Co-ordination of firing activity of neurosecretory cells with cardiac activity in the silkmoth Bombyx mori

Electrocardiograms and electrical action potentials of cerebral neurosecretory cells producing bombyxin (an insulin-related neuropeptide) were simultaneously recorded from male pupae of the silkmoth Bombyx mori. A pupa showed alternations in the flow of haemolymph due to a rhythmic heartbeat reversal: a train of retrograde heartbeat with a slow pulse rate followed a train of anterograde heartbeat with a higher pulse rate. Intervals of heartbeat reversals changed throughout the pupal period. At any stage of the pupal period, firing activity of a population of bombyxin-producing (BP) cells rapidly declined after the start of anterograde heartbeat and an inactive state of cells continued during an anterograde heartbeat period. Analyses of ultradian bursting rhythmicity of a single BP cell revealed that a bursting phase of the cell often delayed at a time when the anterograde cardiac activity occurred at the preceding inter-burst period of firing rhythm. The results support the postulation that firing (secretory) activity of an insect neurosecretory cell system may be co-ordinated with circulation of haemolymph for rapid and pulsatile delivery of the peptides released to target organs.     

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.     

Firing activity of "diapause hormone" producing cells in the male silkmoth,Bombyx mori

Diapause hormone (DH) originally identified to be a factor originating from neurosecretory cells in the suboesophageal ganglion acts on developing ovaries to produce diapause eggs in a female silkmoth, Bombyx mori. A male silkmoth has homologous neurosecretory cells, but little is known of the physiological nature of the cells and actions of their products. We examined the long-term firing activity of putative DH-producing neurosecretory cells and hormonal activity of their products in male pupae that had been experienced different environmental regimens for diapause induction. Firing activity patterns of male labial cells strongly depended on diapause types of pupae: cells in a diapause-type male were active throughout the pupal period, whereas the same cells in a non-diapause-type male were usually inactive during the early two-thirds of the pupal period. A male pupa with electrically active labial cells could induce diapause eggs in a female pupa connected parabiotically to that male. The firing activity of male neurosecretory cells and hormonal action of their products are qualitatively the same as in the female previously examined. We suggest that there is no evident sexual dimorphism in the physiological and biochemical nature of neurosecretory cells producing DH and the amidated peptide DH has different functions in a male.     

Post-eclosion diuresis in a flightless insect, the silkmoth Bombyx mori

Abstract. Although the silkmoth, Bombyx mori L., has lost the ability to fly, it has retained a post-eclosion diuresis. In moths removed from their cocoons before eclosion, or in those which failed to spin cocoons as larvae, the weight loss due to diuresis was 14% of the eclosion body weight in males. Moths which used labial fluid to escape from their cocoons showed a correspondingly smaller diuresis (5%). Both urine and labial fluid had high potassium and low sodium concentrations. Unlike post-eclosion diuresis in butterflies, however, the urine was isosmotic to the haemolymph. In vitro preparations of B.mori Malpighian tubules were stimulated by cyclic AMP, B.mori brain extracts and Manduca sexta diuretic peptide (Mas-DP I).     

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.     

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.     

Prostaglandin signaling and ovarian follicle development in the silkmoth, Bombyx mori

Previous work on in vitro culturing of silkmoth (Bombyx mori) ovarian follicles has shown that starting from middle vitellogenesis, follicles develop according to an endogenous developmental program that does not require the presence of extra-ovarian factors. In this paper, we are reporting on our investigation for a possible involvement of autocrine/paracrine signaling by prostaglandins in the control of silkmoth ovarian follicle development. Using an initial rapid test that evaluates the formation of a protective eggshell around the oocyte, we are showing that aspirin and indomethacin, potent inhibitors of prostaglandin biosynthesis, block the transition of cultured vitellogenic follicles into choriogenesis. More detailed studies involving analyses of temporal expression patterns of genes known to be expressed in follicular epithelium cells at specific stages of ovarian development revealed that inhibition of prostaglandin biosynthesis arrests stages of follicle development from middle vitellogenesis to late choriogenesis. The arrest could be reversed by the addition of exogenous prostaglandins or cAMP into the culture media leading to the conclusion that the production of prostaglandins triggers cAMP-mediated intracellular signaling that allows the developmental progression of the follicles. Finally, because neither prostaglandins nor cAMP is capable of rescuing a developmental block effected at mid-vitellogenesis by the ecdysone agonist tebufenozide, we are proposing that prostaglandins have a role in the maintenance of normal physiological homeostasis in the ovarian follicles rather than a more specific role in developmental decision-making at distinct stages of follicle development.     

