STUDIES ON THE POSTERIOR SILK GLAND OF THE SILKWORM, BOMBYX MORI : I. Growth of Posterior Silk Gland Cells and Biosynthesis of Fibroin During the Fifth Larval Instar

Growth of the posterior silk gland and biosynthesis of fibroin during the fifth larval instar of the silkworm, Bombyx mori, have been studied. In accordance with the exponential increase in the wet weight of the gland, the amounts of DNA, RNA, protein, and lipids per animal increased rapidly in the early stage of the fifth instar (0–96 hr). Biosynthesis of fibroin, on the contrary, mainly proceeds in the later stage of the fifth instar (120–192 hr). Electron microscopical observations have shown that, in the very early stage (0–12 hr), a number of free ribosomes exist in the cytoplasm. Rough endoplasmic reticulum (ER) with closely spaced cisternae was also observed. Then rough ER starts to proliferate rapidly, and at the same time lamellar ER is rapidly or gradually transformed into vesicular or tubular forms. In the later stage of the fifth instar (120–192 hr), the cytoplasm is mostly filled with tubular or vesicular ER. Golgi vacuoles, free vacuoles (fibroin globules), and mitochondria are also observed. It is concluded that in the early stage of the fifth instar the cellular structures necessary for the biosynthesis of fibroin are rapidly formed, while in the later stage the biosynthesis of fibroin proceeds at a maximum rate and utilizes these structures.     

Ultrastructural changes of the posterior silk gland cells in the early larval instars of the silkworm, Bombyx mori

The posterior silk gland cells in the first three larval instars show characteristic changes during growth that are essentially similar to those undergone in the fourth larval instar. In the feeding stage, when the cells grow rapidly, vesico-tubular rough endoplasmic reticulum and a number of Golgi vacuoles occur in the cytoplasm and the glandular lumen is filled with fibrous materials, probably fibroin. In the moulting cycle when the cells stop growing, a series of degenerative changes occur such as the appearance of autophagosomes, autolysosomes, and large vacuoles. Fibrous materials disappear from the glandular lumen. These cyclic changes are discussed in relation to hormonal changes. Intercellular junctions and the tracheal system of the silk gland are described.     

Studies on the posterior silk gland of the silkworm Bombyx mori. V. Electron microscope localization of fibroin in the posterior silk gland at the later stage of the fifth instar

Electron microscope observations of thin sections of epoxy resin- embeded posterior silk gland cells at the later stage of the fifth instar revealed that the Golgi vacuoles and the secretory granules (fibroin globules) in the cytoplasm and the glandular lumen contain fine fibrous materials. In frozen thin sections these structures appear as electron-dense granules and electron-dense blocks, or a column, respectively. Immunoelectron microscopy has shown that ferritin particles or products of the peroxidase reaction are localized on these structures. It was concluded that the fine fibrous materials most probably represent native fibroin molecules or their aggregates.     

Ultrastructure of fibroin in the silk gland of larval Bombyx mori

The fibroin molecules stored in Golgi vacuoles in the posterior silk gland cells of 72-h-old, fifth instar larvae of Bombyx mori L. were observed electron-microscopically. The fibers which float in the Golgi vacuoles often have their ends attached to the limiting membrane. The fibers are helical bundles about 130 Å in diameter composed of 5–7 threads, each 20–30 Å thick.     

Localization of the genes for silk fibroin in silk gland cells of Bombyx mori.

Radioactive iodinated silk fibroin messenger RNA and ribosomal RNA have been used as probes to localize their genes in tissue sections of Bombyx mori by in situ hybridization. From filter hybridization experiments it is inferred that the majority of the grains produced by in situ hybridization with fibroin mRNA represents specific hybridization to fibroin genes. Sections of the posterior silk gland where silk is synthesized have been compared with those of the middle gland which does not synthesize fibroin. Glands have been analyzed from the second through the fifth (last) larval instar during feeding and moulting periods. During later stages when the gland cells increase their DNA content by polyploidization, serial sections were required to follow the distribution of grains through entire nuclei. At all stages, both ribosomal DNA and fibroin genes are distributed randomly throughout the nuclei without a preferred relationship to any nuclear structure.     

