Studies on embryonic diapause in thepnd mutant of the silkworm,Bombyx mori

Summary Two-dimensional gel electrophoresis has been used to analyse patterns of proteins synthesized in the eggs from theBombyx mutantpnd, whose homozygous embryo never enters diapause owing to a genetic defect. At the middle to late stage of gastrulation the diapause type of the heterozygous embryo, derived from a homozygouspnd female mated to a wild-type male, synthesizes eight proteins which are not detected in the homozygouspnd embryo. To examine the relationship between embryonic diapause and the appearance of the heterozygote-specific proteins, the pattern of proteins synthesized in the heterozygotes of the diapause type was compared with that in heterozygotes which were artificially altered so that they would continue development. Only one of the eight heterozygote-specific proteins was constitutively synthesized according to the embryonic genome, irrespective of their developmental state, whereas appearance of the remaining seven proteins was exclusively dependent on their developmental nature. This finding strongly suggests that the unique protein might result from the expression of thepnd ⁺ gene, and the other proteins might be synthesized along with diapause initiation in the heterozygotes. The possible role of the putativepnd ⁺ gene-specific protein at the onset of embryonic diapause is discussed in relation to the action of the diapause factor, which predetermines embryonic diapause by affecting the developing oocytes.     

Distinct effects of different low temperatures on the induction of NAD-sorbitol dehydrogenase activity in diapause eggs of the silkworm,Bombyx mori

Summary Diapause eggs of the silkworm, Bombyx mori, exposed to 5°C and 0.5°C from 2 or 30 days after oviposition, were examined for changes in contents of glycogen, sorbitol and glycerol. Cold acclimation did not alter the profile of accumulation of sorbitol from that in eggs kept continuously at 25°C. However, acclimation at 5°C resulted in conversion of sorbitol to glycogen, while acclimation at 0.5°C was not accompanied by the utilization of sorbitol. NAD-sorbitol dehydrogenase (NAD-SDH; EC 1.1.1.14) activity was examined in the cold-acclimated eggs. The activity was induced by acclimation at 5°C but not at 0.5°C. Incubation at 0.5°C suppressed any further increase in the activity that had been induced. Temperature-directed changes in NAD-SDH activity paralleled those in sorbitol content. Hatching of the diapause eggs was monitored after cold acclimation for various periods of time and subsequent transfer to 25°C. Incubation at 0.5°C was less effective than 5°C at breaking diapause. The time required for the eggs to hatch in synchrony after acclimation at 5°C coincided with that required for the induction of NAD-SDH activity. These results show that different effects result from acclimation at 5°C and near 0°C with respect to the control of NAD-SDH activity, that utilization of sorbitol is controlled by NAD-SDH activity, and that induction of this activity is temperature-dependent. Furthermore, induction of NAD-SDH activity is involved in the termination of diapause in B. mori.Abbreviations DH diapause hormone - NAD nicotinamide-adenine-dinucleotide - NAD-SDH NAD-sorbitol-dehydrogenase     

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.     

Studies on the utilization of single cell protein by the silkworm Bombyx mori

Final-instar larvae of Bombyx mori fed mulberry leaves, supplemented with Spirulina fusiformis (Woronichin) as a source of single cell protein (SCP), required 6 days to attain a maximum larval weight of 2090 mg; control group larvae needed 9 days to attain a final larval weight of 1470 mg. Quantity of feeding, assimilation and conversion efficiencies increased substantially in the SCP-fed group. Significant improvements in the economic characters such as cocoon, pupal, and shell weights were obtained in the SCP supplemented larvae in comparison to the normal leaf fed larvae. About 15% of the labelled S. fusiformis was directly incorporated into larval tissue. Presence of SCP in the gut facilitated better conversion of consumed leaf protein.

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Etudes sur l'utilisation des protéines de cellules isolées par le ver à soie, Bombyx mori

Résumé Des chenilles du dernier stade de Bombyx mori, alimentées sur mûrier additionné de Spirulina fusiformis comme source de protéine de cellule isolée (SCP), atteignent en 6 jours le poids larvaire maximum de 2090 mg; les chenilles témoins consommaient pendant 9 jours pour obtenir leur poids larvaire final de 1470 mg. Les quantités consommées, les coefficients d'assimilation et de conversion ont augmenté substantiellement chez les chenilles avec SCP. Des augmentations significatives de critères économiques, comme les poids de cocon, de nymphe et de cogul, ont été observées avec l'addition de SCP par rapport aux témoins. Environ 15% du S. fusiformis marqué a été incorporé directement dans les tissus larvaires. La présence de SCP dans l'intestin a permis une meilleure conversion des protéines foliaires consommées.

