Pheromone Biosynthesis Activating Neuropeptide Research Articles

PBAN regulates sex pheromone biosynthesis by Ca2+/CaN/ACC and Ca2+/PKC/HK2 signal pathways in Spodoptera litura.

Sex pheromones emitted by female moths play important roles in mate attraction. The molecular mechanism underlying pheromone biosynthesis activating neuropeptide (PBAN)-regulated sex pheromone biosynthesis has been well elucidated in many moth species, although this mechanism is species-dependent. Spodoptera litura, an important pest, has caused serious economic losses to agricultural production, yet the mechanism for its sex pheromone biosynthesis has not been fully identified. The present study investigates in detail mechanism underlying PBAN-regulated sex pheromone biosynthesis in S. litura. The transcriptome sequencing of S. litura pheromone glands (PGs) was analysed to identify a serial of candidate genes potentially involved in sex pheromone biosynthesis. Further investigation revealed a bimodal pattern in both sex pheromone release and mating frequency. PBAN was found to regulate sex pheromone biosynthesis via its receptor by using Ca2+ as a secondary messenger, as demonstrated by RNA interference and the application of pharmacological inhibitors. Furthermore, PBAN/Ca2+ signalling activated calcineurin (CaN) and acetyl-CoA carboxylase (ACC), which mediated sex pheromone biosynthesis in response to PBAN stimulation. Mostly importantly, hexokinase 2 (HK2) was confirmed to be activated by PBAN/PBANR /Ca2+/PKC signalling via phosphorylation at two specific sites (ser423 and ser434 sites of HK2). Overall, our findings shed light on the intricate processes involved in sex pheromone production in S. litura, in which PBAN regulates sex pheromone biosynthesis through PBAN/PBANR/Ca2+/CaN/ACC and PBAN/PBANR/Ca2+/PKC/HK2 signalling pathways. These insights significantly contribute to our comprehension of the specific mechanisms underlying sex pheromone biosynthesis in this moth species.

Pheromone biosynthesis activating neuropeptide family in insects: a review.

Neuropeptides are involved in almost all physiological activities of insects. Their classification is based on physiological function and the primary amino acid sequence. The pyrokinin (PK)/pheromone biosynthesis activating neuropeptides (PBAN) are one of the largest neuropeptide families in insects, with a conserved C-terminal domain of FXPRLamide. The peptide family is divided into two groups, PK1/diapause hormone (DH) with a WFGPRLa C-terminal ending and PK2/PBAN with FXPRLamide C-terminal ending. Since the development of cutting-edge technology, an increasing number of peptides have been sequenced primarily through genomic, transcriptomics, and proteomics, and their functions discovered using gene editing tools. In this review, we discussed newly discovered functions, and analyzed the distribution of genes encoding these peptides throughout different insect orders. In addition, the location of the peptides that were confirmed by PCR or immunocytochemistry is also described. A phylogenetic tree was constructed according to the sequences of the receptors of most insect orders. This review offers an understanding of the significance of this conserved peptide family in insects.

Identification and characterisation of PRXamide peptides in the western flower thrips, Frankliniella occidentalis.

