Hormone signaling linked to silkmoth sex pheromone biosynthesis involves Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of the insect PAT family protein Bombyx mori lipid storage droplet protein-1 (BmLsd1)

Species-specific sex pheromones released by female moths to attract conspecific male moths are synthesized de novo in the pheromone gland (PG) via the fatty acid biosynthetic pathway. This pathway is regulated by a neurohormone termed pheromone biosynthesis activating neuropeptide (PBAN), a 33-amino acid peptide that originates in the subesophageal ganglion. In the silkmoth, Bombyx mori, cytoplasmic lipid droplets, which store the sex pheromone (bombykol) precursor fatty acid, accumulate in PG cells. PBAN stimulates lipolysis of the stored lipid droplet triacylglycerols (TAGs) and releases the precursor for final modification. PBAN exerts its physiological function via the PG cell-surface PBAN receptor, a G protein-coupled receptor that belongs to the neuromedin U receptor family. The PBAN receptor-mediated signal is transmitted via a canonical store-operated channel activation pathway utilizing Gq-mediated phospholipase C activation (Hull, J. J., Kajigaya, R., Imai, K., and Matsumoto, S. (2007) Biosci. Biotechnol. Biochem. 71, 1993-2001; Hull, J. J., Lee, J. M., Kajigaya, R., and Matsumoto, S. (2009) J. Biol. Chem. 284, 31200-31213; Hull, J. J., Lee, J. M., and Matsumoto, S. (2010) Insect Mol. Biol. 19, 553-566). Little, however, is known about the molecular components regulating TAG lipolysis in PG cells. In the current study we found that PBAN signaling involves phosphorylation of an insect PAT family protein named B. mori lipid storage droplet protein-1 (BmLsd1) and that BmLsd1 plays an essential role in the TAG lipolysis associated with bombykol production. Unlike mammalian PAT family perilipins, however, BmLsd1 activation is dependent on phosphorylation by B. mori Ca(2+)/calmodulin-dependent protein kinase II rather than protein kinase A.     

Sex pheromone production in the silkworm, Bombyx mori, is mediated by store-operated Ca2+ channels

In most female moths, pheromone biosynthesis activating neuropeptide (PBAN) regulates sex pheromone production by stimulating an influx of extracellular Ca(2+). Little is known about the plasma membrane channel or how the PBAN stimulus is communicated to the channel. Fluorescent Ca(2+) imaging techniques confirmed PBAN-induced Ca(2+) influx in the silkworm, Bombyx mori, and showed that the PBAN response is reduced with repeated stimulation. Compounds known to impact Ca(2+) signaling were examined for their effects on sex pheromone production. These experiments demonstrated that the PBAN signal is likely mediated by a store-operated channel (SOC). SOC blockers, SKF-96365 and 2-aminoethoxydiphenyl borate, abolished sex pheromone production, as did flufenamic acid, a blocker of transient receptor potential (TRP) channels. Thapsigargin mimicked the pheromonotropic effects of PBAN. Similar results were seen when PBAN-induced lipase activity was assayed. Conversely, 1-oleoyl-2-acetyl-sn-glycerol and arachidonic acid, activators of diacylglycerol-dependent Ca(2+) channels, had no effect on bombykol production.     

Involvement of calcineurin in the signal transduction of PBAN in the silkworm, Bombyx mori (Lepidoptera)

