from the day after emergence, and that mating turns off both of these pre-mating activities. In the fruit fly Drosophila melanogaster, a product of the male accessory glands, termed sex peptide (SP), has been identified as being responsible for suppressing female receptivity after transfer to the female genital tract during mating. Juvenile hormone (JH) production is activated in the D. melanogaster corpus allatum (CA) by SP in vitro. We herein demonstrate cross-reactivity of D. melanogaster SP in the H. armigera moth: JH production in photophase virgin female moth CA in vitro is directly activated in a dose-dependent manner by synthetic D. melanogaster SP, and concurrently inhibits pheromone biosynthesis activating neuropeptide (PBAN)-activated pheromone production by isolated pheromone glands of virgin females. Control peptides (locust adipokinetic hormone, AKH-I, and human corticotropin, ACTH) do not inhibit in vitro pheromone biosynthesis. Moreover, SP injected into virgin H. armigera females, decapitated 24 h after eclosion, or into scotophase virgin females, suppresses pheromone production. In the light of these results, we hypothesize the presumptive existence of a SP-like factor among the peptides transmitted to female H. armigera during copulation, inducing an increased level of JH production and depressing the levels of pheromone produced thereafter.

Action of pheromone biosynthesis activating neuropeptide on In vitro pheromone glands of Heliothis armigera females

Abstract Pheromone glands of Heliothis armigera were stimulated in vitro to incorporate 14C from the radioactive precursor sodium acetate, in the presence of synthetic pheromone biosynthesis activating neuropeptide (PBAN). When hexane extracts of the radioactive products were analysed by TLC and HPLC the radioactivity corresponded in retention time to the main pheromone component (Z)-11-hexadecenal. Maximal stimulation, as depicted by TLC analysis, was observed after 4 h of incubation. The PBAN response was shown to be dose dependent, maximal levels, as analysed by TLC, were obtained at a concentration of 5 pmol/gland. This response was shown to be mediated by cAMP.

Common functional elements of Drosophila melanogaster seminal peptides involved in reproduction of Drosophila melanogaster and Helicoverpa armigera females.

Sex peptide (SP) and Ductus ejaculatorius peptide (Dup) 99B are synthesized in the retrogonadal complex of adult male Drosophila melanogaster, and are transferred in the male seminal fluid to the female genital tract during mating. They have been sequenced and shown to exhibit a high degree of homology in the C-terminal region. Both affect subsequent mating and oviposition by female D. melanogaster. SP also increases in vitro juvenile hormone (JH) biosynthesis in excised corpora allata (CA) of D. melanogaster and Helicoverpa armigera. We herein report that the partial C-terminal peptides SP(8-36) and SP(21-36) of D. melanogaster, and the truncated N-terminal SP(6-20) do not stimulate JH biosynthesis in vitro in CA of both species. Both of these C-terminal peptides reduce JH-III biosynthesis significantly. Dup99B, with no appreciable homology to SP in the N-terminal region, similarly lacks an effect on JH production by H. armigera CA. In contrast, the N-terminal peptides - SP(1-11) and SP(1-22) - do significantly activate JH biosynthesis of both species in vitro. We conclude that the first five N-terminal amino acid residues at the least, are essential for allatal stimulation in these disparate insect species. We have previously shown that the full-length SP(1-36) depresses pheromone biosynthesis in H. armigera in vivo and in vitro. We now show that full-length Dup99B and the C-terminal partial sequence SP(8-36) at low concentrations strongly depress (in the range of 90% inhibition) PBAN-stimulated pheromone biosynthesis of H. armigera. In addition, the N-terminal peptide SP(1-22), the shorter N-terminal peptide SP(1-11) and the truncated N-terminal SP(6-20) strongly inhibit pheromone biosynthesis at higher concentrations.

Regulatory Role of PBAN in Sex Pheromone Biosynthesis of Heliothine Moths

Both males and females of heliothine moths utilize sex-pheromones during the mating process. Females produce and release a sex pheromone for the long–range attraction of males for mating. Production of sex pheromone in females is controlled by the peptide hormone (pheromone biosynthesis activating neuropeptide, PBAN). This review will highlight what is known about the role PBAN plays in controlling pheromone production in female moths. Male moths produce compounds associated with a hairpencil structure associated with the aedaegus that are used as short-range aphrodisiacs during the mating process. We will discuss the role that PBAN plays in regulating male production of hairpencil pheromones.

Pyrokinin/PBAN-like peptides in the central nervous system of Drosophila melanogaster.