     

Gene expression profiling between embryonic and larval stages of the silkworm, Bombyx mori

To elucidate the molecular mechanisms associated with metamorphic phenomenon relating to Bombyx mori, an important organism in the sericulture industry, we identified genes that are expressed in the different developmental stages, specifically the embryonic (ES) and larval (LS) stages of B. mori. Of 8230 high-quality ESTs from two full-length enriched cDNA libraries, 3442 of the ES ESTs were coalesced into 1325 clusters, while 4788 were coalesced into 927 clusters. The functional classification of these ESTs based on Gene Ontology showed that the types of genes that are associated with oxidoreductase activity, enzyme inhibition, and larval development were highly observed in LS, whereas the types of genes that are involved in nucleotide binding, enzyme activity, and protein transport activity were highly observed in ES. In addition, when the gene expression profile between ES and LS was examined by counting the EST frequencies in each library, 69 genes were identified as being either up- or down-regulated in the larval stage compared to the embryonic stage (P>0.99) and this was confirmed by semi-quantitative RT-PCR. The results show that genes involved in proteolysis and peptidolysis, and lipid and carbohydrate metabolism were dramatically up-regulated in LS, while those related to protein metabolism, DNA/RNA, and coenzymes were highly down-expressed. In particular, a GO analysis of these genes revealed that genes that are involved in hydrolase activity were observed to be highly expressed in amount as well as diversity in LS, while those involved in nucleic acid binding were highly expressed in ES. These data may contribute to elucidating genetic events that distinguish the developmental stage and to our understanding of the metamorphosis of B. mori.     

Cell cycles in embryos of the silkworm,Bombyx mori: G2-arrest at diapause stage

Summary In the silkworm, Bombyx mori, diapause occurs at a specific embryonic stage, i.e. after formation of the germ band with cephalic lobes and telson and sequential mesoderm segmentation. As long as the eggs are incubated at 25° C, cell divisions and morphological development of the embryos cease. To examine changes in percentage of embryonic cells in the G₁, S and G₂ phases during embryogenesis, nuclear fractions were isolated from embryos, stained with propidium iodide and then subjected to flow cytometric analysis. The percentages of embryonic cells in G₁, S and G₂ were 10, 35 and 55%, respectively, at the stage of formation of cephalic lobes, whilst 98% of cells were in G₂ at diapause stage. After termination of diapause by acclimation at 5° C or by a combination of chilling and HCl, cell division resumed in the embryos. During this period, the cells rapidly entered S phase through G₁ from G₂, suggesting that their G₁ phase was short. In eggs in which diapause was averted by HCl-treatment after incubation at 25° C for 20 h after oviposition, embryonic development proceeded continuously for 9.5 days at 25° C until hatching. Along with this development, the G₁ fraction increased to levels of about 90%. These results indicate that embryonic cells are arrested in G₂ at diapause and suggest that, concomitant with further embryonic development, cell cycles become slower in proportion to an increasing length of G₁. Finally, most of the cells may be arrested in G₁, while there is only a small fraction of cells continuously cycling. Offprint requests to: T. Yaginuma     

G protein-coupled receptor for diapause hormone, an inducer of Bombyx embryonic diapause

Bombyx diapause hormone was the first chemical substance identified as a maternal control factor that arrests offspring development. However, the molecular mechanisms by which the hormone transduces the signal to the oocyte that induces embryonic diapause immediately after mesoderm segmentation are not fully understood. Here, we describe a cDNA for a G protein-coupled diapause hormone receptor with seven transmembrane domains. Its amino-acid sequence shows a high level of similarity to the receptors of mammalian neuromedin U and insect regulatory peptide, an FXPRL-amide C-terminus. When expressed in a Xenopus oocyte system, the receptor exhibited the highest affinity (EC(50), approximately 70nM) for diapause hormone, when compared with other Bombyx FXPR/KL-amide peptides. Diapause hormone without amidation at the C-terminus, which never induces embryonic diapause in vivo, had no effect in this heterologous expression system. The mRNA is expressed in the ovaries during Bombyx pupal-adult development. These results strongly indicate that the cDNA encodes the diapause hormone receptor.     

