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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. [ABSTRACT FROM AUTHOR]

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.

Pheromone biosynthesis activating neuropeptide (PBAN) produced in the suboesophageal ganglion stimulates pheromone biosynthesis in the pheromone gland, mediating sexual behaviors. The nucleotide sequences of PBAN and PBAN receptor (PBANr) in the legume pod borer, Maruca vitrata , were obtained from the transcriptome of the head and the pheromone gland, respectively. PBAN was expressed in all developmental stages. In contrast, PBANr was only detected in all developmental stages except for male adult. To examine the pheromonotropic activity of PBAN (Mvi-PBAN) in M. vitrata , Mvi-PBAN was chemically synthesized and injected into female adult. The pheromone production was increased in the dose-dependent manner and exhibited the maximal increase 2 h post-injection. [ABSTRACT FROM AUTHOR]

To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines constitutively expressing green fluorescent protein chimeras of the recently identified Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants. Fluorescent chimeras included the BommoPBANR-A, B, and C variants and the PsesePBANR-B and C variants. Cell lines expressing non-chimeric BommoPBANR-B and C variants were also generated. Functional evaluation of these transformed cell lines using confocal laser microscopy revealed that a Rhodamine Red-labeled PBAN derivative (RR-C10PBANR2K) specifically co-localized with all of the respective PBANR variants at the plasma membrane. Near complete internalization of the fluorescent RR-C10PBANR2K ligand 30 min after binding was observed in all cell lines except those expressing the BommoPBANR-A variant, in which the ligand/receptor complex remained at the plasma membrane. Fluorescent Ca2+ imaging further showed that, unlike the BommoPBANR-B or BommoPBANR-C cell lines, RR-C10PBANR2K binding failed to mobilize extracellular Ca2+ in the BommoPBANR-A cell line even at concentrations of 10 M. These observations demonstrate a clear functional difference between the BommoPBANR-A variant and the BommoPBANR-B and –C variants in terms of receptor regulation and activation of downstream effector molecules. We also found that, contrary to previous reports, ligand-induced internalization of BommoPBANR-B and BommoPBANR-C in cell lines stably expressing these variants occurred in the absence of extracellular Ca2+.

The fall armyworm, Spodoptera frugiperda, native to the tropical and subtropical areas of the American continent is one of the world's most destructive insect pests. In most insects, sex pheromone production is initiated following the activation of a pheromone-biosynthesis-activating neuropeptide (PBAN) receptor, which belongs to G protein-coupled receptor. We explored expression level of S. frugiperda PBAN receptor (Sf-PBANr) gene and validated the physiological function by assessing the fecundity of adult females subjected to its specific RNA interference (RNAi). Sf-PBANr was predicted from a transcriptome of S. frugiperda. Reverse-transcription polymerase chain reaction assay showed its expression in all developmental stages of S. frugiperda. Specific suppression of Sf-PBANr by RNAi in either sex significantly reduced the total number of laid eggs per adult female. Matings between both RNAi-treated males and female resulted in 63.3% reduction in fecundity. In contrast, the RNAi effect was less 47.5%-49.5% at the matings from single-parent RNAi treatment. These results suggest that the Sf-PBANr is associated with female of S. frugiperda., (© 2022 Wiley Periodicals LLC.)

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: This study describes generation of an anti-PBAN receptor (PBAN-R) antiserum and its employment for the characterization of the PK/PBAN-R(s). The antiserum recognized, in a specific and dose-dependent manner, the presence of PBAN-R in pheromone gland membrane preparations of three female moths: Heliothis peltigera, Helicoverpa armigera and Spodoptera littoralis. It also reacted specifically with the S. littoralis larval receptor in vivo, most likely by competing with the ligand on the binding site and consequently inhibiting cuticular melanization. Despite its ability to react with the receptor of H. peltigera in dot blot experiments, the antiserum did not react with the receptor in vivo and failed to inhibit sex pheromone biosynthesis. The antiserum was also used to develop two microplate binding assays. The Ab described in this study is the first raised against an insect neuropeptide (Np) receptor to be used in vivo, and its employment for characterization of the PK/PBAN-R(s) may thus provide important information on the mode of action of this Np family. The present study adds important information on the difference between the receptors in the two moth species, hints at the possible existence of receptor subtypes, and provides a platform for the development of a high-throughput assay (HTA) for screening of PK/PBAN agonists and antagonists. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Many species of female moths produce sex pheromones to attract conspecific males. To date, sex pheromones from more than 570 moth species have been chemically identified. Most moth species utilize Type I pheromones that consist of straight-chain compounds 10–18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester and usually with several double bonds. In contrast, some moth species use unsaturated hydrocarbons or hydrocarbon epoxides, classified as Type II lepidopteran pheromones, as sex pheromones. Studies over the past three decades have demonstrated that female moths usually produce sex pheromones as multi-component blends where the ratio of the individual components is precisely controlled, thus making it possible to generate species-specific pheromone blends. As for the biosynthesis of Type I pheromones, it is well established that they are de novo synthesized in the pheromone gland (PG) through modifications of fatty acid biosynthetic pathways. However, as many of the molecular components within the PG cells (i.e., enzymes, proteins, and small regulatory molecules) have not been functionally characterized, the molecular mechanisms underlying sex pheromone production in PG cells remain poorly understood. To address this, we have recently characterized some of the molecules involved in the biosynthesis of the sex pheromone bombykol in the silkmoth, Bombyx mori. Characterization of these, and other, key molecules will facilitate our understanding of the precise mechanisms underlying lepidopteran sex pheromone production. [Copyright &y& Elsevier]