Insect CAPA-PVK (periviscerokinin) and pyrokinin (PK) neuropeptides belong to the PRX family peptides and are produced from capa and pyrokinin genes. We identified and characterised the two genes from the western flower thrips, Frankliniella occidentalis. The capa gene transcribes three splice variants, capa-a, -b, and -c, encoding two CAPA-PVKs (EVQGLFPFPRVamide; QGLIPFPRVamide) and two PKs (ASWMPSSSPRLamide; DSASFTPRLamide). The pyrokinin mRNA encodes three PKs: DLVTQVLQPGQTGMWFGPRLamide, SEGNLVNFTPRLamide, and ESGEQPEDLEGSMGGAATSRQLRTDSEPTWGFSPRLamide, the most extended pheromone biosynthesis activating neuropeptide (PBAN) ortholog in insects. Multiple potential endoproteolytic cleavage sites were presented in the prepropeptides from the pyrokinin gene, creating ambiguity to predict mature peptides. To solve this difficulty, we used three G protein-coupled receptors (GPCRs) for CAPA-PVK, tryptophan PK (trpPK), and PK peptides, and evaluated the binding affinities of the peptides. The binding activities revealed each subfamily of peptides exclusively bind to their corresponding receptors, and were significant for determining the CAPA-PVK and PK peptides. Our biological method using specific GPCRs would be a valuable tool for determining mature peptides, particularly with multiple and ambiguous cleavage sites in those prepropeptides. Both capa and pyrokinin mRNAs were strongly expressed in the head/thorax, but minimally expressed in the abdomen. The two genes also were clearly expressed during most of the life stages. Whole-mounting immunocytochemistry revealed that neurons contained PRXamide peptides throughout the whole-body: four to six neurosecretory cells in the head, and three and seven pairs of immunostained cells in the thorax and abdomen, respectively. Notably, the unusual PRXamide profiles of Thysanoptera are different from the other insect groups.

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.

Insect PRXamides: Evolutionary Divergence, Novelty, and Loss in a Conserved Neuropeptide System

The PRXamide neuropeptides have been described in both protostome and deuterostome species, including all major groups of the Panarthropoda. Best studied are the insect PRXamides consisting of three genes: pk/pban, capa, and eth, each encoding multiple short peptides that are cleaved post-translationally. Comparisons of genome and transcriptome sequences reveal that while retaining its fundamental ancestral organization, the products of the pk/pban gene have undergone significant change in the insect Order Diptera. Basal dipteran pk/pban genes are much like those of other holometabolous insects, while more crown species have lost two peptide coding sequences including the otherwise ubiquitous pheromone biosynthesis activating neuropeptide (PBAN). In the genomic model species Drosophila melanogaster, one of the remaining peptides (hugin) plays a potentially novel role in feeding and locomotor regulation tied to circadian rhythms. Comparison of peptide coding sequences of pk/pban across the Diptera pinpoints the acquisition or loss of the hugin and PBAN peptide sequences respectively, and provides clues to associated changes in life history, physiology, and/or behavior. Interestingly, the neural circuitry underlying pk/pban function is highly conserved across the insects regardless of the composition of the pk/pban gene. The rapid evolution and diversification of the Diptera provide many instances of adaptive novelties from genes to behavior that can be placed in the context of emerging selective pressures at key points in their phylogeny; further study of changing functional roles of pk/pban may then be facilitated by the high-resolution genetic tools available in Drosophila melanogaster.

Loop-mediated isothermal amplification of PBAN gene for molecular diagnosis of Bemisia tabaci biotype Q (Hemiptera: Aleyrodidae)

• LAMP assay for PBAN of the whitefly was developed. • The PBAN-based LAMP primer set showed specific amplification of the target region. • The PBAN-based LAMP assay can be applicable for monitoring B. tabaci biotype Q. The tobacco whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), is a serious pest that transmits tomato yellow leaf curl virus (TYLCV) in tomato. The tobacco whitefly exhibits morphological similarity with the greenhouse whitefly, Trialeurodes vaporariorum (Hemiptera: Aleyrodidae) which also damages various plants in the greenhouse. Therefore, it is very important to quickly and accurately diagnose whiteflies for applying pest management strategies. In this study, we used a loop-mediated isothermal amplification (LAMP) assay to quickly and effectively detect B. tabaci . Primer sets were investigated for the specificity of B. tabaci by visible detection of the target DNA fragment. Two primer sets for pheromone biosynthesis activating neuropeptide (PBAN) gene were obtained from transcriptomic analysis of B. tabaci biotype Q. The PBAN-based LAMP primer set showed specific amplification of the target region. The optimal conditions for B. tabaci detection were 60 °C for 60 min with four LAMP primers of PBAN. The minimum amount of genomic DNA required for visible detection was 100 ng. These results suggest that the PBAN-based LAMP assay can be applicable for field monitoring of B. tabaci .

Effect of Ionizing Radiation on Pheromone Biosynthesis Activating Neuropeptide (PBAN) Gene Expression and its Photosensitive Rhythm in Female <i>Spodoptera litura</i> (F.)