Identification and characterization of piggyBac-like elements in the genome of domesticated silkworm, Bombyx mori

piggyBac is a short inverted terminal repeat (ITR) transposable element originally discovered in Trichoplusia ni. It is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in insects and mammals. Many piggyBac-like sequences have been found in the genomes of phylogenetically species from fungi to mammals. We have identified 98 piggyBac-like sequences (BmPBLE1-98) from the genome data of domesticated silkworm (Bombyx mori) and 17 fragments from expressed sequence tags (ESTs). Most of the BmPBLE1-98 probably exist as fossils. A total of 21 BmPBLEs are flanked by ITRs and TTAA host dinucleotides, of which 5 contain a single ORF, implying that they may still be active. Interestingly, 16 BmPBLEs have CAC/GTG not CCC/GGG as the characteristic residues of ITRs, which is a surprising phenomenon first observed in the piggyBac families. Phylogenetic analysis indicates that many BmPBLEs have a close relation to mammals, especially to Homo sapiens, only a few being grouped with the T. ni piggyBac element. In addition, horizontal transfer was probably involved in the evolution of the piggyBac-like elements between B. mori and Daphnia pulicaria. The analysis of the BmPBLEs will contribute to our understanding of the characteristic of the piggyBac family and application of piggyBac in a wide range of insect species.     

Expression of metabolic enzyme genes and heat-shock protein genes during embryonic development in diapause and non-diapause egg of multivoltine silkworm <Emphasis Type="Italic">Bombyx mori</Emphasis>

The expression of metabolic enzyme genes and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm Bombyx mori was quantified by semi-quantitative RT-PCR. The trehalase gene (Tre) was expressed in non-diapause eggs up-to nine days, while in diapause eggs was not up regulated. The glycogen phosphorylase gene (GPase) was expressed in non-diapause eggs, whereas in diapause eggs a high level was observed in early stage, but down regulated in later stage. The phosphofructokinase gene (PFK) and sorbitol dehyrogenase-2 gene (SDH-2) expression was fluctuated in non-diapause eggs, whereas in diapause eggs these were expressed only at early stage and not observed in later stage. The glucose-6-phosphate dehydrogenase gene (G6P-DH) in non-diapause eggs was highly expressed during the differentiation phase and decreased in the organogenesis phase. In contrast to this, expression in diapause eggs was of low level during differentiation phase and of high level observed in the organogenesis phase. In the tissues, PFK and SDH-2 were selectively expressed in cuticle and midgut, whereas Tre expression was high in midgut and ovary of larvae incubated at 15°C. The Hsp (20.4, 20.8, 40, 70, and 90) were expressed in both diapause and non-diapause eggs. Their expression was, however, selective in tissues with Hsp20.4 in midgut and ovary, Hsp40 in head, Hsp70 in cuticle and Hsp90 in ovary and head in high amounts at 15°C. These results suggest that the metabolic enzyme genes studied except Hsp play a major role during embryogenesis of diapause and non-diapause silkworm.     

The programming of silk-gland development in Bombyx mori

Summary The cytological development of the silk gland has been studied by light and electron microscopy in silkworms experimentally starved at different periods of the natural feeding stage during the fifth instar. When newly molted animals are not provided with food, no sign of growth is observed. Starvation initiated early during the obligatory feeding period, stops cell growth and development of the organelles involved in protein synthesis and secretion, whereas it induces the appearance of organelles concerned with autolysis. These effects are reversible if starvation is not prolonged beyond two days. Starvation during the facultative feeding period, at the time of massive fibroin production, results in quantitative and qualitative modifications of organelles related to the decrease of fibroin production and the onset of autolysis.Rough endoplasmic reticulum, responsible for fibroin synthesis, forms transitory whorls. Fibroin transport via the Golgi apparatus and secretion of the protein into the gland lumen decrease parallel to fibroin synthesis, so that no fibroin storage can be detected in any organelle. After food deprivation, autophagosomes and secondary lysosomes rapidly develop in the cytoplasm, and if starvation continues portions of the cytoplasm are sequestered and completely destroyed. If animals are refed, fibroin production is resumed and autolysis declines.These ultrastructural alterations of the silk gland during experimental starvation are very similar to those observed during the periods of physiological starvation (molt and cocoon spinning) and generally considered to be under hormonal control. Our results raise the question of the nature of interactions between alimentary and hormonal factors which control silk-gland development.This study was supported by a grant from the Centre National de la Recherche Scientifique (A.T.P., Contract n 1472) and was partly carried out in the Centre de Microscopie Electronique, CMEABG, Université Claude Bernard, Lyon and the Laboratoire d'Histologie, Service de Quantimétrie, Faculté de Médicine, Université Claude Bernard, Lyon     

Carbohydrate metabolism and restricted oxygen supply in the eggs of the silkworm, Bombyx mori

