Biosynthesis-activating Neuropeptide Research Articles

Functional Analysis of Pheromone Biosynthesis Activating Neuropeptide Receptor Isoforms in Maruca vitrata.

Insect sex pheromones are volatile chemicals that induce mating behavior between conspecific individuals. In moths, sex pheromone biosynthesis is initiated when pheromone biosynthesis-activating neuropeptide (PBAN) synthesized in the suboesophageal ganglion binds to its receptor on the epithelial cell membrane of the pheromone gland. To investigate the function of PBAN receptor (PBANR), we identified two PBANR isoforms, MviPBANR-B and MviPBANR-C, in the pheromone glands of Maruca vitrata. These two genes belong to G protein-coupled receptors (GPCRs) and have differences in the C-terminus but share a 7-transmembrane region and GPCR family 1 signature. These isoforms were expressed in all developmental stages and adult tissues. MviPBANR-C had the highest expression level in pheromone glands among the examined tissues. Through in vitro heterologous expression in HeLa cell lines, only MviPBANR-C-transfected cells responded to MviPBAN (≥5 µM MviPBAN), inducing Ca2+ influx. Sex pheromone production and mating behavior were investigated using gas chromatography and a bioassay after MviPBANR-C suppression by RNA interference, which resulted in the major sex pheromone component, E10E12-16:Ald, being quantitatively reduced compared to the control, thereby decreasing the mating rate. Our findings indicate that MviPBANR-C is involved in the signal transduction of sex pheromone biosynthesis in M. vitrata and that the C-terminal tail plays an important role in its function.

Redundant actions of neuropeptides encoded by the dh-pban gene for larval color pattern formation in the oriental armyworm Mythimnaseparata

The pyrokinin (PK)/pheromone biosynthesis-activating neuropeptide (PBAN) family, which is defined by a conserved C-terminal pentapeptide (FXPRLamide), is involved in many physiological processes in insects. In the oriental armyworm Mythimna separata, the larvae exhibit a variety of color patterns in response to changes in population density, which are caused by melanization and a reddish coloration hormone (MRCH), which is a member of the FXPRLamide neuropeptides. Interestingly, in some lepidopteran insects, MRCH is known as a PBAN, which activates the pheromone gland to produce sex pheromones. PBAN is encoded by a single gene, dh-pban, which encodes additional FXPRLamide neuropeptides, such as the diapause hormone (DH) and subesophageal ganglion neuropeptides (SGNPs). To determine the roles of the dh-pban gene, which produces multiple types of FXPRLamide neuropeptides after post-transcriptional cleavage of the precursor protein, we performed CRISPR/Cas9-mediated targeted mutagenesis in M. separata. We demonstrated that knockout armyworm larvae lost density-dependent cuticular melanization and retained yellow body color, even when reared under crowded conditions. Moreover, our rescue experiments using the synthetic peptides showed that not only PBAN but also β- and γ-SGNPs significantly induce the cuticular melanization in a dose dependent manner. Taken together, our results provide genetic evidence that neuropeptides encoded by the single dh-pban gene act redundantly to control density-dependent color pattern formation in M. separata.

A novel physiological function of pheromone biosynthesis-activating neuropeptide in production of aggregation pheromone

The western flower thrips, Frankliniella occidentalis, is an insect pest, and its aggregation pheromone (AP) plays a crucial role in the recruitment of both sexes. A novel pheromone biosynthesis-activating neuropeptide (PBAN)-like gene is encoded in F. occidentalis genome, but its physiological function has yet to be elucidated. This study hypothesized the physiological role played by PBAN in mediating AP production. AP has been known to be produced only by male adults in F. occidentalis. Surprisingly, our extraction of headspace volatiles contained two AP components in females as well as in males with similar composition. PBAN injection elevated the AP production whereas RNA interference (RNAi) of the gene expression suppressed the AP production in both sexes. A biosynthetic pathway to produce AP components were predicted and the enzymes catalyzing the main steps were confirmed in their expressions. Individual RNAi treatments of these genes significantly suppressed AP production. RNAi of PBAN gene downregulated the expressions of these biosynthesis-associated genes in both sexes. These results suggest that the novel neuropeptide acts as PBAN mediating AP production through stimulating its biosynthetic machinery in F. occidentalis.

