In Situ Hybridization Analysis of the Expression of Futsch, Tau, and MESK2 Homologues in the Brain of the European Honeybee (Apis mellifera L.)

BACKGROUND: The importance of visual sense in Hymenopteran social behavior is suggested by the existence of a Hymenopteran insect-specific neural circuit related to visual processing and the fact that worker honeybee brain changes morphologically according to its foraging experience. To analyze molecular and neural bases that underlie the visual abilities of the honeybees, we used a cDNA microarray to search for gene(s) expressed in a neural cell-type preferential manner in a visual center of the honeybee brain, the optic lobes (OLs). METHODOLOGY/PRINCIPAL FINDINGS: Expression analysis of candidate genes using in situ hybridization revealed two genes expressed in a neural cell-type preferential manner in the OLs. One is a homologue of Drosophila futsch, which encodes a microtubule-associated protein and is preferentially expressed in the monopolar cells in the lamina of the OLs. The gene for another microtubule-associated protein, tau, which functionally overlaps with futsch, was also preferentially expressed in the monopolar cells, strongly suggesting the functional importance of these two microtubule-associated proteins in monopolar cells. The other gene encoded a homologue of Misexpression Suppressor of Dominant-negative Kinase Suppressor of Ras 2 (MESK2), which might activate Ras/MAPK-signaling in Drosophila. MESK2 was expressed preferentially in a subclass of neurons located in the ventral region between the lamina and medulla neuropil in the OLs, suggesting that this subclass is a novel OL neuron type characterized by MESK2-expression. These three genes exhibited similar expression patterns in the worker, drone, and queen brains, suggesting that they function similarly irrespective of the honeybee sex or caste. CONCLUSIONS: Here we identified genes that are expressed in a monopolar cell (Amfutsch and Amtau) or ventral medulla-preferential manner (AmMESK2) in insect OLs. These genes may aid in visualizing neurites of monopolar cells and ventral medulla cells, as well as in analyzing the function of these neurons

Effectiveness of a Teacher Training Program for Students with Symptoms of Developmental Disorders: Data from a Correspondence High School in Japan

In the present study, a teacher training program based on behavioral therapy was conducted for high school correspondence course teachers of adolescents aged between 15 and 18 years who showed developmental difficulties. Participating teachers were assigned to either an immediate treatment (IT; n = 13) or delayed treatment control (DTC; n = 17) group to evaluate the effectiveness of the program, which comprised five 90-min sessions with small groups of three to six participants and was conducted over three months. The results showed significant improvement in students’ behaviors and social responsiveness and in teachers’ confidence among those in the IT group; however, those in the DTC group did not show any such improvement. We discuss the program’s feasibility in terms of developing support resources for teachers in Japanese high schools

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

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

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

Deep sequencing of the prothoracic gland transcriptome reveals new players in insect ecdysteroidogenesis.

Ecdysteroids are steroid hormones that induce molting and determine developmental timing in arthropods. In insect larva, the prothoracic gland (PG) is a major organ for ecdysone synthesis and release. Released ecdysone is converted into the active form, 20-hydroxyecdysone (20E) in the peripheral tissues. All processes from ecdysone synthesis and release from the PG to its conversion to 20E are called ecdysteroidogenesis and are under the regulation of numerous factors expressed in the PG and peripheral tissues. Classical genetic approaches and recent transcriptomic screening in the PG identified several genes responsible for ecdysone synthesis and release, whereas the regulatory mechanism remains largely unknown. We analyzed RNA-seq data of the silkworm Bombyx mori PG and employed the fruit fly Drosophila melanogaster GAL4/UAS binary RNAi system to comprehensively screen for genes involved in ecdysone synthesis and/or release. We found that the genes encoding δ-aminolevulinic acid synthase (CG3017/alas) and putative NAD kinase (CG33156) were highly expressed in the PG of both B. mori and D. melanogaster. Neither alas nor CG33156 RNAi-induced larvae could enter into the pupal stage, and they had a lower abundance of the active form ecdysteroids in their prolonged larval stage. These results demonstrated that alas and CG33156 are indispensable for ecdysteroidogenesis