Proteomic analysis of the silkworm (Bombyx mori L.) hemolymph during developmental stage

We utilized the proteomic approach to investigate the proteome of the fifth instar hemolymph during growth and development, and to improve the understanding of this important bioprocess and gene expression situation. A total of 25 microL of hemolymph was used for 2D analysis, and the separated proteins were visualized by silver staining and analyzed using the ImageMaster 2D software. The report showed as many as 241 of protein spots were expressed in the beginning of the fifth instar. Among them, most were concentrated in pI 3.5-6.5, which reached 76% of the total protein spots. As for the protein molecular sizes, 182 protein spots concentrated between 35 and 90 kDa, which comes to 75% of the total spots. When the larvae grow to the seventh day (total fifth instar duration was 9 days), 298 protein spots were visualized through 2D electrophoresis. Fifty-seven spots were newly expressed compared to the image of the first day in fifth instar. The results implied that these proteins are related to biosynthesis of silk protein and metamorphosis preparation from larva to pupa. In total, 19 protein spots including 6 special spots expressed in seventh day were analyzed through MALDI-TOF-MS. The relations between proteins and growth and development of silkworm were discussed.     

Ultrastructure of Lyonet's gland in the silkworm (Bombyx mori L.)

The gland cells of Lyonet's gland, which is accessory to the silk gland in the silkworm larva, is characterized by the presence of complicated canaliculi bearing microvilli on their inner surface, large numbers of mitochondria and remarkably convoluted basal plasma membrane. On the other hand, the cell lacks the well-developed cytoplasmic membrane system such as rough- and smooth-surfaced endoplasmic reticula and Golgi bodies, though free ribosomes are numerous. Secretory vesicles are absent, and the canaliculi contain no dense material. From such ultrastructural observations, it was suggested that a possible role of the gland may be the exchange of the small molecules such as water and ions, rather than the hitherto supposed secretory role of a cementing sunstance of silk proteins. The lumen of the proximal part of the glandular duct contains a kind of proteinaceous substance which can be demonstrated histochemically and is regarded as similar to one of the silk proteins in the silk gland, not to the real product of the Lyonet's gland.     

FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.     

Cytology of egg maturation in the silkworm (Bombyx mori L.) in artificial parthenogenesis

The orientation of bivalentss in the first meiotic metaphase was studied on the squash preparations and sections of eggs in Bombyx mori L., Antheraea pernyi Guèr and Carpocapsa pomonella L. The reduction fissure of bivalents separating the homologous chromosomes lies in the plane of spindle equator; the equation fissure separating the sister chromatids is parallel to the long spindle axis. Complicated rearrangements are observed in the nuclei of the silkworm eggs stimulated to ameiotic parthenogenesis by the high temperature dissociation of bivalents, full destruction of spindle, formation of a new spindle and metaphase plate. The latter includes univalent chromosomes involved in the equation division. As a result, two genetically identical pronuclei with the somatic set of chromosomes are formed.     

A carotenoid-pheophytin-protein complex from the digestive juice of silkworm (Bombyx mori L.)

• 1.1. A soluble carotenoid-pheophytin-protein complex was purified from the digestive juice of fifth instar silkworm larvae raised on mulberry leaves.
• 2.2. The pigment-protein complex showed absorption maxima at 276, 429, 453, 481 and 670 nm. Major pigment components were identified as α-carotene, pheophytin a and b.
• 3.3. This complex has an acidic protein component having an isoelectric point of 4.6. The molecular weight was estimated to be 68,000 with four identical subunits.

     

The biochemical effects of potassium chloride on the silkworm, （ Bombyx mori L.）

The supplementation with 50, 100 and 150μg/mL potassium chloride to the fifth instar larvae of the silkworm Bombyx mori on fat body glycogen, protein, total lipids and haemolymph protein and trehalose were analyzed. The fat body glycogen and protein and haemolymph protein were increased significantly in all the treated groups; whereas fat body total lipids increased only in 100 and 150μg/mL and haemolymph trehalose increased only in 150μg/mL potassium chloride-treated groups when compared with those of the corresponding parameters of the carrier controls.     

From genome to proteome: great progress in the domesticated silkworm (Bombyx mori L.)

As the only truly domesticated insect, the silkworm not only has great economic value, but it also has value as a model for genetics and molecular biology research. Genomics and proteomics have recently shown vast potential to be essential tools in domesticated silkworm research, especially after the completion of the Bombyxmori genome sequence. This paper reviews the progress of the domesticated silkworm genome, particularly focusing on its genetic map, physical map and functional genome. This review also presents proteomics, the proteomic technique and its application in silkworm research.     

Effect of chronic low-intensity gamma irradiation on embryogenesis in the silkworm (Bombyx mori L.)

Gamma irradiation of a grain during embryogenesis at an intensity only 100 times exceeding that of the natural radioactive background reduces by 4-7 h the average time of embryogenesis for different species and hybrids of thesilworm embryo. The 10- and 40-time increase in the radiation intensity decreases the stimulatory effect and leads to the delay in the development.     

Effect of low-dose gamma-irradiation of silkworm eggs on the development of the mulberry silkworm (Bombyx mori L.)

Chronic gamma-irradiation of an egg at a dose-rate 100, 1000 and 4000 times as high as that of the natural radiation background significantly accelerates the development of Bombyx mori L. The caterpillar development may also be stimulated by a single exposure of the egg to 2 Gy radiation. The acceleration of the caterpillar growth promotes the increase in the cocoon weight and the raw-silk mass.     

Carbohydrate metabolism during starvation in the silkworm Bombyx mori

The effect of starvation on carbohydrate metabolism in the last instar larvae of the silkworm Bombyx mori was examined. Trehalose concentration in the hemolymph increased slightly during the first 6 h of starvation and decreased thereafter, whereas glucose concentration decreased rapidly immediately after diet deprivation. Starvation-induced hypertrehalosemia was completely inhibited by neck ligation, suggesting that starvation stimulates the release of a hypertrehalosemic factor(s) from the head. The percentage of active glycogen phosphorylase in the fat body increased within 3 h of starvation and its glycogen content decreased gradually. These observations suggest that production of trehalose from glycogen is enhanced in starved larvae. However, hypertrehalosemia during starvation cannot be explained by the increased supply of trehalose into hemolymph alone, as similar changes in phosphorylase activity and glycogen content in the fat body were observed in neck-ligated larvae, in which hemolymph trehalose concentration did not increase but decreased gradually. When injected into larvae, trehalose disappeared from hemolymph at a rate about 40% lower in starved larvae than neck-ligated larvae. The hemolymph lipid concentration increased during starvation, suggesting that an increased supply of lipids to tissues suppresses the consumption of hemolymph trehalose and this is an important factor in hypertrehalosemia.