cDNA CLONING AND SEQUENCE DETERMINATION OF THE PHEROMONE BIOSYNTHESIS ACTIVATING NEUROPEPTIDE FROM THE SEABUCKTHORN CARPENTERWORM,Holcocerus hippophaecolus (LEPIDOPTERA: COSSIDAE)

The PBAN (pheromone biosynthesis activating neuropeptide)/pyrokinin peptides comprise a major neuropeptide family characterized by a common FXPRL amide at the C‐terminus. These peptides are actively involved in many essential endocrine functions. For the first time, we reported the cDNA cloning and sequence determination of the PBAN from the seabuckthorn carpenterworm, Holcocerus hippophaecolus, by using rapid amplification of cDNA ends. The full‐length cDNA of Hh‐DH‐PBAN contained five peptides: diapause hormone (DH) homolog, α‐neuropeptide (NP), β‐NP, PBAN, and γ‐NP. All of the peptides were amidated at their C‐terminus and shared a conserved motif, FXPR (or K) L. Moreover, Hh‐DH‐PBAN had high homology to the other members of the PBAN peptide family: 56% with Manduca sexta, 66% with Bombyx mori, 77% with Helicoverpa zea, and 47% with Plutella xylostella. Phylogenetic analysis revealed that Hh‐DH‐PBAN was closely related to PBANs from Noctuidae, demonstrated by the relatively higher similarity compared with H. zea. In addition, real‐time quantitative PCR (qRT‐PCR) analysis showed that Hh‐DH‐PBAN mRNA expression peaked in the brain–subesophageal ganglion (Br–SOG) complex, and was also detected at high levels during larval and adult stages. The expression decreased significantly after pupation. These results provided information concerning molecular structure characteristics of Hh‐DH‐PBAN, whose expression profile suggested that the Hh‐DH‐PBAN gene might be correlated with larval development and sex pheromone biosynthesis in females of the H. hippophaecolus.     

A cDNA, from Agrotis ipsilon, that encodes the pheromone biosynthesis activating neuropeptide (PBAN) and other FXPRL peptides.

A cDNA encoding the prohormone of the pheromone biosynthesis activating neuropeptide (PBAN) in the moth Agrotis ipsilon was isolated. The cDNA contains 834 nucleotides, coding for a 193-amino acid protein that exhibits 89% identity with PBAN prohormones of other moths. The prohormone contains five potential peptides belonging to the FXPRL family. The peptide corresponding to the Bombyx mori diapause hormone exhibits an extra residue, and the C-terminal leucine is replaced by an isoleucine, introducing a new type of variability in this family of peptides. Northern blot analysis revealed expression in suboesophagal ganglion complexes. Constitutive heterologous expression of Agi-PBAN cDNA in yeast, using three different antibodies, did not produce PBAN-immunoreactive material.     

Cloning and expression of the cDNA encoding the FXPRL family of peptides and a functional analysis of their effect on breaking pupal diapause in Helicoverpa armigera

Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are encoded by a single mRNA in the suboesophegeal ganglion (SG) and are responsible for induction of embryonic diapause in Bombyx mori and sex pheromone biosynthesis in lepidopteran insects. PBAN cDNA analyses revealed that the DH-like peptide is present in several species that have a pupal diapause. However, the function of the DH-like peptide remains unknown. In the present study, we cloned the cDNA encoding DH-PBAN in Helicoverpa armigera utilizing the rapid amplification of the cDNA ends method. The nucleotide se quence analysis revealed that the longest open reading frame of this cDNA encodes a 194-amino acid precursor protein that con tains a 33-aa PBAN, a 24-aa DH-like peptide, and three other neuropeptides, all of which have a common C-terminal pentapeptide motif FXPR/KL ( X=G, T, S). A homology search showed that H. armigera DH-like and PBAN are highly homologous to those from other insects. Northern blot analysis demonstrated a single message RNA corresponding to the size of Har-DH-PBAN cDNA from pupal SG with significantly higher expression in the SG of nondiapause pupae than diapausing pupae. Western blot analysis showed DH-like peptide expression from SG of both males and females. When DH-like peptide was injected into nondiapause larvae and pupae, it did not induce diapause, but rather efficiently broke pupal diapause in H. armigera. The ED(50) of DH to terminate pupal diapause is 20 pmol/pupae. The other four FXPRLamide neuropeptides from the DH-PBAN polyprotein precursor have cross activity for diapause termination. These observations therefore suggest a potential role for these FXPRL family peptides in promoting continuous development in several noctuid species. The high expression of this gene in pharate adults and adults indicates that the FXPRL family peptides may have multiple physiological functions.     

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．     

cDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of the silkworm, Bombyx mori.

We have identified the cDNAs encoding pheromone biosynthesis activating neuropeptide (PBAN) using PCR technique. The nucleotide sequence showed that the PBAN gene encodes, besides PBAN, diapause hormone and three putative amidated peptides. These four peptides share with PBAN the C-terminal pentapeptide amide which is corresponding to the shortest fragment with pheromonotropic activity. The organization of the PBAN gene is characteristic of several short neuropeptides and has some degree of similarity to that of the gene for the insect neuropeptide FMRFamide. Thus, the PBAN gene products construct a family of structurally related peptides and have various biological functions.     

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...     