In this study, the effect of ionizing radiation was studied on the pheromone biosynthesis activating neuropeptide (PBAN) gene expression in female moths of a noctuid pest Spodoptera litura (F.), that would trigger pheromone production and its release needed for calling behaviour. The PBAN gene expression in the radiosterilized female moths (at 130 Gy) showed a decline with respect to control (unirradiated moths), and PBAN expression was drastically reduced at higher dose, 200 Gy. The photosensitivity and diel rhythm of PBAN gene expression, indicating higher expression during peak hours of scotophase and lowest expression during photophase was maintained at 130Gy, which deemed this gamma dose a suitable sterilizing dose for female moths which seemed to retain reproductive competence. This study might support the radiosterilized female moths for their combined release with substerilized males, using 130Gy to effectively operate the F1 sterility technique proposed to control S. litura

Mosaic Evolution of Molecular Pathways for Sex Pheromone Communication in a Butterfly.

Unraveling the origin of molecular pathways underlying the evolution of adaptive traits is essential for understanding how new lineages emerge, including the relative contribution of conserved ancestral traits and newly evolved derived traits. Here, we investigated the evolutionary divergence of sex pheromone communication from moths (mostly nocturnal) to butterflies (mostly diurnal) that occurred ~119 million years ago. In moths, it is the females that typically emit pheromones to attract male mates, but in butterflies males emit pheromones that are used by females for mate choice. The molecular bases of sex pheromone communication are well understood in moths, but they have remained relatively unexplored in butterflies. We used a combination of transcriptomics, real time qPCR, and phylogenetics to identify genes involved in the different steps (i.e., production, regulation, and reception) of sex pheromone communication of the butterfly Bicyclus anynana. Our results show that the biosynthesis and reception of sex pheromones relies both on moth-specific gene families (reductases) and on more ancestral insect gene families (desaturases, olfactory receptors, odorant binding proteins). Interestingly, B. anynana appears to use what was believed to be the moth-specific neuropeptide Pheromone Biosynthesis Activating Neuropeptide (PBAN) for regulating sex pheromone production. Altogether, our results suggest that a mosaic pattern best explains how sex pheromone communication evolved in butterflies, with some molecular components derived from moths, and others conserved from more ancient insect ancestors. This is the first large-scale investigation of the genetic pathways underlying sex pheromone communication in a butterfly.

Octopamine terminates sex pheromone biosynthesis by suppressing PBAN signal in moths.

The biosynthesis and termination of insect sex pheromones should be accurately regulated. In most moths, the biosynthesis and release of sex pheromones are regulated by a class of neuropeptides known as pheromone biosynthesis activating neuropeptides (PBANs). However, endogenous mechanisms underlying the termination of sex pheromone biosynthesis in moths remain elusive. In the present study, Helicoverpa armigera was employed as a model to investigate the role of octopamine (OA) in the inhibition of sex pheromone biosynthesis. Results demonstrated that the release of sex pheromones decreased with an increase in OA titres in older female moths. Moreover, OA treatment led to a significant decrease in sex pheromone production, female capability to attract male counterparts and subsequent female acceptance, indicating its inhibitory role in sex pheromone release. Subsequent qPCR and RNAi analyses revealed that OctβR was a key receptor of OA that regulated sex pheromone biosynthesis. In addition, the OA/OctβR signal suppressed intracellular Ca2+ levels and attenuated PBAN-mediated increase in the enzyme activities of calcineurin and acetyl-CoA carboxylase as demonstrated by OA treatment and OctβR-RNAi. Altogether, these results revealed a mechanism underlying the inhibition of sex pheromone production by OA via suppression of PBAN signalling in moths.

Suppression of PBAN receptor expression reduces fecundity in the fall armyworm, Spodoptera frugiperda.

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.

Trehalase is required for sex pheromone biosynthesis in Helicoverpa armigera.

