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.     

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.     

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.     

Neuroendocrine regulation of seasonal morph development in a bivoltine race (Daizo) of the silkmoth, Bombyx mori L

We investigated the neuroendocrine regulation of the development of seasonal morphs in a bivoltine race (Daizo) of the silkmoth, Bombyx mori, by decerebration, the transplantation of brain-suboesophageal ganglion (Br-SG) complexes and the injection of active neuropeptides. When brains were removed from fresh pupae destined to develop into summer morphs (SD pupae) by embryonic and larval exposures to short days at low temperature, the pupae developed into autumn or intermediate morphs. However, in pupae destined to develop into autumn morphs (LD pupae), the operation did not show an effect on seasonal morph development. Br-SG complexes were excised from fifth-instar LD and fifth-instar SD larvae 2 days after larval ecdysis and were transplanted into the abdomen of SD larvae of the same age. The Br-SG complexes of LD larvae, but not the Br-SG complexes of SD larvae, shifted the host's seasonal morph development toward the autumn morph. Furthermore, when treated with crude pupal SGs extract and diapause hormone (DH), fresh SD pupae developed into autumn or intermediate morphs, respectively. Possibly the development of seasonal morphs in the silkmoth, B. mori, is regulated by a novel function of DH. Alternatively, DH may act on the imaginal wing disks at an earlier stage than on the ovaries.     

家蚕滞育的分子机理2.蛹期滞育激素基因的表达与滞育决定

滞育激素是由食道下神经节分泌的重要昆虫神经肽,诱导昆虫的滞育。选择具有ＲＮＡ聚合酶能够识别的启动子的质粒载体,将滞育激素ｃＤＮＡ克隆进去,在体外大量合成单一的滞育激素ｃＲＮＡ为参照,测定食道下神经节分泌滞育激素ｍＲＮＡ量来确定滞育激素的分泌量。结果证明食道下神经节分泌的滞育激素的数量决定昆虫的滞育。     

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．     

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.     

Molecular mechanism of diapause in insect (I)

The embryonic diapause of the silkworn,Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expreon is a direct response to the environmend stimulus, such as high incubation temperature. The cDNA from the embryonic stage wa cloned and sequenm analysis showed the cDNA encoding DH. Expmion patterns of the DH gene in embryonic stage are different ar incubation temperatures 15°C and 25°C, suggesting that the incubation tempcreturt as an environmental signal is kept within the body to control the DH gene expmion at the pupal stage, so that the embryonic diapause of next generation can be determined.     

Molecular mechanism of diapause in insect (I) --Expression of diapause hormone gene and incubation temperature in embryonic stage of Bombyx mori

The embryonic diapause of the silkworm, Bombyx mori, is induced by the diapause hormone (DH) which is secreted from the suboesophageal ganglion of pupae. The diapause nature of bivoltine strains uses environmental stimuli as the initial signal to determine the diapause nature. The experiments showed that DH gene expression is a direct response to the environmental stimulus, such as high incubation temperature. The cDNA from the embryonic stage was cloned and sequence analysis showed the cDNA encoding DH. Expression patterns of the DH gene in embryonic stage are different at incubation temperatures 15℃ and 25℃, suggesting that the incubation temperature as an environmental signal is kept within the body to control the DH gene expression at the pupal stage, so that the embryonic diapause of next generation can be determined.     

家蚕滞育激素－性信息素合成激活肽基因的表达

家蚕滞育激素－性信息素合成激活肽基因的表达徐卫华（中国农业科学院蚕业研究所,江苏镇江,２１２０００）山下兴亚（名古屋大学农学院,日本名古屋,４６４－０１）关键词滞育激素－性信息素合成激活肽基因;发育阶段;表达;家蚕昆虫是地球上最繁盛的物种,占地球上生...     

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.     

cDNA CLONING AND SEQUENCE DETERMINATION OF THE PHEROMONE BIOSYNTHESIS ACTIVATING NEUROPEPTIDE FROM THE SEABUCKTHORN CARPENTERWORM,Holcocerus hippophaecolus (LEPIDOPTERA: COSSIDAE)