In several moth species sex pheromone production in the pheromone gland is regulated by a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori it is suggested that PBAN, after binding to the cell-surface receptor, primarily activates a plasma membrane receptor-activated Ca2+ channel to increase cytosolic levels of Ca2+, and Ca2+/calmodulin complex directly or indirectly activates a phosphoprotein phosphatase, which in turn elicits activation of acyl CoA reductase (the key enzyme under PBAN control) through dephosphorylation, resulting in pheromone (bombykol) production. The effect of cyclosporin A (CsA) and FK 506, specific inhibitors of calcineurin (phosphoprotein phosphatase 2B) was studied on the sex pheromone production, in B. mori. The in vitro experiments showed that both chemicals exerted a dose-dependent inhibitory action when they were co-incubated with TKYFSPRL amide (Hez-PBAN fragment peptide). Practically, no difference was detected between the two chemicals in the tested doses (0.025-1250 microM). When effects of CsA or FK 506 were studied on cell-free production of bombykol by using microsomal fraction no inhibition was detected. Since microsomal fraction contains the acyl CoA synthetase, the rate-limiting acyl CoA reductase and the precursor, bombykol is produced if supplied with CoA, ATP and NADPH. Thus, the inhibitory action of CsA and FK506 under in vitro conditions should occur before the step of acyl group reduction and the effect is likely to be attributable to the inhibition of calcineurin in the signal transduction cascade mechanism of PBAN, in B. mori. The existence of calcineurin in the pheromone gland by using Western blot analysis is also demonstrated.     

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.     

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.     

PBAN stimulation of pheromone biosynthesis by inducing calcium influx in pheromone glands of Helicoverpa zea

Isolated pheromone glands of Helicoverpa zea were utilized to investigate the physiological action of pheromone biosynthesis activating neuropeptide (PBAN) with regard to the role of calcium ions in stimulating pheromone biosynthesis under various incubation conditions. Incubation of glands with 1 microM or 1 nM PBAN produced a significant amount of pheromone after a 5 min incubation period and reached maximum pheromone production after 30 min. Glands incubated with PBAN for 1 min, and then without PBAN for 30 min, produced pheromone whether or not extracellular calcium was present during the first 1 min. The presence of lanthanum as a calcium channel blocker did not affect pheromone production if present during the first 1 min of incubation with PBAN. However, if calcium was absent or lanthanum ion was present during the 30 min of incubation, no pheromone was produced. A maximum amount of pheromone was reached when glands were incubated for 1 min with PBAN and for 10 min without PBAN, and repeated three times. The present results indicate that a time interval exists between PBAN binding to a receptor and opening of extracellular calcium channels. Calcium influx into the cytosol from extracellular stores is required for PBAN to stimulate pheromone production. This could be achieved by PBAN either binding periodically to the receptor or the plasma membrane calcium channel could remain activated for a period of time after the initial activation.     

Molecular cloning of pheromone biosynthesis activating neuropeptide in silkworm, Bombyx mori

Pheromone biosynthesis activating neuropeptide (PBAN) is a suboesophageal ganglion secretory polypeptide of insect, which activates the pheromone gland to produce sex pheromone biosynthesis in female silkworm, Bombyx mori. A Bombyx genomic library was screened by the method of plaque hybridization using the 32P-labeled BomDH cDNA as a probe. The genomic sequence encoding PBAN has been cloned and its structure is analyzed. The PBAN gene comprises two exons interspersed by a single intron 697 bp in length. Preceding the PBAN amino acid sequence is a 32-amino acid sequence containing two FXPRL amide peptides, which are α-SGNP (Ile-Ile-Phe-Thr-Pro-Lys-Leu) and β-SGNP (Ser-Val-Ala-Asn-Pro-Arg-Thr-His-Glu-Ser-Leu-Glu-Phe-Ile-Pro-Arg-Leu), which is followed by a Gly-Arg processing site. Immediately, after the PBAN amino acid sequence is a Gly-Arg processing site and a FXPRL amide peptide γ-SGNP (Thr-Met-Ser-Phe-Ser-Pro-Arg-Leu). It is suggested that besides PBAN, 7-, 8-, and 17-residue amidated peptides wer     

昆虫神经肽PBAN的细胞内信号转导

昆虫性信息素多数为长链的不饱和醇、醋酸酯、醛或酮类,链长一般为10-20碳,主要在性信息素腺体内由乙酰辅酶A经过脂肪酸合成、碳链缩短、去饱和以及碳酰基的还原修饰等步骤合成的;而性信息素合成激活肽(pheromone biosynthesis activating neuropeptide,PBAN)是由昆虫食管下神经节中的部分神经细胞合成和分泌的神经肽,通常由33个氨基酸组成,在C-末端有一个相同的五肽序列,主要调控性信息素的生物合成。有关PBAN的细胞内信号转导是近几年的研究热点,研究显示 PBAN首先与性信息素腺体细胞表面的G蛋白偶联受体结合,随后依据昆虫种类的不同,其细胞内信号转导方式主要有三种:(1)以cAMP信号传导途径进行信号转导;(2)以cAMP和磷脂酰肌醇信号传导途径共同进行信号转导;(3)主要以Ca2 为第二信使进行信号传导。     

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

Background

The 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.