Trehalase (Treh) hydrolyzes trehalose to generate glucose and it plays important role in many physiological processes. Acetyl-CoA, the precursor of sex pheromone biosynthesis in the pheromone gland (PG) of Helicoverpa armigera, originates from glucose during glycolysis. However, the function of Treh in sex pheromone biosynthesis remains elusive. In the present study, H. armigera was used as a model to investigate the function of two Trehs (Treh1 and Treh2) in sex pheromone biosynthesis. Results demonstrated that knockdown of HaTreh1 or HaTreh2 in female PGs led to significant decreases in Z11-16:Ald production, female ability to attract males, and successful mating proportions. Pheromone biosynthesis activating neuropeptide (PBAN) treatment triggered HaTreh1 and HaTreh2 activities in the isolated PGs and Sf9 cells. However, the activities of HaTreh1 and HaTreh2 triggered by PBAN were offset by H-89, the specific inhibitor of protein kinase A (PKA). Furthermore, the H-89 treatment significantly decreased the phosphorylation level of Trhe2, which was induced by PBAN. In addition, sugar feeding (5% sugar) increased the enzyme activities of Treh1 and Treh2. In summary, our findings confirmed that PBAN activates Treh1/2 activities by recruiting cAMP/PKA signalling, promotes glycolysis to ensure the supply of acetyl-CoA, and ultimately facilitates sex pheromone biosynthesis and mating behaviour.

Hexokinase Is Required for Sex Pheromone Biosynthesis in Helicoverpa armigera.

Simple SummarySex pheromone is an essential chemical signal for insect reproduction. Acetyl-CoA, which is generated from glycolysis, is the precursor of sex pheromone biosynthesis in Helicoverpa armigera. As the first speed-limited enzyme in glycolysis, the function of Hexokinase (HK) in sex pheromone biosynthesis remains elusive. This article employed H. armigera as a model. Results exhibited that the transcription profile of HaHK in female moth PGs was consistent with the release trend of sex pheromone. Knockdown of HaHK in female PGs caused significant decreases in acetyl-CoA content, sex pheromone production, and mating behaviors. Sugar feeding increased the transcription and enzymatic activity of HK. PBAN signal phospho-activated HaHK in PGs and Sf9 cells via PKA. In general, our study confirmed that PBAN/cAMP/PKA signal activated HaHK activity, and HaHK is required for sex pheromone biosynthesis in H. armigera.Acetyl-CoA, the precursor of sex pheromone biosynthesis in Helicoverpa armigera, is generated from glycolysis. As the first speed-limited enzyme in glycolysis, Hexokinase (HK) plays an important role in acetyl-CoA production. However, the function of HK in sex pheromone production remains unclear. This study employed H. armigera as material to explore the role of HK in sex pheromone production. Results demonstrated that the transcription profile of HaHK in female moth pheromone glands (PGs) was consistent with the release fluctuation of sex pheromone. Interference of HaHK prevented the increase of acetyl-CoA content induced by PBAN. Therefore, knockdown of HaHK in female PGs caused significant decreases in (Z)-11-hexadecenal (Z11-16:Ald) production, female capability to attract males, and mating rate. Furthermore, sugar feeding (5% sugar) increased the transcription and enzymatic activity of HK. Pheromone biosynthesis activating neuropeptide (PBAN) signal phospho-activated HaHK in PGs and Sf9 cells via protein kinase A (PKA), as shown by pharmacological inhibitor analysis. In general, our study confirmed that PBAN/cAMP/PKA signal activated HaHK, in turn promoted glycolysis to ensure the supply of acetyl-CoA, and finally facilitated sex pheromone biosynthesis and subsequent mating behavior.

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.

Pyruvate Kinase Is Required for Sex Pheromone Biosynthesis in Helicoverpa armigera.