Transcriptome Analysis of Ostrinia furnacalis Female Pheromone Gland: Esters Biosynthesis and Requirement for Mating Success.

Female moths use sex pheromones to attract males, and corresponding regulatory mechanism underlying sex pheromone biosynthesis is species-dependent. However, the detailed mechanism involved in sex pheromone biosynthesis in Ostrinia furnacalis has not yet been fully addressed. In the present study, transcriptome sequencing of O. furnacalis pheromone glands screened a serials of candidate genes involved in sex pheromone biosynthesis. Our analysis showed that sex pheromone release in O. furnacalis females arrives its peak at the 2nd scotophase, consistent with its mating behavior. Pheromone biosynthesis-activating neuropeptide (PBAN) was confirmed to regulate sex pheromone biosynthesis, and Ca2+ is the secondary messenger of PBAN signaling in O. furnacalis. The functional analysis of candidate genes demonstrated that the decreased mRNA levels or activities of calcineurin (CaN) and acetyl-CoA carboxylase (ACC) led to significant decrease in sex pheromone production and female capability to attract males, as demonstrated by RNAi-mediated knockdown and pharmacological inhibitor assay. Most importantly, the activities of CaN and ACC depend on the activation of PBAN/PBANR/Ca2+. Furthermore, fatty-acyl reductase 14 was involved in PBAN-mediated sex pheromone biosynthesis. Altogether, our results demonstrated that PBAN regulates sex pheromone biosynthesis through PBANR/Ca2+/CaN/ACC pathway to promote sex pheromone biosynthesis in O. furnacalis and provided a reference for non-model organism to study neuropeptide signal transduction.

Pheromone biosynthetic pathway and chemoreception proteins in sex pheromone gland of Eogystia hippophaecolus

The moth Eogystia hippophaecolus (Hua et al.) is a major threat to sea buckthorn plantations in China. Specific and highly efficient artificial sex pheromone traps have been developed and used to control this pest species. However, the biosynthesis of sex pheromones Z7-14: Ac and E3-14:Ac remains poorly understood. We investigated the female pheromone gland transcriptome of E. hippophaecolus and identified two pheromone biosynthesis-activating neuropeptides (PBANs), two pheromone biosynthesis-activating neuropeptide receptors (PBANrs), five acetyl-CoA carboxylases (ACCs), six fatty acid synthases (FASs), 16 Acyl-CoA desaturases (DESs), 26 reductases (REDs), 13 acetyltransferases (ACTs), one fatty acid transport protein (FATP), one acyl-CoA-binding protein (ACBP), and five elongation of very long-chain fatty acid proteins (ELOs) in pheromone biosynthesis pathways. Additionally, we identified 11 odorant-degrading enzymes (ODEs) and 16 odorant-binding proteins (OBPs), 14 chemosensory proteins (CSPs), two sensory neuron membrane proteins (SNMPs), three odorant receptors (ORs), seven ionotropic receptors (IRs), and six gustatory receptors (GRs). 77 unigenes involved in female pheromone biosynthesis, 31 chemoreception proteins and 11 odorant degradation enzymes were identified, which provided insight into the regulation of the pheromone components and pheromone recognition in the sex pheromone gland, and knowledge pertinent to new integrated pest management strategy of interference pheromone biosynthesis and recognition.

Distribution of Pheromone Biosynthesis-Activating Neuropeptide in the Central Nervous System of Plutella xylostella (Lepidoptera: Plutellidae).