Pheromone-biosynthesis-activating neuropeptide (PBAN) regulates sex pheromone production in many female moths. PBAN-like peptides, with common FXPRLamide C-terminals are found in other insect groups where they have other functions. The ubiquity and multifunctional nature of the pyrokinin/PBAN family of peptides suggests that the PBAN receptor proteins could also be present in a variety of insect tissues with alternative functions from that of sex pheromone biosynthesis. Previously we showed the presence of the PBAN-R in Helicoverpa armigera at the protein level. In the present study we confirm the similarities between the two Helicoverpa species: armigera and zea by (1) demonstrating the presence of the receptor protein in Sf9 cells, cloned to express the HezPBAN receptor, as compared with the endogenous receptor protein, previously shown in H. armigera pheromone glands, and (2) by identifying the nucleotide sequence of the PBAN-R from mRNA of H. armigera pheromone glands. Sequences of the two Helicoverpa spp. are 98% identical with most changes taking place in the 3′-end. We demonstrate the spatial distribution of the PBAN receptor protein in membranes of H. armigera brain (Br), thoracic ganglion (TG) and ventral nerve cord (VNC). We also demonstrate the presence and differential expression of the PBAN receptor gene (using reverse transcription–polymerase chain reaction and reverse transcription–quantitative real-time polymerase chain reaction, respectively) in the neural tissues (Br, TG and VNC) of adult H. armigera female moths as compared with its presence in pheromone glands. Surprisingly, the gene for the PBAN receptor is also detected in the male tissue homologous to the female pheromone gland, the aedeagus, although the protein is undetectable and PBAN does not induce physiological (pheromone production) or cellular (cyclic-adenosine monophosphate production) responses in this tissue. Our findings indicate that PBAN or PBAN-like receptors are present in the neural tissues and may represent a neurotransmitter-like function for PBAN-like peptides. In addition, the surprising discovery of the presence of the gene encoding the PBAN receptor in the male homologous tissue, but its absence at the protein level, launches opportunities for studying molecular regulation pathways and the evolution of these G protein coupled receptors (GPCRs). [ABSTRACT FROM AUTHOR]

Abstract: In noctuid moths cuticular pigmentation is regulated by the pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family, which also mediates a variety of other functions in moths and other insects. Numerous studies have shown that these neuropeptides exert their functions through activation of the PBAN receptor (PBAN-R), with subsequent Ca2+ influx, followed by either activation of cAMP or direct activation of downstream kinases. Recently, several PBAN-Rs have been identified, all of which are from the pheromone gland of adult female moths, but evidence shows that functional PK/PBAN-Rs can also be expressed in insect larvae, where they mediate melanization and possibly other functions (e.g., diapause). Here, we identified a gene encoding a G-protein-coupled receptor from the 5th instar larval tissue of the moth Spodoptera littoralis. The cDNA of this gene contains an open reading frame with a length of 1050 nucleotides, which translates to a 350-amino acid, 42-kDa protein that shares 92% amino acid identity with Helicoverpa zea and Helicoverpa armigera PBAN-R, 81% with Bombyx mori PBAN-R and 72% with Plutella xylostella PBAN-R. The S. littoralis PBAN-R gene was stably expressed in NIH3T3 cells and transiently in HEK293 cells. We show that it mediates the dose-dependent PBAN-induced intracellular Ca2+ response and activation of the MAP kinase via a PKC-dependent but Gαi-independent signaling mechanism. Other PK/PBAN family peptides (pheromonotropin and a C-terminally PBAN-derived peptide PBAN28–33NH2) also triggered MAP kinase activation. This receptor, together with the previously cloned PBAN-R, may facilitate our understanding of the cell-specific responses and functional diversities of this diverse neuropeptide family. [Copyright &y& Elsevier]

Abstract: Pheromone biosynthesis-activating neuropeptide (PBAN) is a peptide used by a variety of moths to regulate pheromone production. Pyrokinins are peptides that activate muscle contraction in a variety of insects. These peptides have a common FXPRLamide C-terminal ending that is required for activity. Receptors have been identified from a moth and Drosophila as belonging to the rhodopsin family of G-protein coupled receptors (GPCRs) with sequence similarity to neuromedin U receptors from vertebrates. No insect GPCR has been characterized with regard to role of extracellular domains required for peptide binding and receptor activation. To begin characterizing these GPCRs we created chimera receptors using a PBAN-receptor from a moth and pyrokinin-receptors from Drosophila where extracellular domains were swapped. The N-terminal of the moth GPCR has two N-glycosylation sites that when replaced with glutamines, activity was reduced but not absent, indicating these sites contribute to receptor stability. Activity was greatly reduced by replacing the 2nd extracellular loop that has an N-glycosylation site and a cysteine that can form a disulfide bridge with a cysteine at the beginning of the 3rd transmembrane domain. Exchange of the 3rd extracellular loop between the moth and Drosophila receptor resulted in differential activation by PBAN or a diapause hormone peptide. This result indicates that the 3rd extracellular loop is directly involved in peptide ligand recognition. Results are discussed in context of the structural features of insect GPCRs that are required for receptor activation as compared to vertebrate receptors. [Copyright &y& Elsevier]

