Physiological differentiation of DH-PBAN-producing neurosecretory cells in the silkworm embryo

Embryonic diapause of the silkworm, Bombyx mori, is induced by a neuropeptide hormone, the diapause hormone (DH), which is secreted from a limited number of neurosecretory cells in the subesophageal ganglion (SG) at the maternal generation. We examined the developmental fate of the hormone-producing cell (DH-pheromone biosynthesis activating neuropeptide [PBAN]-producing cell) in the embryonic stage at the level of gene expression and cell biology. The DH-PBAN gene expression started at the histogenesis stage and gradually increased toward hatching. DH is an amidated peptide belonging to FXPRLamide family. The immunoreactive somata against anti FXPRLamide antiserum were found in the SG from blastokinesis. Immunoreactive neural processes with varicosites were also found on the corpus cardiacum and the corpus allatum. The implantation of a part of a developing embryo including the SG into the pupae with the SG removed induced diapause eggs in the progeny. These results were obtained from eggs incubated under diapause-averting conditions as well as diapause-inducing conditions. Thus, a neurosecretory system responsible for biosynthesis of FXPRLamide neuropeptides is established as early as histogenesis, although the system to regulate the secretion of neuropeptide hormones has not been fully formed by that time.     

The diapause hormone-pheromone biosynthesis activating neuropeptide gene of Helicoverpa armigera encodes multiple peptides that break, rather than induce, diapause

FXPRLamide peptides encoded by the DH-PBAN (diapause hormone-pheromone biosynthesis activating neuropeptide) gene induce embryonic diapause in Bombyx mori, but terminate pupal diapause in Helicoverpa armigera (Har). Here, we explore the mechanisms of terminating pupal diapause by the FXPRLamide peptides. Using quantitative RT-PCR, we observed that expression of Har-DH-PBAN mRNA in the SG of nondiapause-type pupae was significantly higher than in diapause-type pupae. Immunocytochemical results indicated that the level of FXPRLamide peptides and axonal release are related to the diapause decision. Ecdysteroidogenesis in prothoracic glands (PGs) was stimulated by synthetic Har-DH in vivo and in vitro, and labeled Har-DH bound to the membrane of the PG, thus suggesting that DH breaks diapause by activating the PG to synthesize ecdysone. Furthermore, the response of DH in terminating diapause was temperature dependent. Decerebration experiments showed that the brain can control pupal development through the regulation of DH, and DH can terminate diapause and promote development without the brain. This result suggests a possible mechanism of response for the signals of DH and other FXPRLamide peptides in H. armigera.     

Effect of the brain and suboesophageal ganglion on pupal development in Helicoverpa armigera through regulation of FXPRLamide neuropeptides

Recent studies in Helicoverpa armigera report a novel role for diapause hormone (DH), pheromone biosynthesis activating neuropeptide (PBAN) and three other FXPRLamide neuropeptides secreted from suboesophageal ganglion (SG) in terminating pupal diapause. In the present paper, we investigated the role of these five FXPRLamide family neuropeptides on pupal development. Although removal of SG could not make nondiapause-destined pupae enter diapause-like status, it did make them eclose approximately 0.6-1.2 days later when compared with the controls. The results of competitive ELISAs showed a high level of FXPRLamide titer in the hemolymph of the SG-removed pupae and this may be due to the expression of the DH-PBAN gene in tissues other than SG. DH-PBAN mRNA and peptides were also detected in the thoracic ganglia (TGs) by RT-PCR and immunocytochemistry. The expression of DH-PBAN gene in the TGs of the SG-removed pupae is significantly higher than that in normal pupae by quantitative PCR and immunocytochemistry. Decerebration experiments proved that the decerebrated pupae could enter diapause-like status through down-regulation of FXPRLamide titer in hemolymph. Our studies confirm that the brain plays an important role in the determination of pupal development by regulating the synthesis and release of FXPRLamide neuropeptides in H. armigera. Thus, the function of FXPRLamide peptides in H. armigera is closely correlated with pupal development.     