Insect neuropeptides in the pyrokinin/pheromone biosynthesis-activating neuropeptide (PBAN) family are actively involved in many essential endocrinal functions and serve as potential targets in the search for novel insect control agents. Here, we dissect the nervous system of larval, pupal, and adult Plutella xylostella (L.) (Lepidoptera: Plutellidae) and describe the ganglion morphology and localization of PBAN during different insect developmental stages. Our results show that the central nervous system (CNS) of this species consists of four types of ganglia: cerebral ganglia (brain), subesophageal ganglion (SEG), thoracic ganglia, and abdominal ganglia. A two-lobed brain is connected to the reniform SEG with a nerve cord in larvae and prepupae, whereas in the late pupae and adults, the brain and SEG are fused, forming a brain-SEG complex. The larvae and prepupae have eight abdominal ganglia each, whereas the late pupae and adults each have four abdominal ganglia. Furthermore, all life stages of P. xylostella had similar patterns of PBAN immunoreactivity in the CNS, and the accumulation of PBAN was similar during all life stages except in adult males. PBAN immunoreactive signals were observed in the brain and SEG, and fluorescence signals originating in the SEG extended the entire length of the ventral nerve cord, ending in the terminal abdominal ganglia. Our results provide morphological data that inform the development and evolution of the CNS. In addition, they indicate that the nervous system contains PBAN, which could be used to control P. xylostella populations.

Sex-specific spatial and temporal gene expressions of Pheromone biosynthesis activating neuropeptide (PBAN) and binding proteins (PBP/OBP) in Spoladea recurvalis

Spoladea recurvalis is one of the most destructive insect pests of amaranth, a leafy vegetable in both Asia and Africa. The present study characterized the pheromone biosynthesis-activating neuropeptide (DH-PBAN) and pheromone/odorant binding proteins in S. recurvalis. The open reading frame of 600 base pairs encodes a 200-amino acid protein possessing five neuropeptide motifs (DH, PBAN, α-, β-, and γ- subesophageal ganglion neuropeptides) and shares a characteristic conserved C-terminal pentapeptide fragment FXPRL. The full-length genome of Spre-DH-PBAN was 4,295 bp in length and comprised of six exons interspersed by five introns. Sequence homology and phylogenetic analysis of Spre-DH-PBAN have high similarity to its homologs in Crambidae of Lepidopteran order. We quantitatively measured the relative expression level (qRT_PCR) of Spre-DH-PBAN gene, the binding proteins such as odorant binding proteins (OBPs) and pheromone binding protein (PBPs) at different developmental stages. The results confirmed their role in recognition and chemoreception of sex pheromone components, and they were distinct, tissue- and sex-specific. This is the first report on the molecular analysis of PBAN gene and binding proteins, which can improve the understanding of molecular mechanisms of growth, development, and reproductive behavior of S. recurvalis, and may become effective targets for controlling this insect.

A class-A GPCR solubilized under high hydrostatic pressure retains its ligand binding ability

The effect of high hydrostatic pressure (HHP) on the solubilization of a class-A G protein-coupled receptor, the silkmoth pheromone biosynthesis-activating neuropeptide receptor (PBANR), was investigated. PBANR was expressed in expresSF+ insect cells as a C-terminal fusion protein with EGFP. The membrane fraction was subjected to HHP treatment (200MPa) at room temperature for 1–16h in the presence of 0–2.0% (w/v) n-dodecyl-β-D-maltopyranoside (DDM). The solubilization yield of PBANR-EGFP in the presence of 0.6% (w/v) DDM increased to ~1.5-fold after 1h HHP treatment. Fluorescence-detection size-exclusion chromatography demonstrated that the PBANR-EGFP ligand binding ability was retained after HHP-mediated solubilization. The PBANR-EGFP solubilized with 1.0% DDM under HHP at room temperature for 6h retained ligand binding ability, whereas solubilization in the absence of HHP treatment resulted in denaturation.

