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Abstract: The diazepam binding inhibitor (DBI) or the acyl-CoA-binding protein (ACBP) is a 9–10 kDa highly conserved multifunctional protein that plays important roles in GABAA receptor activity regulation, lipid absorption and steroidogenesis in various organisms. To study the functions of DBI/ACBP in insect development or diapause, we cloned the cDNA from Helicoverpa armigera (Har) utilizing rapid amplification of cDNA ends (RACE). By homology search, Har-DBI/ACBP is conserved with the DBI/ACBPs known from other insects. Northern blot analysis showed that DBI/ACBP gene expressed in nonneural and neural tissues. RT-PCR combined Southern blot analysis revealed that DBI/ACBP mRNA in the brain of nondiapause individual was much higher than that in the brain of diapausing insects. At early and middle stages of 6th instar larvae, the level of DBI/ACBP mRNA was higher in the midgut of diapause type than that in nondiapause type and low at late 6th instar larval stage and early pupal stage in both types. In the prothoracic gland (PG), DBI/ACBP expression appeared at a high level at middle and late stages of 6th larval instar in both nondiapause and diapause types, and declined after pupation. In vitro experiments revealed that DBI/ACBP mRNA in PG could be stimulated by synthetic H. armigera diapause hormone (Har-DH), suggesting that Har-DH may stimulate the PG to produce ecdysteroids by the DBI/ACBP signal pathway. By in vitro assay, we also found that FGIN-1-27, which has similar functions to DBI/ACBP in ecdysteroidogenesis, could induce PG ecdysteroidogenesis effectively, suggesting that DBI/ACBP regulates biosynthesis of ecdysteroids in PG. Thus, DBI/ACBP indeed plays a key role in metabolism and development in H. armigera. [Copyright &y& Elsevier]

Abstract: We have cloned the gene encoding the diapause hormone and the pheromone biosynthesis activating neuropeptide in Helicoverpa armigera (Har-DH-PBAN). The Har-DH-PBAN gene contains six exons and five introns that fall in the same positions as in the Bombyx mori DH-PBAN gene (Bom-DH-PBAN). The transcription initiation site lays 29 bp upstream of the translation initiation site. Southern blot analysis suggests that a single copy of this gene is present per haploid genome. A structural comparison of DH-PBAN promoters between H. armigera and B. mori show similarities in the TATA box and in a potential binding site for a POU family transcription factor, POU-M2. However, testing of these DNA regions for factor binding in vitro and transcription assays in cell culture highlight significant differences in their regulation particularly in reference to the POU-M2 sites. Our results uncover common and different regulatory mechanisms at work in the control of DH-PBAN gene expression in H. armigera and B. mori. [Copyright &y& Elsevier]

FXPRLamide peptides encoded by the DH-PBAN (diapause hormone-pheromone biosynthesis activating neuropeptide) gene induce embryonic diapause in Bombyx mori, but terminate pupal diapause in Helicoverpa armigera (Har). Here, we explore the mechanisms of terminating pupal diapause by the FXPRLamide peptides. Using quantitative RT-PCR, we observed that expression of Har-DH-PBAN mRNA in the SG of nondiapause-type pupae was significantly higher than in diapause-type pupae. Immunocytochemical results indicated that the level of FXPRLamide peptides and axonal release are related to the diapause decision. Ecdysteroidogenesis in prothoracic glands (PGs) was stimulated by synthetic Har-DH in vivo and in vitro, and labeled Har-DH bound to the membrane of the PG, thus suggesting that DH breaks diapause by activating the PG to synthesize ecdysone. Furthermore, the response of DH in terminating diapause was temperature dependent. Decerebration experiments showed that the brain can control pupal development through the regulation of DH, and DH can terminate diapause and promote development without the brain. This result suggests a possible mechanism of response for the signals of DH and other FXPRLamide peptides in H. armigera. [Copyright &y& Elsevier]

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 ED50 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. [Copyright &y& Elsevier]

