Kunihiro Shiomi

PHE-X-PRO-ARG-LEU-NH2 PEPTIDE PRODUCING CELLS IN THE CENTRAL NERVOUS SYSTEM OF THE SILKWORM, BOMBYX MORI

Members of the neuropeptide family having Phe-X-Pro-Arg-Leu-NH(2) (FXPRLamide; X=Ser, Thr, Val, or Gly) at the C-terminus serve as regulators of oviduct and visceral muscle contraction, sex pheromone production, and diapause induction. Antibody raised against Bombyx mori diapause hormone recognized a variety of FXPRLamide peptides. Using this antibody, the antigen was immunocytochemically localized in the central nervous system (CNS) of the silkworm, Bombyx mori. Immunoreactive somata were observed in all ganglia of the CNS including the brain. Twelve somata localized at the midline of the suboesophageal ganglion (SG) were most intensely stained, and their neurite projections reached the retrocerebral complex. Thus, these cells in the SG exhibited typical features of neuroendocrine neurons. Marked reduction in immunoreactivity was observed in a pair of neurosecretory cells in the labial neuromere in SG of diapause type pupae, which indicates an active release of FXPRLamide peptides from these cells. No clear connection to neurohemal sites were observed in immunoreactive cells in the brain, thoracic or abdominal ganglia, suggesting that the immunoreactive peptides in these organs are likely to serve as neurotransmitters or neuromodulators.

Establishment of a Sandwich ELISA System to Detect Diapause Hormone, and Developmental Profile of Hormone Levels in Egg and Subesophageal Ganglion of the Silkworm, Bombyx mori

Abstract In the silkworm Bombyx mori, diapause hormone (DH) is produced in the female subesophageal ganglion (SG) and induces embryonic diapause by targeting developing ovaries. DH is processed from a precursor protein consisting of DH, pheromone biosynthesis activating neuropeptide (PBAN) and three other neuropeptides (SGNPs). Because these five neuropeptides share a common sequence, FXPRLamide, at the C-terminus, a direct and specific assay for DH itself is required in order to understand the profile of concentration changes. In this study, we produced a mouse monoclonal antibody (anti-DH[N] mAb) against the N-terminal region of DH and developed a sandwich enzyme-linked immunosorbent assay using the anti-DH[N] mAb and a rabbit polyclonal antibody against the C-terminus of DH. This procedure enabled us to specifically quantify the DH molecule at femtomolar levels (equivalent to 1/10 of SG). We then plotted DH levels in eggs and SGs during embryonic and post-embryonic development. DH was present in late-stage embryos that had been destined for the production of both diapause and nondiapause eggs. DH levels in SG gradually increased in both types during larval development and peaked at the early pupal stage. At the middle pupal stage, DH levels in SG and SG-brain complex decreased markedly in the diapause-egg producing type, thus indicating active release of DH into the hemolymph. From 5th instar larva to adult, no sexual differences in DH levels were observed in SGs or SG-brain complexes from diapause and nondiapause egg-producing types.

Myocyte enhancer factor 2 (MEF2) is a key modulator of the expression of the prothoracicotropic hormone gene in the silkworm, Bombyx mori.

Prothoracicotropic hormone (PTTH) plays a central role in controlling molting, metamorphosis, and diapause termination in insects by stimulating the prothoracic glands to synthesize and release the molting hormone, ecdysone. Using Autographa californica nucleopolyhedrovirus (AcNPV)‐mediated transient gene transfer into the central nervous sytem (CNS) of the silkworm, Bombyx mori, we identified two cis‐regulatory elements that participate in the decision and the enhancement of PTTH gene expression in PTTH‐producing neurosecretory cells (PTPCs). The cis‐element mediating the enhancement of PTTH gene expression binds the transcription factor Bombyx myocyte enhancer factor 2 (BmMEF2). The BmMEF2 gene was expressed in various tissues including the CNS. In brain, the BmMEF2 gene was expressed at elevated levels in two types of lateral neurosecretory cells, namely PTPCs and corazonin‐like immunoreactive lateral neurosecretory cells. Overexpression of BmMEF2 cDNA caused an increase in the transcription of PTTH. Therefore, BmMEF2 appears to be particularly important in the brain where it is responsible for the differentiation of lateral neurosecretory cells, including the enhancement of PTTH gene expression. This is the first report to identify a target gene of MEF2 in the invertebrate nervous system.