STUDIES ON THE POSTERIOR SILK GLAND OF THE SILKWORM, BOMBYX MORI : III. Ultrastructural Changes of Posterior Silk Gland Cells in the Fourth Larval Instar

The development of the cells in the posterior silk gland of the silkworm, Bombyx mori, during the fourth larval instar has been studied. In the early stages of this instar, the wet weight of the gland and the amounts of RNA, DNA, and protein per animal increase logarithmically until they reach a stationary state at about 72 hr. At around 96 hr of the fourth instar, the larvae enter the molting state, which lasts for about 24 hr until the fourth ecdysis. Towards the end of the molt stage, the growth of the silk gland is resumed. Electron microscopical observation shows that in the early intermolt stage the cytoplasm is filled with free ribosomes and with rough endoplasmic reticulum (ER), first of the lamellar type (0–6 hr) and then of the vesicular or tubular type. The Golgi apparatus also is well developed. At the beginning of the molt stage (90–96 hr), however, most of the ER becomes lamellar in type, concentric lamellar structures being occasionally observed, and the Golgi vacuoles disappear. Autophagosomes and lysosomes increase markedly and the apical portion of the cytoplasm becomes extensively vacuolated; this suggests that the secretory activities are completely depressed, and pronounced degenerative changes appear during the molt stage. Towards the end of the molt stage, large lamellar ER elements are fragmented into smaller lamellae and there is a pronounced increase in the number of free ribosomes.     

Studies on the posterior silk gland of the silkworm Bombyx mori. VI. Distribution of microtubules in the posterior silk gland cells

There are two microtubule systems in the posterior silk gland cells. One is a radial microtubule system in which the microtubules run radially from the basal to the apical cytoplasm and in which fibroin globules (secretory granules of fibroin) and mitochondria are arranged along these microtubules, thus composing a "canal system" which is assumed to be responsible for the intracellular transport of fibroin globules. The other is a circular microtubule system in the apical cytoplasm which is composed of bundles of microtubules and microfilaments running in a circular arrangement around the glandular lumen at an interval of approximately 4 mum at the end of the fifth instar. This system is presumably concerned with secretion and/or intraluminal transport of fibroin.     

Predominant synthesis of fibroin heavy and light chains on the membrane-bound polysomes prepared from the posterior silk gland of the silkworm, Bombyx mori

Membrane-bound polysomes were prepared from the posterior silk gland of the silkworm, Bombyx mori, on the fourth to fifth day in the fifth larval instar. The polysomes, when supplemented with a soluble fraction from the posterior silk gland, exhibited the elongation reaction of the growing polypeptide-chains, but the initiation reaction of polypeptide synthesis was not demonstrated in this system. The predominant products synthesized on the membrane-bound polysomes were fibroin heavy chain (H-chain) and light chain (L-chain), while polypeptides of heterogeneous size classes were synthesized on the 105,000 X g-sedimentable polysomes. A substantial fraction of the fibroin L-chain synthesized was bound to the H-chain by disulfide bond. Most of the newly synthesized fibroin H- and L-chains on the membrane-bound polysomes were proved to be present within microsomal membrane vesicles because of their insensitivity to digestion with proteases in the absence of Triton X-100.     

Tissue-specific and periodic changes in the nuclease sensitivity of the fibroin gene chromatin in the silkworm Bombyx mori

Nuclei of substantial purity were isolated from the middle or posterior silk glands of the silkworm Bombyx mori larvae. Both the fibroin H- and L-chain gene sequences in the isolated nuclei from the posterior silk glands of the fifth instar larvae, where the genes are transcribed actively, are extremely sensitive to the digestion with DNaseI; on the other hand, these sequences in the middle silk gland nuclei from the same larvae, where the genes are not expressed, are markedly resistant to the digestion. The H-chain gene sequences in the posterior silk gland nuclei from the fifth instar larvae are also highly susceptible to the digestion with micrococcal nuclease, HinfI, and HhaI. The digestion products with micrococcal nuclease show a continuous size distribution. The H-chain gene sequences in the middle silk gland nuclei or the posterior silk gland nuclei from the fourth molting stage are cleaved partially into nucleosome dimer to oligomer sizes upon digestion with higher concentrations of micrococcal nuclease, suggesting that the inactive forms of the H-chain gene chromatin are constructed by folding of the chromatin fiber containing a regular array of nucleosomes. Hypersensitive sites to micrococcal nuclease are present near both ends of the second exon, a major body of the fibroin H-chain gene, in both the active and inactive forms of the chromatin. The DNaseI or micrococcal nuclease sensitivity of the H-chain gene chromatin in the posterior silk gland nuclei shows periodical changes corresponding to the intermolt-molt-intermolt cycle.     

Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.     

Biochemical Studies on the Formation of the Silkprotein

The progression of fibroin in the inside of the silkgland during growth of the fifth instar was elucidated by tracing radioactive fibroin which was made of C¹⁴ glycine. Each fibroin synthesized by the posterior division at different period of the fifth instar moves, in an ordered fashion, in the inside of the gland along the middle and anterior division during growth of the silkworm. Furthermore, fibroin present in the middle division of the silkworm just before spinning is due to the successive disposition, in the order synthesized, of each portion of fibroin synthesized at different periods of the fifth stage.     