Recent studies in Helicoverpa armigera report a novel role for diapause hormone (DH), pheromone biosynthesis activating neuropeptide (PBAN) and three other FXPRLamide neuropeptides secreted from suboesophageal ganglion (SG) in terminating pupal diapause. In the present paper, we investigated the role of these five FXPRLamide family neuropeptides on pupal development. Although removal of SG could not make nondiapause-destined pupae enter diapause-like status, it did make them eclose approximately 0.6–1.2 days later when compared with the controls. The results of competitive ELISAs showed a high level of FXPRLamide titer in the hemolymph of the SG-removed pupae and this may be due to the expression of the DH-PBAN gene in tissues other than SG. DH-PBAN mRNA and peptides were also detected in the thoracic ganglia (TGs) by RT-PCR and immunocytochemistry. The expression of DH-PBAN gene in the TGs of the SG-removed pupae is significantly higher than that in normal pupae by quantitative PCR and immunocytochemistry. Decerebration experiments proved that the decerebrated pupae could enter diapause-like status through down-regulation of FXPRLamide titer in hemolymph. Our studies confirm that the brain plays an important role in the determination of pupal development by regulating the synthesis and release of FXPRLamide neuropeptides in H. armigera. Thus, the function of FXPRLamide peptides in H. armigera is closely correlated with pupal development. [Copyright &y& Elsevier]

Abstract: The diapause hormone (DH)-pheromone biosynthesis activating neuropeptide (PBAN) gene encodes five neuropeptides, DH, PBAN, α-SGNP, β-SGNP, and γ-SGNP (subesophageal ganglion neuropeptide). All share the C-terminal pentapeptide FXPRLamide sequence and are produced in the subesophageal ganglion (SG). Expression of the DH–PBAN gene in the central nervous system of embryonic, larval, pupal, and adult Helicoverpa armigera (Har) was studied using in situ hybridization, whole-mount immunocytochemistry, and competitive ELISA. Both Har-DH–PBAN mRNA and protein are localized in the mandibular, maxillary, and labial cell clusters of the SG and a pair of ventral midline neurons of each thoracic ganglion. The FXPRLamide titers in hemolymph are significantly higher in diapause-destined larvae during the fifth and sixth instar than in similar nondiapause-destined individuals. In contrast, the FXPRLamide titers in diapause-destined pupae are significantly lower than in nondiapause-destined pupae. The results from immunocytochemistry and in situ hybridization are consistent with changes of FXPRLamide titers as measured by ELISA. These data suggest that the expression of DH–PBAN might be correlated with diapause induction at the larval stage of diapause-destined individuals and continuous development at pupal stage of nondiapause-destined individuals. Thus, the DH–PBAN gene may play an important regulatory role in aspects of insect development besides diapause termination and pheromone biosynthesis. The transport pathways of FXPRLamide neuropeptides suggest that humoral route is involved in their regulation of development. [Copyright &y& Elsevier]

Abstract: Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are two crucial neuropeptides which regulate insect development and sex pheromone biosynthesis respectively. These peptides are encoded by a single gene, termed DH-PBAN gene. In this study, we characterized the promoter of the DH-PBAN gene in Helicoverpa armigera (Har). Transient transfection assays using a series of stepwise deletion fragments linked to the luciferase reporter gene indicate that the promoter contains multiple regulator domains that can activate and repress reporter gene expression. The fragment spanning −467 to −371 bp of the DH-PBAN promoter is an activator domain of transcription, whereas the region from −965 to −534 bp represses the promoter activity in the insect cell line BmN. Electrophoretic mobility shift assays demonstrate that at least two nuclear protein factors from the nuclear protein extracts of H. armigera suboesophageal ganglion, Har-DHMBP-1 and-2 (DH-modulator-binding protein) can specifically bind to the activating region. Furthermore, we characterized in detail that the nuclear protein factor Har-DHMBP-3 can specifically bind to a classical E-box, CAGCTG localized at positions −360 to −355 bp, a potential site for interaction with basic helix–loop–helix transcription factors. Mutation of this E-box results in a significant reduction of the promoter activity, suggesting it can modulate the previously identified activator domain. Taken together, multipartite cis-elements and transcription factors in the DH-PBAN promoter are involved in regulation of the gene expression. [Copyright &y& Elsevier]