The role of PTTH in the initiation of adult development.

<p>(A) The effect of brain extract (Br-ex) injection. The Br-CC-CA or brain alone (Br) was removed from post-diapause pupae 12 h after warming and 12 h later, brain extract (2 units of PTTH equivalents) was injected. As a negative control, saline was injected into the brain-deficient pupae. (B) The effect of PTTH removal from the brain extract. The brains were removed from post-diapause pupae 12 h after warming, and the debrained pupae were injected with the brain extract pre-treated with an anti-<i>Mab</i>PTTH or control antibody. As a negative and a positive controls, saline and the same dose (3 units of PTTH equivalents) of the brain extract were injected, respectively (n = 6). The values shown are the means (± SEM) of three independent determinations. (C) The Effect of the implantation of PTTH-containing gel into the Br-CC-CA-deficient pupae. The Br-CC-CA was removed from post-diapause pupae immediately after warming, and the PTTH-containing gel (+PTTH) was implanted into the Br-CC-CA-deficient pupae (n = 8). Control pupae received the gel without PTTH (-PTTH).</p

The role of the Br-CC-CA in post-diapause development.

<p>(A) The effects of brain removal and implantation on adult development of post-diapause pupae. Diapausing pupae were chilled for 8 weeks and the Br-CC-CA was removed immediately after warming. A Br-CC-CA collected from another previously chilled pupa was implanted into a Br-CC-CA-deficient pupa. As a positive control, pupae were only injured (sham-operated). For comparison, a Br-CC-CA collected from an unchilled pupa 5 weeks after pupation (*) was implanted into a Br-CC-CA-deficient pupa. Percentage values were determined with 6–12 pupae per operation. The values shown are the means (±SEM) of three independent determinations. (B) Time-dependent effects of brain removal on the development of post-diapause pupae. The Br-CC-CA was removed from post-diapause pupae at various times after warming (n = 12). (C) The effect of CC-CA removal (allatectomy) on adult development of post-diapause pupae. The CC-CA was removed immediately after warming. Percentage values were determined with 6–12 pupae per operation. The values shown are the means (±SEM) of three independent determinations.</p

Signaling pathways involved in PDF-induced ecdysone biosynthesis.

<p>(A) Effect of PDF on the transcript level of ecdysone biosynthesis-related enzymes in the PGs. PGs of V7 larvae were treated with or without PDF. The transcript levels of <i>nvd</i>, <i>nm-g</i>, <i>spo</i>, <i>phm</i>, <i>dib</i> and <i>sad</i> were quantified with Q-PCR. Each datum point represents the mean ±SEM (n = 3). (B-D) Effect of (B) transcript inhibitor (actinomycin D: ActD, 10 µM), (C) PKA inhibitor (H-89, 0.1 mM) and (D) translation inhibitor (cycloheximide: CHX, 0.2 mM) on PDF-induced ecdysone biosynthesis. Each datum point represents the mean ±SEM (n = 10). (E, F) Effect of (E) PI3K inhibitor (LY294002: LY, 50 µM) and (F) TOR inhibitor (rapamycin: Rap, 10 µM) on PDF-induced ecdysone biosynthesis. Each datum point represents the mean ±SEM (n = 3 and 4). Statistically significant differences were evaluated by Student's <i>t</i>-test (***P<0.001, **P<0.01). (G) Effect of PDF on the levels of p-ERK and p-4E-BP in cultured PGs. The phosphorylated proteins were examined by immunoblotting, and α-tubulin was used as a loading control. (H, I) Effect of (H) PI3K inhibitor (LY294002: LY, 50 µM) and (I) PKA inhibitor (H-89, 0.1 mM) on p-4E-BP levels in cultured PGs. The phosphorylated proteins were examined by immunoblotting, and α-tubulin was used as a loading control.</p

The changes in PTTH titers in the hemolymph.

<p>PTTH titers in the hemolymph of post-diapause pupae after warming (A) and nondiapause pupae after pupal ecdysis (B) were determined by TR-FIA. The values are the means ±SEM (n = 8). Different letters above the bars indicate a significant difference (ANOVA followed by Tukey-Kramer multiple-comparison test, p <0.05).</p

Developmental changes in <i>PTTH</i> gene expression and PTTH content in the brain.

<p>(A) <i>PTTH</i> gene expression in the brains of day-0 nondiapause pupae and post-diapause pupae at various times after warming was analyzed by qRT-PCR. The values shown are the means (± SEM) of three independent determinations. (B) PTTH content in the brains of day-0 nondiapause pupae and post-diapause pupae at various times after warming was determined by TR-FIA. The values are the means ±SEM (n = 6). Different letters above the bars indicate a significant difference (ANOVA followed by Tukey-Kramer multiple-comparison test, p <0.05).</p

qRT-PCR analysis of <i>MabTorso</i> expression in the PGs.