The nucleotide sequence of a major glycine transfer RNA from the posterior silk gland of Bombyx mori L.

The nucleotide sequence of tRNA1Gly isolated from the posterior silk gland of Bombyx mori has been determined. This transfer RNA is present in high amounts in the posterior silk gland during the fifth larval instar. It has a GCC anticodon, capable of decoding a major glycine codon in the fibroin messenger RNA, GGU. Structural features of Bombyx tRNA1Gly and its homology to other eukaryotic glycine tRNAs are discussed.     

Secretory Mechanism of Fibroin,a Silk Protein,in the Posterior Silk Gland Cells of Bombyx mori

There are two microtubule-microfilament systems in the posterior silk gland cells of Bombyx mori. One is a radial microtubule system; the other is a circular microtubule-microfilament system. These two systems are presumably concerned with the intra-cellular transport of secretory granules of fibroin and the secretion of fibroin into the lumen, respectively. Conventional and scanning electron microscopic observations of the two microtubule-microfilament systems in the posterior silk gland cells are reported. Scanning electron micrographs showed that a number of parallel linear cytoplasmic processes ran circularly on the luminal surface of the posterior silk gland cells. These processes were assumed to correspond to the circular microtubule-microfilament systems. The effects of cytochalasin (B or D), a secretion stimulating agent of fibroin, on the intracellular recording of membrane potential from the posterior silk gland cells are also reported. Exposure to cytochalasin resulted in depolarization of the membrane potential of the gland cells. Possible functional roles of the two microtubule-microfilament systems in the secretory mechanism of fibroin are discussed with reference to the effects of antimitotic reagents and cytochalasin on these two systems.     

Histochemical and ultrastructural evidence of lipid secretion by the silk gland of the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae)

The silk gland in Lepidoptera larvae is responsible for the silk production used for shelter or cocoon construction. The secretion of fibroin and sericin by the different silk gland regions are well established. There are few attempts to detect lipid components in the insect silk secretion, although the presence of such element may contribute to the resistance of the shelter to wet environment. This study characterizes the glandular region and detects the presence of lipid components in the secretion of the silk gland of Diatraea saccharalis(Fabricius). The silk gland was submitted to histochemical procedure for lipid detection or conventionally prepared for ultrastructural analyses. Lipid droplets were histochemically detected in both the apical cytoplasm of cell of the anterior region and in the lumen among the microvilli. Ultrastructural analyses of the anterior region showed lipid material, visualized as myelin-like structures within the vesicular Golgi complex and in the apical secretory globules, mixed up with the sericin; similar material was observed into the lumen, adjacent to the microvilli. Lipids were not detected in the cells neither in the lumen of the posterior region. Our results suggest that the silk produced by D. saccharalis has a minor lipid content that is secreted by the anterior region together with the sericin.     

Action of estradiol-17beta on the synthetic activity of the silk gland in Bombyx mori L

The effects of estradiol-17beta (E(2)) were studied on several metabolic parameters in the silk gland of Bombyx mori L. race Nistari. Topical application of different doses (0.05-4.0&mgr;g/g body weight) of E(2) on the first and second day of the fifth instar larvae showed a dose dependent effect when studied on the fifth day. A significant increase in silk gland weight and fibroin content was observed between the doses 0.05 and 0.1, and 0.1 and 1.0&mgr;g/g of E(2). A similar pattern of dose-dependent rise in DNA and RNA content of posterior silk gland (PSG) was observed with the doses of E(2) when the contents were expressed per pair of PSG. Higher doses of E(2) (2.0 or 4.0&mgr;g/g) demonstrated relatively less increase, unchanged level or a decrease in the above parameters in comparison to the control values. The glutamate-pyruvate transaminase of PSG showed a significant increase from 0.1 to 2.0&mgr;g/g of E(2) doses in comparison to the control value. Simultaneous injection of ICI-182780 (1.0&mgr;g/g), a very pure and specific antiestrogenic compound, with E(2) (1.0&mgr;g/g) caused a significant counteraction of E(2)-induced increase in silk gland activity, which was reflected in DNA and RNA content of PSG, wet weight and fibroin content of silk gland, and on glutamate-pyruvate transaminase activity. Cycloheximide (0.5&mgr;g/g), a protein synthesis blocker, caused a significant inhibition of the E(2) (1.0&mgr;g/g)-induced silk gland activity when treated along with estradiol. From this study it appears that estradiol has a specific effect on silk gland function and that it may act in a nuclear mediated way.