Methods

A 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.

Results

Homology 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.

Conclusions

The 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 significance

The 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.     

家蚕滞育激素-性信息素合成激活肽基因表达的调节

滞育激素和性信息素合成激活肽是两个重要的昆虫神经肽,这两个神经肽由一个基因编码．利用分子杂交和ＲＴ－ＰＣＲ技术,确定了滞育激素－性信息素合成激活肽基因表达的调节不属于转录后的调节,推定为翻译后形成一个大的前体多肽再剪接为几个成熟的神经肽分子．     

Identification of lipases involved in PBAN stimulated pheromone production in Bombyx mori using the DGE and RNAi approaches

Background

Pheromone biosynthesis activating neuropeptide (PBAN) is a neurohormone that regulates sex pheromone synthesis in female moths. Bombyx mori is a model organism that has been used to explore the signal transduction pattern of PBAN, which is mediated by a G-protein coupled receptor (GPCR). Although significant progress has been made in elucidating PBAN-regulated lipolysis that releases the precursor of the sex pheromone, little is known about the molecular components involved in this step. To better elucidate the molecular mechanisms of PBAN-stimulated lipolysis of cytoplasmic lipid droplets (LDs), the associated lipase genes involved in PBAN- regulated sex pheromone biosynthesis were identified using digital gene expression (DGE) and subsequent RNA interference (RNAi).

Results

Three DGE libraries were constructed from pheromone glands (PGs) at different developed stages, namely, 72 hours before eclosion (−72 h), new emergence (0 h) and 72 h after eclosion (72 h), to investigate the gene expression profiles during PG development. The DGE evaluated over 5.6 million clean tags in each PG sample and revealed numerous genes that were differentially expressed at these stages. Most importantly, seven lipases were found to be richly expressed during the key stage of sex pheromone synthesis and release (new emergence). RNAi-mediated knockdown confirmed for the first time that four of these seven lipases play important roles in sex pheromone synthesis.

Conclusion

This study has identified four lipases directly involved in PBAN-stimulated sex pheromone biosynthesis, which improve our understanding of the lipases involved in releasing bombykol precursors from triacylglycerols (TAGs) within the cytoplasmic LDs.     

Neuroendocrine control of pheromone production in moths

In many moth species regulation of pheromone production has been attributed to the timely release of a pheromone biosynthesis activating neuropeptide (PBAN). The gene encoding PBAN has been sequenced in two moth species. Immunochemical studies as well asin situ hybridization and Northern analysis of PBAN encoding mRNA have localized the neuroendocrine cells responsible for the production of PBAN and have traced the neuronal network of PBAN immunoreactivity. Release into the bloodstream has been demonstrated, the target tissue delineated, and the signal transduction pathway and its modulation analyzed. This paper reviews the current status of research concerning the neuroendocrine control of pheromone production in Lepidopterans and presents some recent developments concerning the receptors involved in the pheromonotropic activity. In this study, we report on the use of a biologically active photoaffinity-biotin-labeled derivative of PBAN N-[N-(4-azido-tetrafluorobenzoyl-biocytinyloxyl-succinimide) and show the presence of a protein (estimated molecular weight of 50 kDa) which specifically binds to PBAN in membrane preparations of pheromone glands. Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No.2279-E, 1997 series     

Mechanisms involved in the control of pheromone production in female moths: recent developments