Abstract: Internalization of the Bombyx mori pheromone biosynthesis activating neuropeptide receptor (PBANR) has been attributed to the presence of a 67 amino acid C-terminal extension absent in PBANRs from Helicoverpa. To identify the structural motif(s) responsible for internalization, a series of truncation mutants fused with enhanced green fluorescent protein were constructed and transiently expressed in insect Sf9 cells. Confocal microscopy analyses revealed that truncation at Gly357 severely inhibited internalization while truncation at Gln367 did not, indicating that the PBANR internalization motif resides between Gly357-Gln367. Alanine substitution studies suggest that Tyr360 and Leu363 may constitute a YXXL endosomal targeting motif that facilitates endocytosis, however, this motif does not appear to be the primary determinant; an indication that multiple sites are involved. Furthermore, we determined that internalization of the PBANR proceeds via a clathrin-dependent pathway, is dependent on the influx of extracellular calcium, and likely does not involve a G protein-coupled receptor kinase. [Copyright &y& Elsevier]

This review describes the development of a new integrated approach to the generation of a novel type of insect neuropeptide (Np) antagonists and putative insect control agents based on conformationally constrained compounds. The new approach, termed insect Np-based antagonist insecticide (INAI), was applied to the insect pyrokinin (PK)/pheromone biosynthesis-activating Np (PBAN) family as a model and led to the discovery of a potent linear lead antagonist and several highly potent, metabolically stable backbone cyclic (BBC) conformationally constrained antagonists that were devoid of agonistic activity and inhibited sex pheromone biosynthesis in female moths in vivo. This review summarizes the above approach, briefly describes the PK/PBAN Np family, presents data on the in vivo activity of the antagonists, summarizes data on the PK/PBAN receptor, and introduces the advantages of this method for generation of Np antagonists as a basis for the design of insect control agents. [ABSTRACT FROM AUTHOR]

The presence of the pyrokinin (PK)/ Pheromone biosynthesis activating neuropeptide (PBAN) receptor in pheromone gland cells of Heliothis peltigera females was demonstrated, and its spatial distribution in the ovipositor was visualized with two photo-affinity biotinilated ligands: BpaPBAN1-33NH2 and BpaArg27-PBAN28-33NH2. Light microscopy histological studies revealed that the gland is contained within the inter-segmental membrane (ISM) between the 8th and 9th abdominal segments. The gland was found to be composed of a single layer of columnar epithelial cells positioned under the inter-segmental cuticle. Similar epithelial cells were also found in the dorsal and ventral regions of the 9th abdominal segment. All regions containing the glandular cells bound both ligands, indicating presence of the PK/PBAN receptor. The patterns obtained with both ligands were similar, hinting at the possibility that either both ligands bind to the same receptor, or, that if there are two distinct receptors, their spatial distribution throughout the gland is very similar. [Copyright &y& Elsevier]

To facilitate further evaluation of pheromone biosynthesis activating neuropeptide receptor (PBANR) functionality and regulation, we generated cultured insect cell lines constitutively expressing green fluorescent protein chimeras of the recently identified Bombyx mori PBANR (BommoPBANR) and Pseudaletia separata PBANR (PsesePBANR) variants. Fluorescent chimeras included the BommoPBANR-A, -B, and -C variants and the PsesePBANR-B and -C variants. Cell lines expressing non-chimeric BommoPBANR-B and -C variants were also generated. Functional evaluation of these transformed cell lines using confocal laser microscopy revealed that a Rhodamine Red-labeled PBAN derivative (RR-C10PBAN(R2K)) specifically co-localized with all of the respective PBANR variants at the plasma membrane. Near complete internalization of the fluorescent RR-C10PBAN(R2K) ligand 30 min after binding was observed in all cell lines except those expressing the BommoPBANR-A variant, in which the ligand/receptor complex remained at the plasma membrane. Fluorescent Ca(2+) imaging further showed that the BommoPBANR-A cell line exhibited drastically different Ca(2+) mobilization kinetics at a number of RR-C10PBAN(R2K) concentrations including 10 μM. These observations demonstrate a clear functional difference between the BommoPBANR-A variant and the BommoPBANR-B and -C variants in terms of receptor regulation and activation of downstream effector molecules. We also found that, contrary to previous reports, ligand-induced internalization of BommoPBANR-B and BommoPBANR-C in cell lines stably expressing these variants occurred in the absence of extracellular Ca(2+).