The pyrokinin/pheromone biosynthesis activating neuropeptide (PBAN) family of peptides found in insects is characterized by a 5-amino-acid C-terminal sequence, FXPRLamide. The pentapeptide is the active core required for diverse physiological functions, including stimulation of pheromone biosynthesis in female moths, stimulation of muscle contraction, induction of embryonic diapause in Bombyx mori, and stimulation of melanization in some larval moths. Recently, this family of peptides has been implicated in accelerating the formation of the puparium in a dipteran. Using bioassay and immunocytochemical techniques, we demonstrate the presence of pyrokinin/PBAN-like peptides in the central nervous system of Drosophila melanogaster. Pheromonotropic activity was shown in the moths Helicoverpa zea and Helicoverpa armigera by using dissected larval nervous systems and adult heads and bodies of D. melanogaster. Polyclonal antisera against the C-terminal ending of PBAN revealed the location of cell bodies and axons in the central nervous systems of larval and adult flies. Immunoreactive material was detected in at least three groups of neurons in the subesophageal ganglion of 3rd instar larvae, pupae, and adults. The ring gland of both larvae and adults contained immunoreactivity. Adult brain-subesophageal ganglion complex possessed additional neurons. The fused ventral ganglia of both larvae and adults contained three pairs of neurons that sent their axons to a neurohemal organ connected to the abdominal nervous system. These results indicate that the D. melanogaster nervous system contains pyrokinin/PBAN-like peptides and that these peptides could be released into the hemolymph.

Down regulation of pheromone biosynthesis: Cellular mechanisms of pheromonostatic responses

The biochemical second messenger system during pheromonotropic and pheromonostatic activities was assessed and compared. The involvement of G-proteins was implicated by the stimulatory action of sodium fluoride (NaF), at a range of 1–2 mM, on both pheromone biosynthesis and intracellular cAMP levels in isolated intersegmental membranes of Helicoverpa armigera. However, cholera toxin did not mimic the pheromonotropic response of PBAN. The stimulatory action of NaF was significantly inhibited by adrenergic agonists (tyramine and clonidine) as was observed at low levels of PBAN. At high levels of PBAN, although cAMP production was inhibited, pheromone biosynthesis was unaffected by clonidine. A similar phenomenon was observed with the ionophore, thapsigargin, in which adrenergic agonists did not inhibit pheromone biosynthesis but reduced intracellular cAMP to basal levels. Thus pheromonotropic activity exhibited both cAMP-independent and cAMP-dependent stimulatory responses. The calcium calmodulin inhibitor, W7, inhibited pheromone biosynthesis and intracellular cAMP production which was induced either by Hez-PBAN, NaF or thapsigargin. The pheromonostatic activity by clonidine was prevented in the presence of pertussis toxin, thereby indicating the involvement of inhibitory G-protein (Gi) in the inhibitory action of adrenergic agonists on the activity of Hez-PBAN. From the results we hypothesized that negative regulation of pheromonotropic activity occurs at the membrane receptor level by the interaction of an adrenergic receptor.

Modulation of the PBAN-stimulated of pheromonotropic activity in Helicoverpa armigera

An extensive study of the effect of octopamine on pheromonotropic activity was initiated. Octopamine (1 mM), tyramine (1mM) and K+(100 mM) were observed to inhibit the pheromonotropic action due to PBAN in pheromone gland incubations. The biogenic amine inhibition was reversed in the presence of the antagonists phentolamine and yohimbine. The pheromonostatic action of octopamine was dependent on the PBAN concentration in the incubation medium. Octopamine, at concentrations of 0.1 and 1 mM, significantly inhibited the pheromonotropic action of 1 and 5μM PBAN after a minimum of 2 h incubation in vitro. This inhibitory action was evident on the production of Z11-hexadecenal, the main pheromone component, (analysed by thin-layer and gas chromatography) as well as the fatty acid fraction (analysed by thin-layer chromatography) in the intersegmental membrane portion of pheromone gland incubations. No significant pheromonotropic or pheromonostatic effect was observed in incubations of the 8th segment alone. The pheromonostatic action was also evident at the cellular level (inhibition of the production of intracellular cAMP) but only in the intersegmental membrane portion of pheromone gland incubations. On the other hand, in incubations of the 8th segment alone, octopamine stimulated intracellular cAMP levels. Pulse-chase experiments, using the precursor 14C sodium acetate, showed that the pheromonostatic activity was not due to an increase in the release or degradation rate of the pheromone component but due to an inhibition in biosynthesis. The pheromonostatic action of octopamine was confirmed by experiments in vivo. The physiological significance of the action of octopamine is discussed.