Identification and expression profiling of pheromone biosynthesis activating neuropeptide in Chlumetia transversa (Walker)

Insect neuropeptides (NPs) in the pyrokinin/pheromone biosynthesis-activating neuropeptide (PBAN) family are actively involved in many essential endocrine functions. These peptides are potential targets in the search for novel insect control agents. This is the first report on the cloning and sequence determination of Chlumetia transversa (Walker) PBAN (Ct-PBAN) using rapid amplification of cDNA ends. The open reading frame of Ct-PBAN was 588bp in length and encoded 195 amino acids, which were assembled into five putative neuropeptides (diapause hormone homolog, α-neuropeptide, β-neuropeptide, PBAN, and γ-neuropeptide). These peptides were amidated at C-terminus and shared the conserved pentapeptide motif FXPR (or K) L. Moreover, Ct-PBAN had high homology to PBANs in Helicoverpa zea (84.1%), Helicoverpa armigera (83.5%), Helicoverpa assulta (83%), and Heliothis virescens (82.6%). Phylogenetic analysis showed that Ct-PBAN was closely related to its orthologs in the family Noctuidae. In addition, real-time quantitative polymerase chain reaction assays showed that the expression of Ct-PBAN peaked in the female head and was also detected at high levels in 1-d-old adults. These results suggested that Ct-PBAN is associated with sex pheromone biosynthesis in female C. transversa and could be used for developing C. transversa control systems based on molecular techniques.

Identification of differentially expressed genes in the pheromone glands of mated and virgin Bombyx mori by digital gene expression profiling.

BackgroundMating decreases female receptivity and terminates sex pheromone production in moths. Although significant progress has been made in elucidating the mating-regulated inactivation of pheromone biosynthesis-activating neuropeptide (PBAN) secretion, little is known about the mating induced gene expression profiles in pheromone glands (PGs). In this study, the associated genes involved in Bombyx mori mating were identified through digital gene expression (DGE) profiling and subsequent RNA interference (RNAi) to elucidate the molecular mechanisms underlying the mating-regulated gene expression in PGs.ResultsEight DGE libraries were constructed from the PGs of mated and virgin females: 1 h mating (M1)/virgin (V1) PGs, 3 h mating (M3)/virgin (V3) PGs, 24 h mating (M24)/virgin (V24) PGs and 48 h mating (M48)/virgin (V48) PGs (M48 and V48). These libraries were used to investigate the gene expression profiles affected by mating. DGE profiling revealed a series of genes showing differential expression in each set of mated and virgin female samples, including immune-associated genes, sex pheromone synthesis-associated genes, juvenile hormone (JH) signal-associated genes, etc. Most interestingly, JH signal was found to be activated by mating. Application of the JH mimics, methoprene to the newly-emerged virgin females leaded to the significant reduction of sex pheromone production. RNAi-mediated knockdown of putative JH receptor gene, Methoprene tolerant 1 (Met1), in female pupa resulted in a significant decrease in sex pheromone production in mature females, suggesting the importance of JH in sex pheromone synthesis.ConclusionA series of differentially expressed genes in PGs in response to mating was identified. This study improves our understanding of the role of JH signaling on the mating-elicited termination of sex pheromone production.

Identification of Functionally Important Residues of the Silkmoth Pheromone Biosynthesis-activating Neuropeptide Receptor, an Insect Ortholog of the Vertebrate Neuromedin U Receptor