EcR expression in the prothoracicotropic hormone-producing neurosecretory cells of the Bombyx mori brain

The steroid hormone 20-hydroxyecdysone (20E) initiates insect molting and metamorphosis through binding with a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and ultraspiracle (USP). Expression of the specific isoforms EcR-A and EcR-B1 governs steroid-induced responses in the developing cells of the silkworm Bombyx mori. Here, analysis of EcR-A and EcR-B1 expression during larval-pupal development showed that both genes were up-regulated by 20E in the B. mori brain. Whole-mount in situ hybridization and immunohistochemistry revealed that EcR-A and EcR-B1 mRNAs and proteins were exclusively located in two pairs of lateral neurosecretory cells in the larval brain known as the prothoracicotropic hormone (PTTH)- producing cells (PTPCs). In the pupal brain, EcR-A and EcR-B1 expression was detected in tritocerebral cells and optic lobe cells in addition to PTPCs. As PTTH controls ecdysone secretion by the prothoracic gland, these results indicate that 20E-responsive PTPCs are the master cells of insect metamorphosis.     

Effect of diazinon exposure on antioxidant reactions in the silkmoth,Bombyx mori

We investigated the effects of exposure to diazinon, an organophosphate insecticide, on the expression of antioxidant and detoxification factors in the silkmoth. Exposure to diazinon resulted in induction of mRNA encoding manganese containing superoxide dismutase (SOD) and omega‐class glutathione S‐transferases (GST), whereas no changes were observed in catalase, other class of GST and acetylcholinesterase. Liquid chromatography showed that the amount of reactive oxygen species was increased, whereas the amount of glutathione was decreased in the silkmoth fat body after exposure to diazinon. These results suggest that SOD and omega‐class GSTs can contribute to organophosphate resistance in Lepidopterans.     

Mitochondrial genome nucleotide substitution pattern between domesticated silkmoth, Bombyx mori, and its wild ancestors, Chinese Bombyx mandarina and Japanese Bombyx mandarina

Bombyx mori and Bombyx mandarina are morphologically and physiologically similar. In this study, we compared the nucleotide variations in the complete mitochondrial (mt) genomes between the domesticated silkmoth, B. mori, and its wild ancestors, Chinese B. mandarina (ChBm) and Japanese B. mandarina (JaBm). The sequence divergence and transition mutation ratio between B. mori and ChBm are significantly smaller than those observed between B. mori and JaBm. The preference of transition by DNA strands between B. mori and ChBm is consistent with that between B. mori and JaBm, however, the regional variation in nucleotide substitution rate shows a different feature. These results suggest that the ChBm mt genome is not undergoing the same evolutionary process as JaBm, providing evidence for selection on mtDNA. Moreover, investigation of the nucleotide sequence divergence in the A+T-rich region of Bombyx mt genomes also provides evidence for the assumption that the A+T-rich region might not be the fastest evolving region of the mtDNA of insects.     

Pheromone-producing cells in the silkmoth, Bombyx mori: identification and their morphological changes in response to pheromonotropic stimuli

A method to isolate functional clusters of viable pheromone gland cells of Bombyx mori was developed. The 8th-9th intersegmental invaginated membrane corresponding to the pheromone gland was dissected, trimmed and separated into two distinct layers, the outer and inner layers, by enzymatic digestion with papain. The outer layer mainly consists of cuticle, while the inner layer consists of homogeneous cells with many refractile granules. The solubilized microsome fraction prepared from the inner layer retained the ability to produce bombykol in vitro, whereas the outer layer fraction did not produce bombykol. Moreover, in tissue incubations, the inner layer - but not the outer layer - produced bombykol in response to the pheromonotropic peptide TKYFSPRLamide, ionomycin and calcium ionophore A23187. These results indicate that the inner-layer cells are indeed the pheromone-producing cells, which retain their functional integrity after separation with papain. These cells could be cultured successfully in Grace's medium for at least 5days.The presence or absence of pheromonotropic stimuli prior to dissection greatly influenced the size, number and distribution of refractile granules in the cytoplasm of the pheromone-producing cells. Staining with Nile Red proved that these refractile granules were lipid droplets. When pheromone production was studied under normal conditions or stimulated in decapitated females with pheromone-biosynthesis-activating neuorpeptide (PBAN) charge, the size of lipid droplets observed in the pheromone-producing cells reduced prominently and their number increased dramatically with time. By contrast, when pheromone production was suppressed by decapitation, the size and number of the lipid droplets remained constant. Lipid droplets observed in the pheromone-producing cells could be carriers of pheromone precursors and/or the pheromone bombykol. The present results suggest that the isolated cell preparation can be used for quantitative visualization of the cellular dynamics during pheromone production in B. mori.     