Mechanism of neuronal coordination of the rhythmic activities of developing flight muscles and a neurosecretory cell population in the silkmoth, Bombyx mori

Ultradian rhythmic firing activity (period of 40-90 min) of a population of neurosecretory cells (NSCs) producing FXPRLamide peptides in the subesophageal ganglion (SG) of the silkmoth, Bombyx mori, is closely coordinated with periodically occurring electrical activity of developing flight muscles (FMs) during metamorphosis. To examine neuronal mechanisms and pathways that mediate the coordination of the NSC and flight motor systems, the ventral nerve cord (VNC) or circumesophageal connectives were transected. Transection of the VNC between the SG and thoracic ganglia greatly shortened the period of activity rhythm of the FMs (5-15 min) with no effect on the rhythmicity of NSCs. Bilateral transsection of the circumesophageal connectives between the brain and SG abolished the rhythmic activity of NSCs, thereby suggesting that the coordination of the two systems may be mediated by a common oscillatory mechanism in the brain. Further, bisection of the brain in the midline failed to abolish the ultradian rhythmicity of FMs. Thus, each brain hemisphere may have an ultradian oscillator that activates the NSC system in the SG, and modulates the short-period oscillation of the flight motor system located in the thoracic ganglia.     

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

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.     

Corazonin and corazonin-like substances in the central nervous system of the Pterygote and Apterygote insects

Antisera against corazonin were used to investigate distribution of immunoreactive cells in the central nervous system (CNS) of representatives of six insect orders: Ctenolepisma lineata (Zygentoma), Locusta migratoria (Orthoptera), Oxya yezoensis (Orthoptera), Gryllus bimaculatus (Orthoptera), Pyrrhocoris apterus (Hemiptera), Arge nigrinodosa (Hymenoptera), Athalia rosae (Hymenoptera), Bombyx mori (Lepidoptera) and Anomala cuprea (Coleoptera). Corazonin-like immunoreactive (CLI) cells were detected in the brain and ventral ganglia of all insects studied except for the albino strain of L. migratoria and the beetle A. cuprea. Implantation of the brain or different ganglia from insects with detected immunoreactivity induced dark coloration in the albino locust, providing further evidence for the presence of authentic corazonins [His(7)- and Arg(7)-isoforms] in these insects. The protocerebral lateral neurosecretory cells projecting into the ipsilateral retrocerebral neurohemal organs and bilateral longitudinal tracts extending and branching throughout the entire CNS seem to be a well-conserved part of the corazonin system in insects. The bilateral longitudinal tracts were formed by species-specific numbers of bilateral interneurons segmentally distributed in the ventral ganglia. Additional immunoreactive somata, mostly interneurons, were detected in the CNS of various insects. The distribution of corazonin in the cephalic neurosecretory system and in the bilateral interneurons suggests that corazonin acts as a hormone as well as a neurotransmitter or a neuromodulator. An ancient origin of corazonin is suggested by the presence of a corazonin-like substance in the primitive insect, C. lineata. These results support previous findings on the common occurrence of corazonin among insects, except for the albino strain of L. migratoria and the Coleoptera.     

PBAN/pyrokinin peptides in the central nervous system of the fire ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

The pyrokinin/pheromone-biosynthesis-activating neuropeptide (PBAN) family of peptides found in insects is characterized by a 5-amino-acid C-terminal sequence, FXPRLamide. The pentapeptide is the active core required for diverse physiological functions, including the stimulation of pheromone biosynthesis in female moths, muscle contraction, induction of embryonic diapause, melanization, acceleration of puparium formation, and termination of pupal diapause. We have used immunocytochemical techniques to demonstrate the presence of pyrokinin/PBAN-like peptides in the central nervous system of the fire ant, Solenopsis invicta. Polyclonal antisera against the C-terminal end of PBAN have revealed the location of the peptide-producing cell bodies and axons in the central nervous system. Immunoreactive material is detectable in at least three groups of neurons in the subesophageal ganglion and corpora cardiaca of all adult sexual forms. The ventral nerve cord of adults consists of two segmented thoracic ganglia and four segmented abdominal ganglia. Two immunoreactive pairs of neurons are present in the thoracic ganglia, and three neuron pairs in each of the first three abdominal ganglia. The terminal abdominal ganglion has no immunoreactive neurons. PBAN immunoreactive material found in abdominal neurons appears to be projected to perisympathetic organs connected to the abdominal ganglia. These results indicate that the fire ant nervous system contains pyrokinin/PBAN-like peptides, and that these peptides are released into the hemolymph. In support of our immunocytochemical results, significant pheromonotropic activity is found in fire ant brain-subesophageal ganglion extracts from all adult fire ant forms (queens, female and male alates, and workers) when extracts are injected into decapitated females of Helicoverpa zea. This is the first demonstration of the presence of pyrokinin/PBAN-like peptides and pheromonotropic activity in an ant species. This research was supported in part by a US-Israel Binational Science Foundation Grant (no. 2003367).     

