Prothoracicotropic activities of PDF <i>in vitro</i>.

<p>(A) Effect of PDF (1 µM) and DH31 (1 µM) on ecdysone biosynthesis in the PGs of V7 larvae (n = 14 and 12). (B) Dose-response curves for PDF and PTTH on ecdysone biosynthesis in the PGs of V7 larvae. Closed circles indicate PDF (n = 4–24) and open squares indicate PTTH (n = 10–38). (C, D) Developmental changes in PDF responsiveness on (C) ecdysone biosynthesis (n = 8–14) and (D) intracellular cAMP level (n = 5–6). (E) Effect of extracellular Ca²⁺ on ecdysone biosynthesis (n = 6). (F) Effect of extracellular Ca²⁺ on the change in intracellular cAMP levels (n = 4–8). Statistically significant differences were evaluated by Student's <i>t</i>-test (***P<0.001, **P<0.01, *P<0.05).</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

Changes in ecdysteroid titers in the hemolymph.

<p>Ecdysteroid titers in the hemolymph of post-diapause pupae (A) and nondiapause pupae (B) were determined by ELISA. The values are the means ±SEM (n = 6).</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

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

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

The responsiveness of the PGs to PTTH in post-diapause pupae.

<p>(A) The PGs of post-diapause pupae were dissected at various times after warming and incubated <i>in vitro</i> with or without PTTH (0.2 units/ml) for 2 h. The amount of ecdysteroid secreted into the medium was determined by ELISA. The values are the means ± SEM (n = 6). (B) Activation ratios were calculated from the data in A. (C, D) The PGs of post-diapause pupae 12 h after warming (C) and of day-1 nondiapause pupae (D) were dissected and incubated <i>in vitro</i> with or without PTTH at various concentrations (0.01 to 2 units/ml) for 2 h, and the amount of ecdysteroid secreted into the medium was determined by ELISA. The values are the means ± SEM (n = 8). (E) The responsiveness to PTTH of the PGs of post-diapause pupae and nondiapause pupae was expressed as activation ratio, which was calculated from the data in C and D. *, P < 0.05, Student t-test.</p

Endocrine Mechanisms Regulating Post-Diapause Development in the Cabbage Armyworm, <i>Mamestra brassicae</i>

<div><p>Diapause, a programmed developmental arrest at a specific stage, is common in insects and is regulated by hormones. It is well established that in pupal diapause, cessation of ecdysteroid secretion from the prothoracic glands (PGs) after pupal ecdysis leads to diapause initiation, while resumption of its secretion induces post-diapause development. However, what regulates the activity of the glands is poorly understood, especially for the glands of diapause-terminated pupae. In the present study, we investigate the mechanisms by which post-diapause development is regulated in the cabbage armyworm <i>Mamestra brassicae</i>. We demonstrate that the brain is necessary for the initiation of post-diapause development and that the factor in the brain responsible for the activation of the PGs is the prothoracicotropic hormone (PTTH). Further, through measuring the hemolymph PTTH titers by time-resolved fluoroimmunoassay, we show that PTTH is actually released into the hemolymph prior to the activation of the PGs. Although its peak titer is much lower than expected, this low concentration of PTTH is most likely still effective to activate the PGs of post-diapause pupae, because the responsiveness to PTTH of the glands at this stage is very high compared to that of nondiapause pupal PGs. These results strongly suggest that in <i>M</i>. <i>brassicae</i>, PTTH serves as a trigger to initiate pupa-adult development after diapause termination by stimulating the PGs to secrete ecdysteroid.</p></div