Immunoreactive intensity of FXPRL amide neuropeptides in response to environmental conditions in the silkworm, Bombyx mori

In the silkworm Bombyx mori, the diapause hormone-pheromone biosynthesis activating neuropeptide gene, DH-PBAN, is a neuropeptide gene that encodes a polypeptide precursor consisting in five Phe-X-Pro-Arg-Leu-NH₂ (FXPRL) amide (FXPRLa) neuropeptides; DH (diapause hormone), PBAN (pheromone-biosynthesis-activating neuropeptide) and α-, β- and γ-SGNPs (subesophageal ganglion neuropeptides). These neuropeptides are synthesized in DH-PBAN-producing neurosecretory cells contained within three neuromeres, four mandibular cells, six maxillary cells, two labial cells (SLb) and four lateral cells of the subesophageal ganglion. DH is solely responsible, among the FXPRLa peptide family, for embryonic diapause. Functional differentiation has been previously suggested to occur at each neuromere, with the SLb cells releasing DH through brain innervation in order to induce embryonic diapause. We have investigated the immunoreactive intensity of DH in the SLb when thermal (25°C or 15°C) and light (continuous illumination or darkness) conditions are altered and following brain surgery that induces diapause or non-diapause eggs in the progeny. We have also examined the immunoreactivity of the other FXPRLa peptides by using anti-β-SGNP and anti-PBAN antibodies. Pupal SLb somata immunoreactivities seem to be affected by both thermal and light conditions during embryogenesis. Thus, we have been able to identify a close correlation between the immunoreactive intensity of neuropeptides and environmental conditions relating to the determination of embryonic diapause in B. mori.     

Structure and activity of Bombyx PBAN

Two structurally related molecular species of pheromone biosynthesis activating neuropeptides (PBANs), PBAN-I and -II, were isolated from adult heads of the silkworm, Bombyx mori, and characterized. PBAN-I is a carboxyl-terminally amidated 33-residue peptide. Structure-activity relationship studies revealed that 1) its carboxyl-terminal pentapeptide is the smallest size showing activity, 2) the carboxyl-terminal amide is indispensable for activity, and 3) oxidation of three Met residues in PBAN-I to Met(O) (methionine sulfoxide) caused marked enhancement of activity, and the three Met(O) residues contribute equally to the enhancement of activity. Molecular design of PBAN analogs using a carboxyl-terminal hexapeptide showed that modification of the amino-terminal amino group brought about a dramatic increase in activity. This increase was presumed to be mainly due to the increased stability in hemolymph. PBANs share the common carboxyl-terminal sequence, -Phe-Xaa-Pro-Arg-Leu-NH₂, with myotropic peptides isolated from locust and cockroach. Examination of cross-activity of these two groups of peptides revealed that PBAN and its analogs exhibited myotropic activity comparable to myotropic peptides, while myotropic peptides showed extremely high pheromonotropic activity. In B. mori, PBAN activates sex pheromone (bombykol) production presumably by promoting the reduction reaction from acyl to alcohol, which is the last step in the biosynthesis of bombykol. © 1994 Wiley-Liss, Inc.     

Discovery of a linear lead antagonist to the insect pheromone biosynthesis activating neuropeptide (PBAN)

We report the discovery of a linear lead antagonist for the insect pheromone biosynthesis activating neuropeptide (PBAN) which inhibits sex pheromone biosynthesis in the female moth Heliothis peltigera. Two approaches have been used in attempting to convert PBAN agonists into antagonists. The first involved omission of the C-terminal amide and reduction of the sequence from the N-terminus in a linear library based on PBAN 1-33NH(2.) The second involved replacement of L amino-acids by the D hydrophobic amino acid D-Phe in a linear library based on PBAN28-33NH(2.) Screening of the two libraries for pheromonotropic antagonists resulted in the disclosure of one compound out of the D-Phe library (Arg-Tyr-Phe-D-Phe-Pro-Arg-Leu-NH(2)) which inhibited sex pheromone production by 79 and 64% at 100 pmol in two moth colonies and exhibited low agonistic activity. Omission of the C-terminal amide in PBAN 1-33NH(2) and its shorter analogs did not lead to the discovery of an antagonistic compound.     

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.     

PBAN/pyrokinin神经肽类及其基因的研究进展

昆虫在其生长发育过程中,如胚胎发育、蜕皮变态、滞育、迁飞、代谢、生殖等都离不开神经肽的调控。信息素合成激活肽(pheromone biosynthesis activating neuropeptide,PBAN)和Pyrokinin神经肽是C端具有五肽FXPRL(X=S,V,T,G等)(苯丙-X-脯-精-亮氨酸)序列的一类神经肽,在昆虫的生长发育中起重要的生理功能,如性信息素的合成、控制表皮色素、促进胚胎滞育和刺激内脏肌肉收缩等重要的生理功能。因此近几年对PBAN/pyrokinin神经肽的鉴定、加工、作用和降解方式的研究成为研究的热点,为研制高效、低毒、专一性强、无公害的杀虫剂提供了思路。介绍了PBAN/pyrokinin神经肽类及其基因的研究进展,并对PBAN/pyrokinin神经肽在害虫防治中的应用进行了展望。     

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.     

Identification and Determination of the Partial cDNA Encoding the Pheromone Biosynthesis Activating Neuropeptide in Helicoverpa armigera

Sex pheromone biosynthesis is induced in many moths by a neuropeptide, PBAN, consisting of 33-amino acid amidated at the C-terminus. The present study is concerned with cloning and characterizing the partial sequence of Hea-PBAN cDNA which is isolated from the brain and suboesophageal ganglion complex (Br-Sg) of Helicoverpa armigera adults. From the cDNA sequence, it can be predicted that the cDNA has a PBAN domain with 33 amino acids, LSDDMPATPADQEMYRQDPEQIDSRTKYFSPRL, with FSPRL amidated at the C-terminus. The amino acid sequence of predicted peptides including the PBAN, is identical to that of H. zea and H. assulta.