Pyrokinin/PBAN-like peptides in the central nervous system of Drosophila melanogaster

The pyrokinin/pheromone biosynthesis activating neuropeptide (PBAN) family of peptides found in insects is characterized by a 5-amino-acid C-terminal sequence, FXPRLamide. The pentapeptide is the active core required for diverse physiological functions, including stimulation of pheromone biosynthesis in female moths, stimulation of muscle contraction, induction of embryonic diapause in Bombyx mori, and stimulation of melanization in some larval moths. Recently, this family of peptides has been implicated in accelerating the formation of the puparium in a dipteran. Using bioassay and immunocytochemical techniques, we demonstrate the presence of pyrokinin/PBAN-like peptides in the central nervous system of Drosophila melanogaster. Pheromonotropic activity was shown in the moths Helicoverpa zeaand Helicoverpa armigera by using dissected larval nervous systems and adult heads and bodies of D. melanogaster. Polyclonal antisera against the C-terminal ending of PBAN revealed the location of cell bodies and axons in the central nervous systems of larval and adult flies. Immunoreactive material was detected in at least three groups of neurons in the subesophageal ganglion of 3rd instar larvae, pupae, and adults. The ring gland of both larvae and adults contained immunoreactivity. Adult brain-subesophageal ganglion complex possessed additional neurons. The fused ventral ganglia of both larvae and adults contained three pairs of neurons that sent their axons to a neurohemal organ connected to the abdominal nervous system. These results indicate that the D. melanogasternervous system contains pyrokinin/PBAN-like peptides and that these peptides could be released into the hemolymph.     

Developmental changes in the localization of protein kinase CK2 in non-diapause and diapause eggs of the silkworm, Bombyx mori

To analyze the role of protein kinase CK2 (CK2) during early embryogenesis in non-diapause and diapause of the silkworm, the distribution and localization of Bombyx mori CK2 (BmCK2) were investigated by an immunohistochemical technique using antibodies against the α- and β-subunits of BmCK2. Both were localized in blastoderm cells of non-diapause and diapause eggs until 24 h after oviposition. More than 24 h after oviposition, however, the distribution of BmCK2 was different in non-diapause and diapause eggs. In non-diapause eggs, BmCK2 was mainly localized in yolk cells. In contrast, in diapause eggs, the localization was mainly observed in germ-band cells. Furthermore, we confirmed that the RNA helicase-like protein that was localized together with BmCK2 in non-diapause eggs was phosphorylated by BmCK2 in vitro. These data suggest that the role of BmCK2 is different in non-diapause and diapause eggs.     

Firing activities of neurosecretory cells producing diapause hormone and its related peptides in the female silkmoth,Bombyx mori. I. labial cells

There are three known clusters of neurosecretory cells expressing a gene encoding diapause hormone (DH) and four related peptides in the suboesophageal ganglion (SOG) of Bombyx mori. Long-term chronic recordings were made from the axonal tract (NCC-3) of a pair of cells localized in the labial (posterior) neuromere of SOG during pupal-adult development. There was a significant difference in firing activity patterns of the labial neurosecretory cells between diapause-egg and non-diapause-egg producers: labial cells in the former were active throughout pupal-adult development, whereas the same cells in the latter usually maintained an inactive state until the last quarter of pupal-adult development, a time at which a secretion of DH seems to be too late to act on the developing ovary for the induction of diapausing eggs. This observation strongly supports the notion that labial cells release DH and are responsible for determination of embryonic diapause in the silkmoth.     