Embryonic thermosensitive TRPA1 determines transgenerational diapause phenotype of the silkworm, Bombyx mori

Significance Diapause has evolved as a specific subtype of dormancy in most insect species and as a seasonal polyphenism that ensures survival under unfavorable environmental conditions and synchronizes populations. In Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. However, the molecular mechanisms involved in the perception of environmental temperature and in linking thermal information to neuroendocrine functions are still unknown. Here, we show that the Bombyx transient receptor potential A1 (TRPA1) could be thermally activated during embryogenesis, and an unknown signaling pathway linked to the release of diapause hormone may then be activated to affect the induction of diapause in progeny. The Bombyx TRPA1 acts as a molecular switch for the development of an alternative phenotype in an animal with seasonal polyphenism. In the bivoltine strain of the silkworm, Bombyx mori, embryonic diapause is induced transgenerationally as a maternal effect. Progeny diapause is determined by the environmental temperature during embryonic development of the mother; however, its molecular mechanisms are largely unknown. Here, we show that the Bombyx TRPA1 ortholog (BmTrpA1) acts as a thermosensitive transient receptor potential (TRP) channel that is activated at temperatures above ∼21 °C and affects the induction of diapause in progeny. In addition, we show that embryonic RNAi of BmTrpA1 affects diapause hormone release during pupal-adult development. This study identifying a thermosensitive TRP channel that acts as a molecular switch for a relatively long-term predictive adaptive response by inducing an alternative phenotype to seasonal polyphenism is unique.

Induction of non-diapause eggs by injection of anti-diapause hormone rabbit serum into the diapause type of the silkworm, Bombyx mori

Abstract Polyclonal antiserum was raised by immunizing rabbits with the synthetic diapause hormone (BomDH-I[19-Cys]) of the silkworm, Bombyx mori . By immunological analyses, the antiserum was demonstrated to specifically recognize the diapause hormone. The antiserum was injected into larvae, pupae and pharate adults of the Daizo strain that were destined to lay diapause eggs, to see whether the serum is able to act in vivo as an anti-hormone agent. Injection of the antiserum at various stages from the fourth larval instar to the early pharate adult stage induced moths to lay non-diapause eggs. The effect of the antiserum injection declined suddenly from the middle of the pharate adult stage, when diapause hormone is secreted actively. The dose-response curve demonstrated the maximal dose to be a 1.0 μl injection of the antiserum and a half-maximal dose of 0.1 μl. When neutralized in vitro with the synthetic diapause hormone, the antiserum lost its ability to induce non-diapause eggs, which indicates that the antiserum inactivates diapause hormone through immunoneutralization. A transplantation experiment using suboesophageal ganglia preexposed to the antiserum indicated that the antiserum had no cytotoxic effects. Following injection of the antiserum trehalase activity and glycogen content in developing ovaries were reduced to the levels found after removal of the suboesophageal ganglion. The results indicate the potential for using rabbit IgG as a simple tool for the control of neuropeptide hormone titers in insect hemolymph.

An FXPRLamide Neuropeptide Induces Seasonal Reproductive Polyphenism Underlying a Life-History Tradeoff in the Tussock Moth

The white spotted tussock moth, Orgyia thyellina, is a typical insect that exhibits seasonal polyphenisms in morphological, physiological, and behavioral traits, including a life-history tradeoff known as oogenesis-flight syndrome. However, the developmental processes and molecular mechanisms that mediate developmental plasticity, including life-history tradeoff, remain largely unknown. To analyze the molecular mechanisms involved in reproductive polyphenism, including the diapause induction, we first cloned and characterized the diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN) cDNA encoding the five Phe-X-Pro-Arg-Leu-NH2 (FXPRLa) neuropeptides: DH, PBAN, and α-, β-, and γ-SGNPs (subesophageal ganglion neuropeptides). This gene is expressed in neurosecretory cells within the subesophageal ganglion whose axonal projections reach the neurohemal organ, the corpus cardiacum, suggesting that the DH neuroendocrine system is conserved in Lepidoptera. By injection of chemically synthetic DH and anti-FXPRLa antibody into female pupae, we revealed that not only does the Orgyia DH induce embryonic diapause, but also that this neuropeptide induces seasonal polyphenism, participating in the hypertrophy of follicles and ovaries. In addition, the other four FXPRLa also induced embryonic diapause in O. thyellina, but not in Bombyx mori. This is the first study showing that a neuropeptide has a pleiotropic effect in seasonal reproductive polyphenism to accomplish seasonal adaptation. We also show that a novel factor (i.e., the DH neuropeptide) acts as an important inducer of seasonal polyphenism underlying a life-history tradeoff. Furthermore, we speculate that there must be evolutionary conservation and diversification in the neuroendocrine systems of two lepidopteran genera, Orgyia and Bombyx, in order to facilitate the evolution of coregulated life-history traits and tradeoffs.