The PBAN (pheromone biosynthesis activating neuropeptide)/pyrokinin peptides comprise a major neuropeptide family characterized by a common FXPRL amide at the C‐terminus. These peptides are actively involved in many essential endocrine functions. For the first time, we reported the cDNA cloning and sequence determination of the PBAN from the seabuckthorn carpenterworm, Holcocerus hippophaecolus, by using rapid amplification of cDNA ends. The full‐length cDNA of Hh‐DH‐PBAN contained five peptides: diapause hormone (DH) homolog, α‐neuropeptide (NP), β‐NP, PBAN, and γ‐NP. All of the peptides were amidated at their C‐terminus and shared a conserved motif, FXPR (or K) L. Moreover, Hh‐DH‐PBAN had high homology to the other members of the PBAN peptide family: 56% with Manduca sexta, 66% with Bombyx mori, 77% with Helicoverpa zea, and 47% with Plutella xylostella. Phylogenetic analysis revealed that Hh‐DH‐PBAN was closely related to PBANs from Noctuidae, demonstrated by the relatively higher similarity compared with H. zea. In addition, real‐time quantitative PCR (qRT‐PCR) analysis showed that Hh‐DH‐PBAN mRNA expression peaked in the brain–subesophageal ganglion (Br–SOG) complex, and was also detected at high levels during larval and adult stages. The expression decreased significantly after pupation. These results provided information concerning molecular structure characteristics of Hh‐DH‐PBAN, whose expression profile suggested that the Hh‐DH‐PBAN gene might be correlated with larval development and sex pheromone biosynthesis in females of the H. hippophaecolus.     

Two nitridergic peptides are encoded by the gene capability in Drosophila melanogaster

A Drosophila gene (capability, capa) at 99D on chromosome 3R potentially encodes three neuropeptides: GANMGLYAFPRV-amide (capa-1), ASGLVAFPRV-amide (capa-2), and TGPSASSGLWGPRL-amide (capa-3). Capa-1 and capa-2 are related to the lepidopteran hormone cardioacceleratory peptide 2b, while capa-3 is a novel member of the pheromone biosynthesis-activating neuropeptide/diapause hormone/pyrokinin family. By immunocytochemistry, we identified four pairs of neuroendocrine cells likely to release the capa peptides into the hemolymph: one pair in the subesophageal ganglion and the other three in the abdominal neuromeres. In the Malpighian (renal) tubule, capa-1 and capa-2 increase fluid secretion rates, stimulate nitric oxide production, and elevate intracellular Ca(2+) and cGMP in principal cells. Capa-stimulated fluid secretion, but not intracellular Ca(2+) concentration rise, is inhibited by the guanylate cyclase inhibitor methylene blue. The actions of capa-1 and capa-2 are not synergistic, implying that both act on the same pathways in tubules. The capa gene is thus the first to be shown to encode neuropeptides that act on renal fluid production through nitric oxide.     

Molecular cloning,developmental expression and tissue distribution of diapause hormone and pheromone biosynthesis activating neuropeptide in the bamboo borer Omphisa fuscidentalis

Diapause, an arrested period of post‐embryonic development in insects, is under the control of hormonal interactions. In the bamboo borer Omphisa fuscidentalis Hampson (Lepidoptera: Crambidae), larvae remain in diapause for as long as 9 months during the dry season, from September to the following June, although the factors that regulate larval diapause are poorly understood. The present study describes the cloning and expression analysis of the diapause hormone and pheromone biosynthesis activating neuropeptide (DH‐PBAN) precursor of O. fuscidentalis (Ompfu‐DH‐PBAN cDNA), aiming to reveal how it may be involved regulating larval diapause in this species in combination with environmental factors. The open reading frame (ORF) of the cDNA encodes a 199‐amino acid precursor protein that contains DH, PBAN and three other neuropeptides, all of which share a conservative C‐terminal pentapeptide motif FXPR/KL (X = G, T or S). The Ompfu‐DH‐PBAN is highly similar (74%) to the DH‐PBAN of the legume pod borer (Maruca vitrata). A quantitative real‐time polymerase chain reaction reveals that Ompfu‐DH‐PBAN mRNA is expressed only in neural tissues and that expression is highest in the suboesophageal ganglion. In addition, the expression level of Ompfu‐DH‐PBAN mRNA in the suboesophageal ganglion is consistently high during the fifth larval instar, increasing moderately in early diapause before reaching a peak during late diapause. After pupation, expression of the Ompfu‐DH‐PBAN precursor decreases to a low level. In addition to endocrine factors, the results demonstrate that photoperiod increases the expression level of Ompfu‐DH‐PBAN mRNA in larval diapause. These results also suggest that the expression of the Ompfu‐DH‐PBAN gene correlates with larval diapause development and may be activated by photoperiod in O. fuscidentalis.     