The biosynthesis of sex pheromone components in many lepidopteran insects is regulated by the interaction between pheromone biosynthesis-activating neuropeptide (PBAN) and the PBAN receptor (PBANR), a class A G-protein-coupled receptor. To identify functionally important amino acid residues in the silkmoth PBANR, a series of 27 alanine substitutions was generated using a PBANR chimera C-terminally fused with enhanced GFP. The PBANR mutants were expressed in Sf9 insect cells, and their ability to bind and be activated by a core PBAN fragment (C10PBAN(R2K)) was monitored. Among the 27 mutants, 23 localized to the cell surface of transfected Sf9 cells, whereas the other four remained intracellular. Reduced binding relative to wild type was observed with 17 mutants, and decreased Ca(2+) mobilization responses were observed with 12 mutants. Ala substitution of Glu-95, Glu-120, Asn-124, Val-195, Phe-276, Trp-280, Phe-283, Arg-287, Tyr-307, Thr-311, and Phe-319 affected both binding and Ca(2+) mobilization. The most pronounced effects were observed with the E120A mutation. A molecular model of PBANR indicated that the functionally important PBANR residues map to the 2nd, 3rd, 6th, and 7th transmembrane helices, implying that the same general region of class A G-protein-coupled receptors recognizes both peptidic and nonpeptidic ligands. Docking simulations suggest similar ligand-receptor recognition interactions for PBAN-PBANR and the orthologous vertebrate pair, neuromedin U (NMU) and NMU receptor (NMUR). The simulations highlight the importance of two glutamate residues, Glu-95 and Glu-120, in silkmoth PBANR and Glu-117 and Glu-142 in human NMUR1, in the recognition of the most functionally critical region of the ligands, the C-terminal residue and amide.

Structural and functional differences between pheromonotropic and melanotropic PK/PBAN receptors

BackgroundThe pyrokinin/pheromone biosynthesis-activating neuropeptide (PK/PBAN) plays a major role in regulating a wide range of physiological processes in insects. The ubiquitous and multifunctional nature of the PK/PBAN peptide family raises many questions regarding the mechanisms by which these neuropeptides elicit their effects and the nature of the receptors that mediate their functions. MethodsA sex pheromone gland receptor of the PK/PBAN family from Heliothis peltigera female moth and a Spodoptera littoralis larval receptor were cloned and stably expressed, and their structural models, electrostatic potentials and cellular functional properties were evaluated. ResultsHomology modeling indicated highly conserved amino-acid residues in appropriate structural positions as experimentally shown for class A G-protein coupled receptors. Structural differences could be proposed and electrostatic potentials of the two receptor models revealed net charge differences. Calcium mobilization assays demonstrated that both receptors were fully functional and could initiate extracellular calcium influx to start PK/PBAN signal transduction. Evaluation of the signaling response of both receptors to PBAN and diapause hormone (DH) revealed a highly sensitive, though differential response. Both receptors responded to PBAN whereas only Spl-PK/PBAN-R exhibited a high response toward DH. ConclusionsThe structural, electrostatic and cellular functional differences indicate that different PK/PBAN in vivo functions may be mediated by different PK/PBAN receptors and elicited by different peptide(s). General significanceThe results advance our understanding of the mode of action of the PK/PBAN family, and might help in exploring novel high-affinity receptor-specific antagonists that can serve as a basis for the development of new families of insect-control agents.

The Arginine Residue within the C-Terminal Active Core of Bombyx mori Pheromone Biosynthesis-Activating Neuropeptide is Essential for Receptor Binding and Activation.

In most lepidopteran insects, the biosynthesis of sex pheromones is regulated by pheromone biosynthesis-activating neuropeptide (PBAN). Bombyx mori PBAN (BomPBAN) consists of 33 amino acid residues and contains a C-terminus FSPRLamide motif as the active core. Among neuropeptides containing the FXPRLamide motif, the arginine (Arg, R) residue at the second position from the C-terminus is highly conserved across several neuropeptides, which can be designated as RXamide peptides. The purpose of this study was to clarify the role of the Arg residue in the BomPBAN active core. We synthesized 10-residue peptides corresponding to the C-terminal part of BomPBAN with a series of replacements at the second position from the C-terminus, termed the C2 position, and measured their efficacy in stimulating Ca2+ influx in insect cells expressing a fluorescent PBAN receptor chimera (PBANR–EGFP) using the fluorescent Ca2+ indicator, Fura Red–AM. The PBAN analogs with the C2 position replaced with alanine (Ala, A), aspartic acid (Asp, D), serine (Ser, S), or l-2-aminooctanoic acid (Aoc) decreased PBAN-like activity. RC2A (SKTRYFSPALamide) and RC2D (SKTRYFSPDLamide) had the lowest activity and could not inhibit the activity of PBAN C10 (SKTRYFSPRLamide). We also prepared Rhodamine Red-labeled peptides of the PBAN analogs and examined their ability to bind PBANR. In contrast to Rhodamine Red-PBAN C10 at 100 nM, none of the synthetic analogs exhibited PBANR binding at the same concentration. Taken together, our results demonstrate that the C2 Arg residue in BomPBAN is essential for PBANR binding and activation.

