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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. [ABSTRACT FROM AUTHOR]

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), Bombyx neuropeptide GPCR-B2 (BNGR-B2). BNGR-B2 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. [ABSTRACT FROM AUTHOR]

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), Bombyx neuropeptide GPCR-B2 (BNGR-B2). BNGR-B2 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. [ABSTRACT FROM AUTHOR]

The creation of functional materials from renewable resources has attracted much interest. We previously reported on the genetic code expansion of the domesticated silkworm Bombyx mori to functionalize silk fiber with synthetic amino acids such as 4-azido-L-phenylalanine (AzPhe). The azido groups act as selective handles for biorthogonal chemical reactions. Here we report the characterization and scaled-up production of azido-functionalized silk fiber for textile, healthcare, and medical applications. To increase the productivity of azido-functionalized silk fiber, the original transgenic line was hybridized with a high silk-producing strain. The F1 hybrid produced circa 1.5 times more silk fibroin than the original transgenic line. The incorporation efficiency of AzPhe into silk fibroin was retained after hybridization. The tensile properties of the azido-functionalized silk fiber were equal to those of normal silk fiber. Scaled-up production of the azido-functionalized silk fiber was demonstrated by rearing circa 1000 transgenic silkworms. Differently-colored fluorescent silk fibers were successfully prepared by click chemistry reactions, demonstrating the utility of the azido-functionalized silk fiber for developing silk-based materials with desired functions. [ABSTRACT FROM AUTHOR]

Ecdysteroids are steroid hormones that control many aspects of development and physiology. During larval development, ecdysone is synthesized in an endocrine organ called the prothoracic gland through a series of ecdysteroidogenic enzymes encoded by the Halloween genes. The expression of the Halloween genes is highly restricted and dynamic, indicating that their spatiotemporal regulation is mediated by their tight transcriptional control. In this study, we report that three zinc finger-associated domain (ZAD)-C 2 H 2 zinc finger transcription factors-Séance (Séan), Ouija board (Ouib), and Molting defective (Mld)-cooperatively control ecdysone biosynthesis in the fruit fly Drosophila melanogaster Séan and Ouib act in cooperation with Mld to positively regulate the transcription of neverland and spookier , respectively, two Halloween genes. Remarkably, loss-of-function mutations in séan , ouib , or mld can be rescued by the expression of neverland , spookier , or both, respectively. These results suggest that the three transcription factors have distinct roles in coordinating the expression of just two genes in Drosophila Given that neverland and spookier are located in constitutive heterochromatin, Séan, Ouib, and Mld represent the first example of a transcription factor subset that regulates genes located in constitutive heterochromatin., (Copyright © 2018 by the Genetics Society of America.)

Ecdysteroids, including the biologically active hormone 20-hydroxyecdysone (20E), play essential roles in controlling many developmental and physiological events in insects. Ecdysteroid biosynthesis is achieved by a series of specialized enzymes encoded by the Halloween genes. Recently, a new class of Halloween gene, noppera-bo ( nobo ), encoding a glutathione S -transferase (GST) in dipteran and lepidopteran species, has been identified and characterized. GSTs are well known to conjugate substrates with the reduced form of glutathione (GSH), a bioactive tripeptide composed of glutamate, cysteine, and glycine. We hypothesized that GSH itself is required for ecdysteroid biosynthesis. However, the role of GSH in steroid hormone biosynthesis has not been examined in any organisms. Here, we report phenotypic analysis of a complete loss-of-function mutant in the γ -glutamylcysteine synthetase catalytic subunit ( Gclc ) gene in the fruit fly Drosophila melanogaster Gclc encodes the evolutionarily conserved catalytic component of the enzyme that conjugates glutamate and cysteine in the GSH biosynthesis pathway. Complete Gclc loss-of-function leads to drastic GSH deficiency in the larval body fluid. Gclc mutant animals show a larval-arrest phenotype. Ecdysteroid titer in Gclc mutant larvae decreases, and the larval-arrest phenotype is rescued by oral administration of 20E or cholesterol. Moreover, Gclc mutant animals exhibit abnormal lipid deposition in the prothoracic gland, a steroidogenic organ during larval development. All of these phenotypes are reminiscent to nobo loss-of-function animals. On the other hand, Gclc mutant larvae also exhibit a significant reduction in antioxidant capacity. Consistent with this phenotype, Gclc mutant larvae are more sensitive to oxidative stress response as compared to wild-type. Nevertheless, the ecdysteroid biosynthesis defect in Gclc mutant animals is not associated with loss of antioxidant function. Our data raise the unexpected hypothesis that a primary role of GSH in early D. melanogaster larval development is ecdysteroid biosynthesis, independent from the antioxidant role of GSH., (Copyright © 2017 by the Genetics Society of America.)