Species‐specific pheromone blends of nocturnal female moths, derived from fatty acid precursors, are produced and released for mate‐finding, and are initiated by the circadian, trophic hormone, Pheromone Biosynthesis Activating Neuropeptide (PBAN). PBAN, produced in the sub‐oesophageal ganglion, is a 33 amino acid neuropeptide with a minimum active core in its FXPRLamide C‐terminal. PBAN acts directly on pheromone gland cells of mature females by binding to a specific G‐protein‐coupled membrane receptor (GPCR), and thereby initiating a signal transduction cascade involving calcium and cAMP. This discussion will review recent developments concerning the identification of the PBAN GPCR, its regulation by juvenile hormone (JH), and its mode of action at the level of the pheromone biosynthetic pathway. The discussion will also include recent developments concerning events occurring as a result of the transfer of pheromonostatic compounds of male origin after mating.     

The identification of an age- and female-specific putative PBAN membrane-receptor protein in pheromone glands of Helicoverpa armigera: possible up-regulation by Juvenile Hormone

The present study was designed to determine the age and female specificity of a membrane protein that binds to a pheromone biosynthesis activating neuropeptide (PBAN) ligand and to elucidate the effect of Juvenile Hormone (JH) on binding as well as pheromone activation. The precise age at which developing adult females of Helicoverpa armigera begin to respond to PBAN was determined. PBAN activates in vitro pheromone biosynthesis as well as its intracellular second messenger, cAMP, only in intersegments of newly emerged adult female pheromone glands (i.e. 1-day-old females). An increase in response was observed in 2-day-old females. Intersegments of female pupae and the homologous tissues of adult males do not respond to PBAN. However, in the presence of Juvenile Hormone II (JH II) PBAN induced a response in females, 1 day before emergence (pharate females), but not in younger female pupae. This phenomenon was also observed after topical applications of the JH analog fenoxycarb (FX). In addition the response to PBAN by intersegments of FX-treated emerged adults increased significantly to the level of 2-day-old females. JH II also stimulated the level of incorporation of (35)S-labelled amino acids in female pupae into membrane proteins that are typical in adult intersegments. Using a photoaffinity-biotin labelled PBAN analog we demonstrate specific binding of a membrane protein (estimated MW: 50 kD) in adult females. This binding was not detected in female pupae 3 days before emergence. However, in such female pupae specific binding of the 50 kD protein by the photoaffinity-biotin labelled PBAN analog was induced after JH II or FX treatments thereby providing evidence that JH may up-regulate this putative receptor protein.     

Ant Trail Pheromone Biosynthesis Is Triggered by a Neuropeptide Hormone

Our understanding of insect chemical communication including pheromone identification, synthesis, and their role in behavior has advanced tremendously over the last half-century. However, endocrine regulation of pheromone biosynthesis has progressed slowly due to the complexity of direct and/or indirect hormonal activation of the biosynthetic cascades resulting in insect pheromones. Over 20 years ago, a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN) was identified that stimulated sex pheromone biosynthesis in a lepidopteran moth. Since then, the physiological role, target site, and signal transduction of PBAN has become well understood for sex pheromone biosynthesis in moths. Despite that PBAN-like peptides (∼200) have been identified from various insect Orders, their role in pheromone regulation had not expanded to the other insect groups except for Lepidoptera. Here, we report that trail pheromone biosynthesis in the Dufour''s gland (DG) of the fire ant, Solenopsis invicta, is regulated by PBAN. RNAi knock down of PBAN gene (in subesophageal ganglia) or PBAN receptor gene (in DG) expression inhibited trail pheromone biosynthesis. Reduced trail pheromone was documented analytically and through a behavioral bioassay. Extension of PBAN''s role in pheromone biosynthesis to a new target insect, mode of action, and behavioral function will renew research efforts on the involvement of PBAN in pheromone biosynthesis in Insecta.     

家蚕滞育激素－性信息素合成激活肽基因的表达

家蚕滞育激素－性信息素合成激活肽基因的表达徐卫华（中国农业科学院蚕业研究所,江苏镇江,２１２０００）山下兴亚（名古屋大学农学院,日本名古屋,４６４－０１）关键词滞育激素－性信息素合成激活肽基因;发育阶段;表达;家蚕昆虫是地球上最繁盛的物种,占地球上生...