Functional impact of silencing the Helicoverpa armigera sex‐peptide receptor on female reproductive behaviour

Female Helicoverpa armigera sex pheromone production is under the control of pheromone biosynthesis-activating neuropeptide (PBAN). After mating, females undergo suppression of sex pheromone production and enhanced oviposition as a result of the transfer of male-derived seminal peptides. In a previous study we identified a putative H. armigera sex-peptide receptor (HeaSP-R) and demonstrated a significant up-regulation in gene expression levels of this receptor in brains and pheromone glands of mated females, thereby implicating a regulatory role for sex peptide in the reproductive behaviour of H. armigera. In the present study, we show that virgin females injected with Drosophila melanogaster SP (DrmSP), in addition to inhibition of pheromone production, also exhibited a suppression of calling behaviour and a significant reduction in the gene expression levels of the PBAN-receptor. In addition, RNA interference (RNAi) silencing of the HeaSP-R expression by 50-60% prevented DrmSP-suppression of pheromone production and calling behaviour. Moreover, mated, silenced females failed to increase their oviposition rates as is normally observed in mated females, and their behaviour did not differ from that of virgin females. However, sex pheromone production by mated, silenced females remained low, comparable to mated, normal females, thereby indicating the probable involvement of additional factors in the suppression of sex pheromone production after mating.

The Use of Mass Isotopomer Distribution Analysis to Quantify Synthetic Rates of Sex Pheromone in the Moth Heliothis virescens

Although there has been much investigation of the steps involved in sex pheromone biosynthesis in moths, little is known about the kinetics of biosynthesis in vivo, primarily because there are few techniques suitable for studying the small amounts of pheromone produced without perturbing a female moth's normal physiology. In this paper, female Heliothis virescens moths fed on U-(13)C-glucose were subjected to mass isotopomer distribution analysis, enabling calculation of fractional (FSR) and absolute (ASR) synthetic rates of the main pheromone component, (Z)-11-hexadecenal, at two different photoperiodic times: during the scotophase (when adults are sexually active) and during the photophase (when adults do not engage in mating behavior). FSRs differed substantially at the two times with, as expected, the greater rate occurring during the scotophase. After determining Z11-16:Ald pool sizes, ASR through the scotophase was calculated to be roughly 20 times greater than ASR in the photophase. These differences are consistent with the release/non-release of the pheromone biosynthesis-activating neuropeptide. This approach should facilitate determination of more quantitative measures of semiochemical production in moths and other sugar-feeding insects that synthesize semiochemicals from glycolytic metabolites.

RNA interference of PBAN receptor suppresses expression of two fatty acid desaturases in female Plutella xylostella

Pheromone biosynthesis-activating neuropeptide (PBAN) stimulates sex pheromone biosynthesis by activating PBAN receptor (PBANr), which triggers a specific signal transduction in the pheromone gland cells. We have shown that RNA interference (RNAi) of PBANr of Plutella xylostella significantly suppressed pheromone biosynthesis and subsequent mating behavior. In order to assess molecular events occurring downstream of PBAN signaling, we cloned partial sequences of Δ9 and Δ11 fatty acid desaturases of P. xylostella. Phylogenetic analysis indicated that these two desaturase genes were highly clustered with other desaturases associated with sex pheromone biosynthesis in other insects. RT-PCR analysis showed that Δ9 desaturase was dominantly expressed in adult females, whereas Δ11 desaturase was expressed in all P. xylostella developmental stages. When PBANr expression was suppressed by PBANr-RNAi, the treated females also showed significant suppression of expression of both desaturases. These results suggest that expressions of the two desaturases are controlled by PBAN and that the two desaturases may be involved as downstream components in sex pheromone biosynthesis of P. xylostella.