Functional analysis of the SGNP I in the pupal diapause of the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae)

Helicoverpa assulta suboesophageal ganglion neuropeptide I (Has-SGNP I) is a 24-amino acids peptide amide, which shows 62.5% similarity with the diapause hormone of Bombyx mori (Bom-DH). It has been demonstrated that embryonic diapause is induced by DH in B. mori. Injection of synthetic amidated Has-SGNP I terminated pupal diapause in a dose-dependent manner. Therefore, Has-SGNP I might be referred to a "diapause termination hormone" in H. assulta (Has-DTH). The maximal dose of Has-DTH for diapause termination was 1.0 microg and the half-maximal dose 0.4 microg. The time required for diapause termination of Has-DTH was 2-3 days longer than that of 20-hydroxyecdysone. During the pupal stage, DTH mRNA content in the SGs of nondiapausing pupae was always higher than in diapausing pupae using the combined method of quantitative RT-PCR and Southern blot. DTH gene also expressed at a low level while diapausing pupae were chilled at 4 degrees C, but increased rapidly and largely after being transferred to 25 degrees C. Using a competitive ELISA, Has-DTH-like immunoreactivity in the haemolymph showed the same pattern as that of Has-DTH gene expression. Those results indicated that Has-DTH gene expression was related to diapause development and could be activated by low temperature. Has-DTH might be useful to elucidate the mechanism of diapause termination in pupal diapause species.     

Neuroendocrine control of diapause hormone secretion in the silkworm,Bombyx mori

To clarify the control mechanism of diapause hormone (DH) secretion in the silkworm Bombyx mori a series of anatomical and pharmacological experiments were carried out. The arrangement of 'diapause' and 'non-diapause' eggs in the ovarioles of the moths was determined by the coloration method to estimate the accumulation of 3-hydroxykynurenine in the eggs. The females destined to lay non-diapause eggs (non-diapause producers) had diapause eggs in their ovaries if their subesophageal ganglions (Sg) had been surgically removed at 2days after larval-pupal ecdysis or later. In contrast when the surgical extirpation extended to the brain and the corpora cardiaca (CC)-corpora allata (CA) complex in addition to the Sg, the non-diapause producers had no diapause eggs. When the Sg was removed from the females destined to lay diapause eggs (diapause-producers), diapause eggs appeared in response to the treatment at 2days after larval-pupal ecdysis, but the appearance of diapause eggs was delayed by 2days when the brain-CC-CA complex was included among the organs removed. These observations suggested that DH is produced in Sg and transferred to the CC-CA complex, and that the secretion of DH from the complex is suppressed in non-diapause producers. The pattern of diapause and non-diapause eggs induced by the transection of the subesophageal connective in diapause and non-diapause producers suggested a regenerative and secretory capacity of the neurosecretory cells after the operation. The appearance of diapause eggs in non-diapause producers with transected protocerebrum of the brain confirmed that there was an inhibitory center in the protocerebrum. Changes in parts of the ovarioles containing diapause and non-diapause eggs with time of injection of gamma-aminobutyric acid (GABA) and picrotoxin suggested that a GABAergic inhibitory mechanism in DH secretion may be active in non-diapause producers but inactive in diapause producers throughout the pupal stage.     

Isolation of the diapause hormone from the silkworm,Bombyx mori

The diapause hormone (DH) secreted from the suboesophageal ganglion is responsible for the embryonic diapause in the silkworm, Bombyx mori. It was extracted from two million mated male adult heads. At least two species of DH were separated from the extracts (a complex lipid fraction) by gel permeation chromatography with Sephadex LH-20 by organic eluants. One of them was further purified by repeating gel permeation column chromatography with Merckogel^® Type OR 6000 in the cold and dark to give finally a single peak with high DH activity. This preparation seems to be chemically pure, and its molecular weight is chromatographically estimated to be between 2000 and 4000. Other characteristics of DH are also presented. Biological activity of the final DH preparation is discussed with reference to other insect hormones such as juvenile hormone and α-ecdysone.     

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.     

Diapausing Colorado potato beetles are devoid of short neuropeptide F I and II

An extract of head ganglia and retrocerebral complexes of nondiapausing and diapausing Leptinotarsa decemlineata was prepared to characterize regulatory neuropeptides involved in adult diapause by using a differential peptidomics approach. To reduce sample complexity, both extracts were roughly separated by means of an identical chromatographic step. MALDI-TOF MS led to the identification of proctolin, an adipokinetic hormone, and short neuropeptide F I and II in the extract of nondiapausing beetles. In combination with nano-ESI-Q-TOF MS(2) evidence was found for the presence of three pyrokinins, the first to be identified in a coleopteran species. Pyrokinins, involved in the induction of embryonic diapause in Bombyx mori, were present in both physiological conditions suggesting that they are of minor importance in the regulation of adult diapause in the Colorado potato beetle. A striking difference, detected by the differential peptidomics approach, between both neuropeptide profiles was the absence of ions corresponding to the short neuropeptide F (sNPF) related peptides, also known as Led-NPF-I and -II, in the extract of diapausing animals. Therefore, we postulate that "short NPFs" are involved in the regulation of adult diapause, displayed by the Colorado potato beetle.