Immunoreactive intensity of FXPRL amide neuropeptides in response to environmental conditions in the silkworm, Bombyx mori

In the silkworm Bombyx mori, the diapause hormone-pheromone biosynthesis activating neuropeptide gene, DH-PBAN, is a neuropeptide gene that encodes a polypeptide precursor consisting in five Phe-X-Pro-Arg-Leu-NH₂ (FXPRL) amide (FXPRLa) neuropeptides; DH (diapause hormone), PBAN (pheromone-biosynthesis-activating neuropeptide) and α-, β- and γ-SGNPs (subesophageal ganglion neuropeptides). These neuropeptides are synthesized in DH-PBAN-producing neurosecretory cells contained within three neuromeres, four mandibular cells, six maxillary cells, two labial cells (SLb) and four lateral cells of the subesophageal ganglion. DH is solely responsible, among the FXPRLa peptide family, for embryonic diapause. Functional differentiation has been previously suggested to occur at each neuromere, with the SLb cells releasing DH through brain innervation in order to induce embryonic diapause. We have investigated the immunoreactive intensity of DH in the SLb when thermal (25°C or 15°C) and light (continuous illumination or darkness) conditions are altered and following brain surgery that induces diapause or non-diapause eggs in the progeny. We have also examined the immunoreactivity of the other FXPRLa peptides by using anti-β-SGNP and anti-PBAN antibodies. Pupal SLb somata immunoreactivities seem to be affected by both thermal and light conditions during embryogenesis. Thus, we have been able to identify a close correlation between the immunoreactive intensity of neuropeptides and environmental conditions relating to the determination of embryonic diapause in B. mori.     

Cloning and expression of the cDNA encoding the FXPRL family of peptides and a functional analysis of their effect on breaking pupal diapause in Helicoverpa armigera

Diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN) are encoded by a single mRNA in the suboesophegeal ganglion (SG) and are responsible for induction of embryonic diapause in Bombyx mori and sex pheromone biosynthesis in lepidopteran insects. PBAN cDNA analyses revealed that the DH-like peptide is present in several species that have a pupal diapause. However, the function of the DH-like peptide remains unknown. In the present study, we cloned the cDNA encoding DH-PBAN in Helicoverpa armigera utilizing the rapid amplification of the cDNA ends method. The nucleotide se quence analysis revealed that the longest open reading frame of this cDNA encodes a 194-amino acid precursor protein that con tains a 33-aa PBAN, a 24-aa DH-like peptide, and three other neuropeptides, all of which have a common C-terminal pentapeptide motif FXPR/KL ( X=G, T, S). A homology search showed that H. armigera DH-like and PBAN are highly homologous to those from other insects. Northern blot analysis demonstrated a single message RNA corresponding to the size of Har-DH-PBAN cDNA from pupal SG with significantly higher expression in the SG of nondiapause pupae than diapausing pupae. Western blot analysis showed DH-like peptide expression from SG of both males and females. When DH-like peptide was injected into nondiapause larvae and pupae, it did not induce diapause, but rather efficiently broke pupal diapause in H. armigera. The ED(50) of DH to terminate pupal diapause is 20 pmol/pupae. The other four FXPRLamide neuropeptides from the DH-PBAN polyprotein precursor have cross activity for diapause termination. These observations therefore suggest a potential role for these FXPRL family peptides in promoting continuous development in several noctuid species. The high expression of this gene in pharate adults and adults indicates that the FXPRL family peptides may have multiple physiological functions.     

Increased urinary C-type natriuretic peptide excretion may be an early marker of renal tubulointerstitial fibrosis

The cDNAs encoding allatotropin (AT) and allatotropin-like peptides (ATLPs) were isolated from the silkworm, Bombyx mori. Similar to those of the tobacco hornworm, Manduca sexta, four peptides (AT, ATLP1, ATLP2, and ATLP3) are present in three different variants generated by alternative splicing. RT-PCR analyses showed that these splice variants are expressed in the central nervous system with differing expression patterns in each ganglion. Immunohistochemistry using an anti-AT antibody confirmed that AT-expressing cells were located in these central nervous ganglia as well as in two large anterior cells of the frontal ganglia. Injection of synthetic AT and ATLP-1 into B. mori larvae increased the latency to feed, indicating that AT and ATLP might function in the regulation of feeding behavior in B. mori.     