Transcription factor fork head regulates the promoter of diapause hormone gene in the cotton bollworm, Helicoverpa armigera, and the modification of SUMOylation

The transcription factor fork head (FoxA) plays important roles in development and metabolism. Here, we cloned a fork head gene in Helicoverpa armigera, and found that the fork head protein is mainly located in the nucleus. This fork head gene belongs to the FoxA subfamily of the Fox transcription factors. The diapause hormone and pheromone biosynthesis-activating neuropeptide (DH-PBAN), which are two well-documented insect neuropeptides that regulate insect development and pheromone biosynthesis, are encoded by a single mRNA. In the present study, fork head was shown to bind strongly to the promoter of H. armigera DH-PBAN gene, and regulate its promoter activity. Furthermore, the effect of SUMOylation of the FH protein on the regulation of Har-DH-PBAN gene was investigated, and we show that the SUMO can modify Har-FH protein and cause down-regulation of DH-PBAN gene expression. These results suggest that SUMOylated FH plays a key role in insect diapause in H. armigera.

Identification and differential expression of a sex-peptide receptor in Helicoverpa armigera

Sex-pheromone production in the night flying female moth, Helicoverpa armigera is under neuroendocrine control due to the timely release of Pheromone Biosynthesis-Activating Neuropeptide (PBAN). Males orient to the females by upwind anemotaxis which usually leads to a successful mating. During copulation insect males transfer seminal peptides, produced in Male Accessory Glands (MAGs) which are implicated in post-mating behavioral changes of the females. These changes include the termination of pheromone biosynthesis and thus females do not re-mate. In previous studies we showed that synthetic Drosophila melanogaster Sex-Peptide ( DrmSP), which is responsible for terminating receptivity in female flies, can terminate PBAN-stimulated pheromone production by pheromone glands of the female moth, H. armigera. In addition, we demonstrated that at least one fraction of the H. armigera MAG extract is both immunoreactive to DrmSP antibody and is pheromonostatic, we also showed that different sets of DrmSP-like immunoreactive peptides are up-regulated in the central nervous system of mated females. In the present study, we identify a putative receptor for sex-peptide (SP-R) in H. armigera on the basis of sequence homologies deposited in the GenBank. In addition, in an attempt to draw some light on the physiological significance of SP-like peptides in this moth, we conducted a differential expression study of this receptor comparing gene expression levels in relation to different photoperiods, sex and mating status of the moth. Photoperiod and mating influence SP-R gene expression levels and sexual dimorphic changes were observed in neural tissues due to the different physiological states. After mating SP-R transcript levels in female neural tissues and pheromone glands are up-regulated. Physiological studies in vivo confirm the up-regulation of gene expression levels in pheromone glands isolated from mated females.

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.

The pyrokinin/ pheromone biosynthesis-activating neuropeptide (PBAN) family of peptides and their receptors in Insecta: evolutionary trace indicates potential receptor ligand-binding domains

The pyrokinin/pheromone biosynthesis-activating neuropeptide (PBAN) family of G-protein-coupled receptors and their ligands have been identified in various insects. Physiological functions of pyrokinin peptides include muscle contraction, whereas PBAN regulates, among other functions, pheromone production in moths which indicates the pleiotropic nature of these peptides. Based on the alignment of annotated genomic sequences, the pyrokinin/PBAN family of receptors have similarity with the corresponding structures of the capa or periviscerokinin receptors of insects and the neuromedin U receptors of vertebrates. In our study, evolutionary trace (ET) analysis on the insect receptor sequences was conducted to predict the putative ligand recognition and binding sites. The ET analysis of four class-specific receptors indicated several amino acid residues that are conserved in the transmembrane domains. The receptor extracellular domains exhibit several class-specific amino acid residues, which could indicate putative domains for activation of these receptors by ligand recognition and binding.