Pyruvate kinase (PYK) is a speed-limited enzyme of glycolysis that catalyzes the formation of pyruvate, and plays an important role in acetyl-CoA synthesis. The acetyl-CoA is the precursor of sex pheromone biosynthesis in Helicoverpa armigera. However, the role of PYK in sex pheromone biosynthesis remains elusive. Here, PYK in H. armigera (HaPYK) was found to be highly expressed in the pheromone glands (PGs). The developmental expression profile of HaPYK was consistent with the fluctuation of sex pheromone release. Function analysis revealed that the knockdown of HaPYK led to a decrease in the levels of pyruvic acid and acetyl-CoA in PGs, which in turn caused a significant decrease in cis-11-hexadecenal (Z11-16: Ald) production, female capability to attract males, and mating frequency. Further study demonstrated that sugar feeding (5% sugar) increased the transcription and enzyme activity of HaPYK, thereby facilitating sex pheromone biosynthesis. Moreover, pheromone biosynthesis activating neuropeptide (PBAN) upregulated HaPYK activity through protein kinase C (PKC), as shown by PKC-specific inhibitor analysis. Altogether, our results revealed that PBAN activated HaPYK by Ca2+/PKC, thereby regulating the synthesis of pyruvate and subsequent acetyl-CoA, ensuring the supply of sex pheromone precursor, and finally facilitating sex pheromone biosynthesis and mating behavior.

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.

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.

Isolation and functional characterization of the pheromone biosynthesis activating neuropeptide receptor of Chinese oak silkworm, Antheraea pernyi

Insect pheromone biosynthesis activating neuropeptide (PBAN) controls the synthesis and actuating of sex pheromones of female adult. In the current examination, the full-length cDNA encoding the PBAN receptor was cloned from the pheromone gland (PG) of Antheraea pernyi (AntpePBANR). The AntpePBANR displayed the characteristic seven transmembrane areas of the G protein-coupled receptor (GPCR) and was closely related to the PBANR from Bombyx mori and Manduca sexta in the phylogenetic tree. The AntpePBANR expressed in mammalian cell lines were enacted by AntpePBAN in a concentration-dependent manner. AntpePBANR activation resulted in the calcium mobilization but did not activate the cAMP elevation pathway. Cells expressing AntpePBANR were profoundly responsive to Antpe-γ-SGNP (suboesophageal ganglion neuropeptides) and Antpe-DH (diapause hormone), different individuals from FXPRLamide (X = T, S or V) family in A. pernyi. Deletion of residues in the C-terminal hexapeptide (FSPRLamide) proved that P, R and L played the key parts in initiating the AntpePBANR, the amination to the last C terminal residues which can also likewise impact the activation of AntpePBAN receptor altogether. The mRNA of the AntpePBANR gene demonstrated the most noteworthy transcript levels in pheromone gland followed by fat body.

Calcineurin is required for male sex pheromone biosynthesis and female acceptance.

Lepidoptera sex pheromone biosynthesis is regulated by pheromone biosynthesis activating neuropeptide (PBAN). PBAN regulates not only female sex pheromone biosynthesis but also male sex pheromone biosynthesis. Previous research has confirmed that PBAN regulates sex pheromone biosynthesis using Ca2+ as a secondary messenger in all examined species to date. However, the downstream signal of Ca2+ has remained elusive. In the present study, calcineurin A (CNA), a downstream signal of Ca2+ , was discovered in Helicoverpa armigera male hairpencil and named HaCNA. Sequence analysis demonstrated that the open reading frame of HaCNA contains 1488 nucleotides encoding 495 amino acid residues. A homology search revealed that HaCNA shares a high amino acid identity with the CNA of other insects. Developmental and spatial expression analyses revealed that the mRNA levels of HaCNA peaked at 24 h after emergence and that HaCNA expression was ubiquitous in all examined tissues. Activity analysis revealed that PBAN activates HaCNA, and a Ca2+ inhibitor, Lacl3 , attenuated the effect of PBAN by decreasing HaCNA activity. Pharmacological inhibitor and RNA interference-mediated knockdown assays revealed that both activity inhibition and decreased mRNA levels of HaCNA led to a significant decrease in the production of the male sex pheromone components [octadecanol and (Z)-11 hexadecanol)] and in the efficacy of female mating acceptance. Our results demonstrate that HaCNA acts as downstream signal of PBAN/Ca2+ and plays an important role in PBAN-induced male sex pheromone biosynthesis and female mating acceptance.