Increased oxygen supply to diapause eggs of the silkworm (O₂-incubation) effectively prevented diapause initiation and induced the same pattern of glycogen, polyol and lactate levels as was observed in normal non-diapause eggs. Sensitivity to oxygen decreased as embryonic development proceeded. After the termination of this sensitive period, accumulation of polyols and lactate followed.Experiments were carried out to test whether changes in the oxygen permeability of the egg membranes are involved in restricting the supply of this gas to eggs at the onset of diapause. Oxygen permeability of the chorion was measured with apparatus especially designed for this purpose. Although the chorion of the diapause egg was less permeable than that of the non-diapause egg, the oxygen permeability of the chorion does not change appreciably during the early developmental stages of the diapause eggs. The changes in rate of water loss through the egg membranes were measured during the early developmental stages of the embryos. The level of water loss decreased gradually as the formation of serosal cuticle proceeded. Moreover, it was observed that the water loss up to the time of formation of serosal cuticle was closely related to the oxygen permeability of the chorion.From these results, we suggest that the formation of the serosal cuticle may be an additional cause of the restricted oxygen supply at the onset of the diapause.     

cDNA cloning and mRNA expression of L-3,4-dihydroxyphenylalanine decarboxylase gene homologue from the silkworm,Bombyx mori

l-3,4-Dihydroxyphenylalanine decarboxylase (DDC) cDNA, from Bombyx mori that contains an open reading frame of 1437 bp encoding 478 amino acids, was cloned and characterized. Expression analyses of B. mori DDC mRNA by Northern and in situ hybridization indicated that expression of silkworm DDC expression is possibly controlled by neuropeptide hormones in tissue- and stage-specific manners.     

Isolation and characterization of sex chromosome rearrangements generating male muscle dystrophy and female abnormal oogenesis in the silkworm, Bombyx mori

In deletion-mapping of W-specific RAPD (W-RAPD) markers and putative female determinant gene (Fem), we used X-ray irradiation to break the translocation-carrying W chromosome (W ^Ze ). We succeeded in obtaining a fragment of the W ^Ze chromosome designated as Ze ^W, having 3 of 12 W-RAPD markers (W-Bonsai, W-Yukemuri-S, W-Yukemuri-L). Inheritance of the Ze ^W fragment by males indicates that it does not include the Fem gene. On the basis of these results, we determined the relative positions of W-Yukemuri-S and W-Yukemuri-L, and we narrowed down the region where Fem gene is located. In addition to the Ze ^W fragment, the Z chromosome was also broken into a large fragment (Z₁) having the + ^sch (1-21.5) and a small fragment (Z₂) having the + ^od (1-49.6). Moreover, a new chromosomal fragment (Ze ^WZ₂) was generated by a fusion event between the Ze ^W and the Z₂ fragments. We analyzed the genetic behavior of the Z₁ fragment and the Ze ^WZ₂ fragment during male (Z/Z₁ Ze ^WZ₂) and female (Z₁ Ze ^WZ₂/W) meiosis using phenotypic markers. It was observed that the Z₁ fragment and the Z or the W chromosomes separate without fail. On the other hand, non-disjunction between the Ze ^WZ₂ fragment and the Z chromosome and also between the Ze ^WZ₂ fragment and the W chromosome occurred. Furthermore, the females (2A: Z/Ze ^WZ₂/W) and males (2A: Z/Z₁) resulting from non-disjunction between the Ze ^WZ₂ fragment and the W chromosome had phenotypic defects: namely, females exhibited abnormal oogenesis and males were flapless due to abnormal indirect flight muscle structure. These results suggest that Z₂ region of the Z chromosome contains dose-sensitive gene(s), which are involved in oogenesis and indirect flight muscle development.     

Identification of the female-determining region of the W chromosome in Bombyx mori

The W chromosome of the silkworm Bombyx mori is devoid of functional genes, except for the putative female-determining gene (Fem). To localize Fem, we investigated the presence of W-specific DNA markers on strains in which an autosomal fragment containing dominant marker genes was attached to the W chromosome. We produced new W-chromosomal fragments from the existing Zebra-W strain (T(W;3)Ze chromosome) by X-irradiation, and then carried out deletion mapping of these and sex-limited yellow cocoon strains (T(W;2)Y-Chu, -Abe and -Ban types) from different Japanese stock centers. Of 12 RAPD markers identified in the normal W chromosomes of most silkworm strains in Japan, the newly irradiated W(B-YL-YS)Ze chromosome contained three, the T(W;2)Y-Chu chromosome contained six, and the T(W;2)Y-Abe and -Ban chromosomes contained only one (W-Rikishi). To investigate the ability of the reduced W-chromosome translocation fragments to form heterochromatin bodies, which are found in nuclei of normal adult female sucking stomachs, we examined cells of the normal type p50 strain and the T(W;2)Y-Chu and -Abe strains. A single sex heterochromatin body was found in nuclei of p50 females, whereas we detected only small sex heterochromatin bodies in the T(W;2)Y-Chu strain and no sex heterochromatin body in the T(W;2)Y-Abe strain. Since adult females of all strains were normal and fertile, we conclude that only extremely limited region, containing the W-Rikishi RAPD sequence of the W chromosome, is required to determine femaleness. Based on a comparison of the normal W-chromosome and 7 translocation and W-deletion strains we present a map of Fem relative to the 12 W-specific RAPD markers.     