Fuel mobilization for energy metabolism in the silkmoth, Bombyx mori, during reproductive processes

Flight muscles of male moth, B. mori seem to utilize carbohydrate preferentially as a source of energy for all its acrobatic movements during the search for female moth. Depletion of triacylglycerol from flight muscles without affecting its level from fat body suggests that this lipid fraction serves as a source of energy in flight muscles during insemination processes. Significant depletion of triacylglycerol and glycogen from flight muscles of female moth after egg laying indicates that they are used to meet the energy requirement of female during oviposition activity. Depletion of proteins from flight muscles of male and female insects suggest that these proteins are transported to the accessory reproductive glands to meet their protein demand.     

BmVMP90, a large vitelline membrane protein of the domesticated silkmoth Bombyx mori, is an essential component of the developing ovarian follicle

We present the characterization of BmVMP90, a vitelline membrane protein (VMP) of the silkmoth Bombyx mori bearing similarities with dipteran VMPs whose existence had recently been suggested by an in silico analysis of the silkmoth genome and follicular cell RNA expression analyses. Using a specific antibody, we determine the presence of BmVMP90 protein in ovarian follicular cell extracts at the end of vitellogenesis and in vitelline membrane extracts but not in the chorion of fractionated eggshells isolated from ovulated follicles. Whole mount follicle immunofluorescence studies reveal a pattern of BmVMP90 deposition matching the ?imprinted? pattern of follicular cells on the vitelline membrane surface. Antisense DNA-directed inhibition BmVMP90 expression in ex?vivo cultures of early vitellogenic follicles produced a phenotype of kidney- or bean-shaped follicles with detached follicular epithelia, suggestive of the importance of BmVMP90 for the integrity of developing follicles and normal deposition of the chorion structure that follows vitelline membrane formation but no adverse effects on the execution of the follicular cell-imprinted program of choriogenesis per se.     

Lesion studies of neural mechanisms underlying the mating dance of the silkmoth,Bombyx mori

Lesion studies were carried out to reveal neural mechanisms controlling a characteristic mating behavior, referred to as ‘mating dance’ in a maleBombyx mori which appears in reaction to the sex pheromone of the conspecific female. The experiments revealed 2 essential neural mechanisms involved in the mating dance. One is a flight motor system in the thorax, which organizes the wing vibration representing the mating dance. The other, a dance command element, is in the head ganglia. Once activated by the female sex pheromone, the latter can maintain its excitation for several minutes without additional stimulation. This long-lasting neuronal excitation was suggested to descend via cervical connectives to the thoracic ganglia and activate the flight motor system into operation.     

Adenine compounds in the male reproductive tract and the spermatophore of the silkmoth, Bombyx mori.

1. Studies were made on the levels of six adenine compounds in various glands of the male reproductive tract before and after ejaculation and their changes with time in the spermatophore of the silkworm, Bombyx mori. 2. The ductus (d.) deferens, ampulla d. deferentis and vesicular seminalis contain high concentrations of cAMP. The total amount of cAMP transferred from the male reproductive tract corresponds to its content in the spermatophore, and is sufficient to induce motility of apyrene spermatozoa. 3. The ATP content of the g. pellucida is much higher than those of the two other adenine nucleotides, indicating a high energy charge. But, in the other male glands, the AMP content is very much higher than that of ATP, indicating low energy charges.     

Odorant-binding proteins and chemosensory proteins in pheromone detection and release in the silkmoth Bombyx mori

The genome of the silkmoth Bombyx mori contains 44 genes encoding odorant-binding proteins (OBPs) and 20 encoding chemosensory proteins (CSPs). In this work, we used a proteomic approach to investigate the expression of proteins of both classes in the antennae of adults and in the female pheromone glands. The most abundant proteins found in the antennae were the 4 OBPs (PBP, GOBP1, GOBP2, and ABP) and the 2 CSPs (CSP1 and CSP2) previously identified and characterized. In addition, we could detect only 3 additional OBPs and 2 CSPs, with clearly different patterns of expression between the sexes. Particularly interesting, on the other hand, is the relatively large number of binding proteins (1 OBP and 7 CSPs) expressed in the female pheromone glands, some of them not present in the antennae. In the glands, these proteins could be likely involved in the solubilization of pheromonal components and their delivery in the environment.