The receptor for diuretic hormone 31 (DH31R) was identified in the silkworm Bombyx mori. A heterologous expression system revealed that an orphan G-protein coupled receptor, BNGR-B1, responded to DH31 and upregulated the intracellular cAMP level. DH31R (BNGR-B1) was predominantly expressed in the anterior silk gland, midgut, and ovary, whereas DH31 was predominantly expressed in the central nervous system and midgut.

In insects, the precise timing of moulting and metamorphosis is strictly guided by ecdysteroids that are synthesised from dietary cholesterol in the prothoracic gland (PG). In the past decade, several ecdysteroidogenic enzymes, some of which are encoded by the Halloween genes, have been identified and characterised. Here, we report a novel Halloween gene, noppera-bo (nobo), that encodes a member of the glutathione S-transferase family. nobo was identified as a gene that is predominantly expressed in the PG of the fruit fly Drosophila melanogaster. We generated a nobo knock-out mutant, which displayed embryonic lethality and a naked cuticle structure. These phenotypes are typical for Halloween mutants showing embryonic ecdysteroid deficiency. In addition, the PG-specific nobo knock-down larvae displayed an arrested phenotype and reduced 20-hydroxyecdysone (20E) titres. Importantly, both embryonic and larval phenotypes were rescued by the administration of 20E or cholesterol. We also confirm that PG cells in nobo loss-of-function larvae abnormally accumulate cholesterol. Considering that cholesterol is the most upstream material for ecdysteroid biosynthesis in the PG, our results raise the possibility that nobo plays a crucial role in regulating the behaviour of cholesterol in steroid biosynthesis in insects.

Batesian mimicry protects animals from predators through resemblance with distasteful models in shape, color pattern, or behavior. To elucidate the wing coloration mechanisms involved in the mimicry, we investigated chemical composition and gene expression of the pale yellow and red pigments of a swallowtail butterfly, Papilio polytes, whose females mimic the unpalatable butterfly Pachliopta aristolochiae. Using LC/MS, we showed that the pale yellow wing regions in non-mimetic females consist of kynurenine and N-β-alanyldopamine (NBAD). Moreover, qRT-PCR showed that kynurenine/NBAD biosynthetic genes were upregulated in these regions in non-mimetic females. However, these pigments were absent in mimetic females. RNA-sequencing showed that kynurenine/NBAD synthesis and Toll signaling genes were upregulated in the red spots specific to mimetic female wings. These results demonstrated that drastic changes in gene networks in the red and pale yellow regions can switch wing color patterns between non-mimetic and mimetic females of P. polytes.

In insects molting and metamorphosis are primarily under the control of two insect hormones, ecdysone and juvenile hormone (JH). Physiological and biochemical studies of insect hormone metabolic pathways suggested the involvement of P450 (CYP) enzymes in the pathways, but molecular details of the enzymes were unclear. In recent years, the genome information and studies using molecular biology and genetics have allowed us to understand enzymes in the ecdysteroid and JH metabolic pathways. Genome sequencing has been accomplished in several insect species, and has shown the presence of 36-180 CYP enzymes. To date, six and one CYP enzymes have been revealed in the biosynthesis and inactivation pathways of 20-hydroxyecdysone (20E), respectively. In the 20E biosynthetic pathway, correlation among the enzymes, substrates and metabolites is elucidated in the late steps, but the enzyme(s) and intermediates in the early steps have not been fully understood and are referred to as the 'Black Box'. The gene expression of some CYP enzymes in the 20E biosynthesis is modulated by neuropeptides and JH. Furthermore, involvement of a CYP enzyme is found in both JH biosynthesis and inactivation pathways. Thus, recent studies have shown the importance of CYP enzymes in insect hormone metabolisms.

Background: Dibenzoylhydrazine analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. A notable feature is their high activity against lepidopteran insects, raising the question as to whether species-specific analogues can be isolated. In this study, the specificity of ecdysone agonists was addressed through a comparative analysis in two important lepidopterans, the silkworm Bombyx mori L. and the cotton leafworm Spodoptera littoralis (Boisd.)., Results: When collections of non-steroidal ecdysone agonists containing different mother structures (dibenzoylhydrazine, acylaminoketone, tetrahydroquinoline) were tested, in vitro reporter assays showed minor differences using cell lines derived from both species. However, when compounds with high ecdysone agonist activity were examined in toxicity assays, larvicidal activity differed considerably. Of note was the identification of three dibenzoylhydrazine analogues with > 100-fold higher activity against Bombyx than against Spodoptera larvae., Conclusion: The present study demonstrated that species-specific ecdysone-agonist-based insecticides can be developed, but their species specificity is not based on differences in the activation of the ecdysone receptor but rather on unidentified in vivo parameters such as permeability of the cuticle, uptake/excretion by the gut or metabolic detoxification.