Effect of different artificial diets on diapause induction under controlled temperature and photoperiod in the silkworm, Bombyx mori L.

ABSTRACT. Embryonic diapause of the silkworm, Bombyx mori , is generally induced by temperature and photoperiod during the egg stage of the previous generation and not in the larval stage. However, when silkworm larvae are reared on an artificial diet instead of mulberry leaves, their diapause is strongly affected by temperature and photoperiod experienced in the larval stage, with a distinct long-day response for diapause induction. Moreover when larvae which have been reared on artificial diet under long-day condition are fed mulberry leaves even for a short period of time, most of the resultant female adults lay diapause eggs. These results suggest that the photoperiodic response of larvae for diapause induction may be strongly suppressed by some components in mulberry leaves.     

The genome sequence of silkworm, Bombyx mori.

We performed threefold shotgun sequencing of the silkworm (Bombyx mori) genome to obtain a draft sequence and establish a basic resource for comprehensive genome analysis. By using the newly developed RAMEN assembler, the sequence data derived from whole-genome shotgun (WGS) sequencing were assembled into 49,345 scaffolds that span a total length of 514 Mb including gaps and 387 Mb without gaps. Because the genome size of the silkworm is estimated to be 530 Mb, almost 97% of the genome has been organized in scaffolds, of which 75% has been sequenced. By carrying out a BLAST search for 50 characteristic Bombyx genes and 11,202 non-redundant expressed sequence tags (ESTs) in a Bombyx EST database against the WGS sequence data, we evaluated the validity of the sequence for elucidating the majority of silkworm genes. Analysis of the WGS data revealed that the silkworm genome contains many repetitive sequences with an average length of <500 bp. These repetitive sequences appear to have been derived from truncated transposons, which are interspersed at 2.5- to 3-kb intervals throughout the genome. This pattern suggests that silkworm may have an active mechanism that promotes removal of transposons from the genome. We also found evidence for insertions of mitochondrial DNA fragments at 9 sites. A search for Bombyx orthologs to Drosophila genes controlling sex determination in the WGS data revealed 11 Bombyx genes and suggested that the sex-determining systems differ profoundly between the two species.     

Photoperiodic induction of diapause in the silkworm, Bombyx mori: location of the photoreceptor using a chemiluminescent paint

ABSTRACT. To locate the photoreceptor involved in the photoperiodic induction of diapause in Bombyx mori L., covering of larval head with black paint or local illumination using chemiluminescent paint was carried out. A silkworm race showing a response of long-day type during the larval stage was employed. The results demonstrated that the photoreceptor is located in the head but is extraocular. The optical properties of the larval body suggest that during the first and second stadia light is admitted through the translucent clypeus of the head, but during later stadia enters over the entire larval body including the head, and that it reaches the cerebral lobe where a photoreceptor is possibly located.     

Possible involvement of ecdysteroids in embryonic diapause of Locusta migratoria

In the Ibaraki population (Japan) of Locusta migratoria, adult locusts produce diapause eggs under short-day (SD) conditions and non-diapause eggs under long-day (LD) conditions. The identity and titre of ecdysteroids in the ovaries and eggs from LD and SD adult females were investigated by RIA/HPLC. Maternal ecdysteroids accumulated in the developing ovaries represented about 90% polar conjugates, 5% free ecdysteroids and 5% non-hydrolyzable metabolites. Before oviposition the quantity of ecdysteroids reached 29.8±1.85 ng 20-hydroxyecdysone equiv. per mg tissue ovaries from LD females and 13.1±3.55 ng 20E equiv./mg in ovaries from SD females. The sum of RIA-positive materials in newly laid eggs was more than three times higher in non-diapause eggs than in diapause eggs. Ecdysteroids present in egg extracts comprised about 85% polar conjugates, 5% free ecdysteroids and 10% non-hydrolyzable metabolites. On the other hand, after diapause termination the amount of ecdysteroids increased drastically. Also, the composition of ecdysteroids differed from that observed during diapause and became comparable to that of non-diapause eggs. The significant differences in the ecdysteroids between non-diapause and diapause eggs may suggest the possible involvement of these compounds in the control of embryonic diapause of this locust.