Metamorphosis of midgut epithelial cells in the silkworm (Bombyx Mori L.) with special regard to the calcium salt deposits in the cytoplasm. I. light microscopy

The morphological changes of the metamorphosing midgut cell in the silkworm were traced light-microscopically. The regenerative cells of the larval midgut proliferate rapidly during larval-pupal molt and finally replace the larval midgut, establishing new pupal midgut tissue composed of only one cell type. Pupal midgut cells contain numerous basophilic granules which are believed on histological grounds to be the deposits of calcium salts. Calcium seems to be transported from hemolymph to the pupal midgut cells and stored there temporarily as insoluble salts such as phosphate or carbonate, and then finally discharged into the lumen in a merocrine fashion. The midgut cells of the adult no longer contain calcium deposits.     

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.     

X-ray-induced recombination during oogenesis in the silkworm (Bombyx mori L.)

Summary The physical induction of recombinational events has been studied in the female silkworm (Bombyx mori), in which crossing-over does not normally occur. Female silkworms heterozygous in the trans type of two egg-color genes,pe (V-0.0) andre (V-31.7), received a single dose of X-rays (1000 R) at various developmental stages. Then they were crossed to marked males homozygous for both genes. The results indicated that X-rays increase the occurrence of recombinational events in silkworm females from first instar larvae to late stage pupae. The spontaneous frequency of exchange type recombinants in the control series was 2.5 x 10^–5, while after irradiation the frequency of these recombinants was up to 38.8 x^10–5. The sensitive stage to X-ray-induced recombinational events was late stage larvae from fourth to fifth instar. Exchange (cross-over) type recombinants were about three times more frequent than interchange types among the 122 recombinants recovered. The biological significance of the present finding is discussed.     

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.     

Purification of glycosylphosphatidylinositol-anchoring aminopeptidase in from the plasma membrane of larval midgut epithelial cells of the silkworm,Bombyx mori

• 1.1. Aminopeptidase N was selectively released from larval midgut of silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C, and purified to a homogeneous state by ion exchange, gel filtration. Con A-Sepharose and 4-aminobenzyl phosphonic acid-agarose column chromatographies.
• 2.2. The purified aminopeptidase N preparation showed 190.8 U/mg of specific activity. Its molecular weight was estimated to be around 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• 3.3. Purified aminopeptidase N molecule preferentially hydrolyzed Leu-, Ala- and Met-p-nitroanilide as substrates. Especially, Leu-p-nitroanilide proved to be the best substrate for aminopeptidase N from larval midgut of silkworm.
• 4.4. By treatment with phosphatidylinositol-specific phospholipase C, two other hydrolases, alkaline phosphatase and alkaline phosphodiesterase I, were also solubilized from silkworm midgut.

     

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.     

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.     

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.

     

Studies on the activity of the alkaline phosphatase in the midgut of infected silkworm, Bombyx mori L.

The effects of several factors on the activity of alkaline phosphatase (ALKP) in the midgut of the silkworm, Bombyx mori L were studied. The ALKP activity varied greatly among the tested strains. There was positive correlation between ALKP activity and cocoon quality. The ALKP activity of the midgut decreased when the silkworm was infected with cytoplasmic polyhedrosis virus (CPV); whereas, infection with nuclear polyhedrosis virus (NPV) did not affect ALKP activity. This suggests that the pathogenic mechanism of CPV differs from that of NPV. Bacillus thuringiensis caused direct damage to the midgut tissues of the silkworm with a rapid decline in ALKP activity. Activation of ALKP by Mg²⁺ was more evident than the other chemicals. Ascorbic acid, Ca²⁺, citric acid and Cl^– were inhibitory to ALKP in vitro .     

Developmental changes of the antenna and its neurons in the silkworm,Bombyx mori,with special regard to larval-pupal transformation

The larval antenna of Bombyx mori has 13 sensilla and about 52 sensory neurons in its distal portion. The axons form two nerve cords which unite in the cranial hemocoel to supply the brain as the olfactory nerve. The antennal imaginal disc, which is a thick pseudostratified epithelium continuous with the antennal epidermis, thickens markedly during the 5th instar by rapid cell proliferation. At the prepupal stage cell proliferation ceases and the disc everts to form a large pupal antenna. Simultaneously, an extensive cell rearrangement occurs in the antennal epidermis and the disc tissue becomes much thinner because of the abrupt expansion of antennal surface area. The two larval nerve cords thin down markedly by degeneration of axons, but they do not disintegrate totally even after the onset of pupation. The epidermis of the larval antenna forms the distal portion of the pupal antenna, while the imaginal disc forms the more basal portion. Development to the adult antenna occurs almost immediately after the onset of pupation; many adult neurons appear in the simple epidermis facing toward the thick outer side of the newly formed pupal cuticle. By 12 hours after the onset of pupation, these neurons align themselves in many transverse rows which are the first sign of the adult antennal configuration. Addition of these neuronal axons to the once-thinned nerve cords causes resumed thickening of the cords during the first 24 hours and thereafter. Differentiation of adult sensilla begins in the next 24 hours and is almost completed at the third day of pupation, which requires a total of 10 days.     

Electron microscopy of hybridoma cells with special regard to monoclonal antibody production

Electron microscopy of mouse hybridoma cell lines shows that the major difference between non, low and high producer cell lines is the amount of endoplasmic reticulum. Vesicular-tubular or cavernous structures of endoplasmic reticulum, which can survive long after cell death, are particularly abundant in producer cell lines. Immunogold labelling with anti-mouse IgG reveals that antibodies are predominantly located in these structures. The cell membrane undergoes structural changes during the late stages of batch culture with the disappearance of microvilli and the appearance of blebs and deep indentations. Necrosis disrupts the cytoplasmic structures and the nucleus is last to degrade.     

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.     

Toxic impact of ingested Jatropherol-I on selected enzymatic activities and the ultrastructure of midgut cells in silkworm, Bombyx mori L.

Abstract:  Jatropherol-I, a phorbol-type diterpene from Jatropha curcas seeds was found highly toxic to third instars silkworm larvae after ingestion with LC₅₀ values 0.5793, 0.2197 and 0.1578 mg/ml at 48, 72 and 120 h respectively. The acute toxicity was associated with changes in activities of several midgut enzymes and pathological changes in midgut epithelial cells. Jatropherol-I caused various fluctuations in activities of different midgut enzymes in third instars larvae of silkworm. Compared with controls, both esterase and carboxyliesterase showed significantly depressed activities at 3 h ( t -test, P < 0.05) and increased activities at 24 h and rapidly decreased activities at 48 h after ingestion of 0.125 and 0.25 mg/ml of Jatropherol-I. Jatropherol-I induced two (E4 and E5) and depressed one (E2) of midgut esterase isozymes analysed 3 h after ingestion. There was no significantly different glutathione S-transferase activity between most of treated silkworm and control ( t -test, P > 0.05). Activities of acetylcholinesterase fluctuated weakly in treated silkworm in 48 h. Jatropherol-I induced a gradual decline in midgut protease activities in silkworm with an obvious dose- and time-dependent effect. Jatropherol-I also caused pathological changes in midgut cells, especially in their endoplasmic reticulum. They were noted slight dilatation on 12 h exposure, while extreme dilatation, vesiculations and shedding of ribosome from membrane were observed in endoplasmic reticulum after a longer time exposure. Other pathological changes in microvilli, lysosome, mitochondria and chromatin were also observed in midgut cell of treated silkworm.