<p>The PGs were dissected from nondiapause and diapause pupae 0 to 3 days after pupal ecdysis (A) or from post-diapause pupae at various times after warming (B), and relative expression levels of the <i>MabTorso</i> gene were determined by qRT-PCR, with the level in day-0 nondiapause pupae being 1. The values shown are the means (± SEM) of three independent determinations.</p

Screening of candidate receptors.

<p>(A) Tissue distribution of <i>BNGR-B2</i>. The expression of <i>BNGR-B2</i> was measured by RT-PCR in the selected tissues from gut-purged fifth instar larvae (p50T strain). PG: prothoracic gland, BR: brain, FB: fat body, MT: Malpighian tubule, ASG: anterior silk gland, MG: midgut, TE: testis and OV: ovary. (B) Developmental profile of <i>BNGR-B2</i> in the PGs. The expression of <i>BNGR-B2</i> was measured by Q-PCR. The timing of molting, gut purge and pupation in our rearing conditions is indicated with arrows. Each datum point represents the mean ±SEM (n = 3). The dashed line indicates the outline of the hemolymph ecdysteroid titer described by Koyama et al., 2004 (4th instar), Sakurai et al., 1998 (5th instar) and Kaneko et al., 2006 (5th instar). (A, B) <i>RpL3</i> was used as an internal standard.</p

Characterization of BNGR-B2.

<p>(A) Phylogenetic relationship of BNGR-B2 and highly homologous receptors. The tree was generated based on the amino acid sequences of selected regions with the neighbor-joining method using the ClustalX multiple alignment program and a bootstrap value of 1000 trials for each branch position. The indicated numbers are the bootstrap values as a percentage of 1000 replicates, and the scale bar indicates 0.05 changes per residue. Bootstrap values greater than 50% are indicated. The <i>Mus musculus</i> calcitonin receptor (CR) was used as an outgroup. (B) Ligand-binding analysis of BNGR-B2 by examining the change in intracellular cAMP levels. BNGR-B2-expressing HEK293 cells were treated with 1 µM of the candidate BNGR-B2 ligands (PDF and DH31). Each datum point represents the mean ±SEM (n = 5). Statistically significant differences were evaluated by Student's <i>t</i>-test (***P<0.001).</p

Integration of the PDF signaling model with the known PTTH signaling pathway.

<p>Solid lines indicate demonstrated or highly likely pathways, and dashed lines indicate hypothetical pathways. Gαs: G protein αs subunit, AC: adenylate cyclase, AMP: adenosine monophosphate, cAMP: cyclic AMP, EPAC: exchange protein directly activated by cAMP, eIF4e: eukaryotic translation initiation factor 4E, 4E-BP: eIF4E binding protein, TOR: target of rapamycin, PKA: protein kinase A, PKC: protein kinase C, PI3K: phosphatidylinositol 3-kinase, AKT: protein kinase B, CREB: cAMP response element-binding protein, MAPK: mitogen-activated protein kinase, ERK: extracellular signal-regulated kinase, MEK: MAP kinase kinase, Raf: MAP kinase kinase kinase, S6: ribosomal protein S6, p70S6K: 70 kDa S6 kinase, PLC: phospholipase C, DAG: diacylglycerol, IP₃: inositol 1,4,5-trisphosphate, IP₃R: IP₃ receptor, CaM: calmodulin.</p

Pigment Dispersing Factor Regulates Ecdysone Biosynthesis via <i>Bombyx</i> Neuropeptide G Protein Coupled Receptor-B2 in the Prothoracic Glands of <i>Bombyx mori</i>

<div><p>Ecdysone is the key hormone regulating insect growth and development. Ecdysone synthesis occurs in the prothoracic glands (PGs) and is regulated by several neuropeptides. Four prothoracicotropic and three prothoracicostatic factors have been identified to date, suggesting that ecdysone biosynthesis is intricately regulated. Here, we demonstrate that the neuropeptide pigment dispersing factor (PDF) stimulates ecdysone biosynthesis and that this novel signaling pathway partially overlaps with the prothoracicotropic hormone (PTTH) signaling pathway. We performed transcriptome analysis and focused on receptors predominantly expressed in the PGs. From this screen, we identified a candidate orphan G protein coupled receptor (GPCR), <i>Bombyx</i> neuropeptide GPCR-B2 (BNGR-B2). <i>BNGR-B2</i> was predominantly expressed in ecdysteroidogenic tissues, and the expression pattern in the PGs corresponded to the ecdysteroid titer in the hemolymph. Furthermore, we identified PDF as a ligand for BNGR-B2. PDF stimulated ecdysone biosynthesis in the PGs, but the stimulation was only observed in the PGs during a specific larval stage. PDF did not affect the transcript level of known ecdysone biosynthetic enzymes, and inhibiting transcription did not suppress ecdysone biosynthesis, suggesting that the effects of PDF might be mediated by translational regulation and/or post-translational modification. In addition, the participation of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K), target of rapamycin (TOR) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) in the PDF signaling pathway was discovered.</p></div