Firing activities of neurosecretory cells producing diapause hormone and its related peptides in the female silkmoth,Bombyx mori. II. mandibular and maxillary cells

A gene encoding a precursor polyprotein of diapause hormone (DH) and four related peptides is expressed by three groups of neurosecretory cells in the suboesophageal ganglion (SOG) of Bombyx mori. Long-term chronic recordings of firing activities were made from a common axonal tract (the maxillary nerve) of two groups of cells localized in the mandibular and maxillary neuromeres of SOG during pupal-adult development. Mandibular and maxillary cells usually produced a cluster of action potentials at an interval of 30-60 min during pupal-adult development and there was no significant difference in the firing activity profile between diapause-egg and non-diapause-egg producers. We suggest that rather than DH secretion, pupal mandibular and maxillary cells are involved in the secretion of DH-related neuropeptides. DH secretion seems to be assigned to the third group of cells (labial cells); hence, there may be different posttranslational processing of the precursor polyprotein in different neurosecretory cell groups.     

Distribution of locustamyotropin-like immunoreactivity in the nervous system of Locusta migratoria.

Locustamyotropin-like immunoreactivity was visualized in the nervous system of Locusta migratoria by means of the peroxidase antiperoxidase method. Highly specific antibodies to the carboxy-terminus of the locustamyotropins were obtained by elution through an affinity column to which Lom-MT II was covalently bound. Specific cells in the nervous system of Locusta migratoria contain substances immunoreactive to anti-locustamyotropin. In total, about 100 cells immunoreactive to the Lom-MT-II antiserum were detected in the head ganglia, in the abdominal neuromeres of the metathoracic ganglion, and in the five free abdominal ganglia. In the brain, immunoreactive cell groups were situated in the inner and outer edge of the tritocerebrum. Prominent axon bundles tightly surround the tractus I to the corpora cardiaca. The corpora allata were innervated by the nervus corporis allati I coming from the corpora cardiaca and by fibers in the nervus corporis allati II originating from cell bodies in the suboesophageal ganglion. Immunoreactive cell bodies in the suboesophageal and abdominal ganglia are distributed along the anterior posterior midline axis, both dorsally and ventrally. The processes of the cell bodies in the abdominal ganglia leave the ganglia and were traced in the respective median nerves into the neurohaemal organs. Since the Lom-MT-II antiserum cross-reacts with all peptides of the locustamyotropin family that have a carboxy-terminus in common, these cells may contain one or several locustamyotropins. The Lom-MT antiserum also recognizes pheromone biosynthesis activating neurohormone, as was revealed by the intensive labeling of suboesophageal cell bodies in Bombyx mori.     

Temperature-dependent activation of ERK/MAPK in yolk cells and its role in embryonic diapause termination in the silkworm Bombyx mori

The silkworm Bombyx mori requires 2-3 months of low temperature (5 degrees C) to terminate embryonic diapause. The molecular mechanisms, however, are unknown. Extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) is temperature-dependently activated in the yolk cells of diapausing eggs after 45 days at 5 degrees C, coincident with the acquisition of developmental competence of the embryos at 25 degrees C. Yolk cell granulation and dissociation also begin in diapause eggs incubated at 5 degrees C for 45 days. We used dechorionated egg culture as a model system of diapause termination and observed that both yolk cell dissociation and embryonic development are inhibited by MAPK-ERK kinase (MEK) inhibitor U0126. Therefore, we suggest that ERK in yolk cells has a role in regulating changes in yolk morphology and termination of embryonic diapause in B. mori.     

家蚕滞育激素-性信息素合成激活肽基因表达的调节

滞育激素和性信息素合成激活肽是两个重要的昆虫神经肽,这两个神经肽由一个基因编码．利用分子杂交和ＲＴ－ＰＣＲ技术,确定了滞育激素－性信息素合成激活肽基因表达的调节不属于转录后的调节,推定为翻译后形成一个大的前体多肽再剪接为几个成熟的神经肽分子．