Disruption of diapause induction by TALEN-based gene mutagenesis in relation to a unique neuropeptide signaling pathway in Bombyx

The insect neuropeptide family FXPRLa, which carries the Phe-Xaa-Pro-Arg-Leu-NH2 sequence at the C-terminus, is involved in many physiological processes. Although ligand–receptor interactions in FXPRLa signaling have been examined using in vitro assays, the correlation between these interactions and in vivo physiological function is unclear. Diapause in the silkworm, Bombyx mori, is thought to be elicited by diapause hormone (DH, an FXPRLa) signaling, which consists of interactions between DH and DH receptor (DHR). Here, we performed transcription activator-like effector nuclease (TALEN)-based mutagenesis of the Bombyx DH-PBAN and DHR genes and isolated the null mutants of these genes in a bivoltine strain. All mutant silkworms were fully viable and showed no abnormalities in the developmental timing of ecdysis or metamorphosis. However, female adults oviposited non-diapause eggs despite diapause-inducing temperature and photoperiod conditions. Therefore, we conclude that DH signaling is essential for diapause induction and consists of highly sensitive and specific interactions between DH and DHR selected during ligand–receptor coevolution in Bombyx mori.

Insights for future studies on embryonic diapause promoted by molecular analyses of diapause hormone and its action in Bombyx mori

Publisher Summary This chapter outlines the current topics on silkworm diapause, especially on the regulation of diapause hormone-pheromone synthesis activating neuropeptide (DH-PBAN) gene expression analyzed by a transgenic Drosophila system, the regulation of diapauses hormone (DH) peptide secretion, and diapause metabolism in eggs that are deficient in glycogen and sorbitol. The recent advance in molecular studies of silkworm diapause presents a scenario for diapause using molecular terms. In this scenario, the key topics are the organization of the DH-PBAN gene, expression of the gene, and its regulation, peptidogenesis and secretion from the neuroendocrine organ, the dynamics in circulation, the reception and expression of the hormonal signal in the target organ, and the regulation of target reactions leading to the metabolic change. Diapause occurs as an alternative developmental program in the lifecycle and is accomplished by the dynamic change of developmental, behavioral, and physiological events. Bombyx mori is a typical insect entering diapause at an early embryonic stage before dermal differentiation is completed. New information on regulatory mechanisms of gene expression and of peptide secretion from the neuroendocrine organ is also provided in the chapter, with a discussion a new diapause-associated metabolism. The chapter also proposes some new directions for future studies of insect diapause.

A hydrophobic peptide (VAP-peptide) of the silkworm, Bombyx mori: a unique role for adult activity proposed from gene expression and production at the terminal phase of metamorphosis

A unique hydrophobic peptide (VAP-peptide) isolated from male adult heads of the silkworm, Bombyx mori, has been shown to act as a synergist to the diapause hormone when administered exogenously. Here, we investigated the true role of the endogenous VAP-peptide on differentiation and development of adult organs in the silkworm. By northern blot analyses, the VAP-peptide gene was shown to be exclusively expressed at the terminal phase of adult development in epithelial tissues, especially in the wing and the thoracic integument. In situ hybridization analysis revealed that the gene was highly expressed in the epidermal cells of the wing vein and the thoracic integument. The stage- and tissue-dependent gene expression were clearly correlated to the accumulation profile of VAP-peptide. In the adult thoracic integument, VAP-peptide was predominantly deposited in the cuticle layer. Affinity chromatography indicated the ability of VAP-peptide to bind to chitin. Based on its expression patterns, localization, and chemical properties, VAP-peptide is conceived to be a structural protein that participates in mechanical strengthening of specific cuticle structures, supporting their physical requirements in the adult life of the silkworm.

A Baculovirus Immediate-Early Gene, ie1, Promoter Drives Efficient Expression of a Transgene in Both Drosophila melanogaster and Bombyx mori

Many promoters have been used to drive expression of heterologous transgenes in insects. One major obstacle in the study of non-model insects is the dearth of useful promoters for analysis of gene function. Here, we investigated whether the promoter of the immediate-early gene, ie1, from the Bombyx mori nucleopolyhedrovirus (BmNPV) could be used to drive efficient transgene expression in a wide variety of insects. We used a piggyBac-based vector with a 3xP3-DsRed transformation marker to generate a reporter construct; this construct was used to determine the expression patterns driven by the BmNPV ie1 promoter; we performed a detailed investigation of the promoter in transgene expression pattern in Drosophila melanogaster and in B. mori. Drosophila and Bombyx belong to different insect orders (Diptera and Lepidoptera, respectively); however, and to our surprise, ie1 promoter-driven expression was evident in several tissues (e.g., prothoracic gland, midgut, and tracheole) in both insects. Furthermore, in both species, the ie1 promoter drove expression of the reporter gene from a relatively early embryonic stage, and strong ubiquitous ie1 promoter-driven expression continued throughout the larval, pupal, and adult stages by surface observation. Therefore, we suggest that the ie1 promoter can be used as an efficient expression driver in a diverse range of insect species.