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.     

Study of gut bacterial diversity of Bombyx mandarina and Bombyx mori through 16S rRNA gene sequencing

The wild silkworm B. mandarina is living in the natural environment has a strong stress resistance and adaptability after harsh natural selection. The indoor rearing or domestication of the wild silkworm under artificial custody for long period deteriorates stress resistance and ecological adaptability. Therefore, we aimed to investigate the effects of artificial domestication and evolutionary pressure on the gut bacterial diversity of B. mandarina and B. mori. The intestinal content of 6th day of fifth instar B. mandarina and B. mori larvae were analyzed by sequencing of the 16S rRNA gene through Illumina miseq sequencing technology. The outcome of the study revealed that abundance of predominant bacteria of phylum Firmicutes were respectively 81.40% and 81.85% in the late fifth instar silkworm larvae (6th day) of B. mandarina and B. mori. In Firmicutes, abundance of predominant bacterial genus Enterococcus in B. mandarina (69.73%) was comparatively higher than B. mori (48.99%). The genus Advenella belongs to phylum Proteobacteria was recorded only in B. mandarina (11.54%). The abundance of Unclassified_Peptostreptococcaceae, Methanobrevibacter, Ignatzschineria, Petrimonas and Proteiniphilum in B. mandarina were between 0.12 and 0.17%, nevertheless, these bacterial genera were not detected in B. mori. The abundance of genera Lactococcus, Bacillus and Pseudomonas in B. mori (17.73%, 5.02%, and 1.61%) were remarkably higher than B. mandarina (0.15%, 0.54% and 0.45%). These results indicated that substantial difference was observed between the intestinal bacteria of B. mori and B. mandarina population, and structure of the intestinal bacteria could be affected by the artificial domestication and evolutionary pressure.     

Ultrastructural observations on the organogenesis of the posterior silk gland of the silkworm, Bombyx mori

In the early stages of development (0 to 48 hr after organogenesis) the posterior silk gland cells of the silkworm, Bombyx mori, have characteristics of undifferentiated cells, that is, there are a number of free ribosomes in the cytoplasm and development of rough endoplasmic reticulum (rER) and Golgi bodies is very poor. Mitotic cells are frequently found. At ～ 60 hr when differentiation of the silk gland to the posterior, middle, and anterior divisions is completed, mitotic cells were no longer observable and the posterior silk gland is now composed of two rows of cells regularly packed forming a tubular structure. Differentiation of the cytoplasm is, however, not yet apparent and only a slight proliferation of rER can be observed. At 84 to 144 hr, proliferation of rER and transformation of rER from lamellar to vesico-tubular configuration are observed and Golgi vacuoles begin to enlarge. Just before hatching, the ultrastructures of cells are very similar to those of the later stage of the fifth instar when fibroin is synthesized extensively; the cytoplasm is filled with vesico-tubular rER, Golgi bodies, free secretory granules of fibroin, and mitochondria. Fibroin is probably synthesized, transported, and secreted in a manner similar to that in the fifth instar larvae.     

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.     

Morphological and electrophysiological differences in tarsal chemosensilla between the wild silkmoth Bombyx mandarina and the domesticated species Bombyx mori

Gustatory and olfactory senses of phytophagous insects play important roles in the recognition of host plants. In the domestic silkmoth Bombyx mori and its wild species Bombyx mandarina, the morphologies and responses of adult olfactory organs (antennae) have been intensely investigated. However, little is known about these features of adult gustatory organs and the influence of domestication on the gustatory sense. Here we revealed that both species have two types of sensilla (thick [T] and slim [S] types) on the fifth tarsomeres of the adult legs. In both species, females have 3.6–6.9 times more T-sensilla than males. Therefore, T-sensilla seem to play more important roles in females than in males. Moreover, gustatory cells of T-sensilla of B. mandarina females responded intensely to mulberry leaf extract in electrophysiological experiments, while T-sensilla of B. mori females (N4 strain) hardly responded to mulberry leaf extract. These results suggest that T-sensilla of B. mandarina females are involved in the recognition of oviposition sites. We also observed that, in three B. mori strains (N4, p50T, and Kinshu × Showa), the densities of sensilla on the fifth tarsomeres were much lower than in B. mandarina. These results indicate that domestication has influenced the tarsal gustatory system of B. mori.     

Determination of the multiplicity of the silk fibroin gene and detection of fibroin gene-related DNA in the genome of Bombyx mori.

The number of silk fibroin genes per genome in the silkworm Bombyx mori has been determined by hybridization using fibroin [¹²⁵I]mRNA. The purified [¹²⁵I]mRNA had an oligonucleotide pattern after RNAase T₁ digestion which was characteristic of fibroin mRNA (Suzuki &; Brown, 1972) and it hybridized specifically to DNA with a G + C content expected for a fibroin gene. Thermal denaturations indicated that these hybrids were mismatched by about 3%, which probably indicates some variation among the sequences encoding the internal repetitions of the fibroin protein.The concentration of fibroin gene sequences in B. mori DNA was measured by saturation hybridization of [¹²⁵I]mRNA to filter bound DNA. The same saturation level of 1.8 × 10^?5 μg mRNA per μg DNA was calculated from data obtained with unfractionated DNA and with fibroin gene sequences which had been separated from bulk B. mori DNA by actinomycin $\text{D}\text{CsCl}$ centrifugation. Scatchard plots of the subsaturation data extrapolated to an identical saturation value. Internal reiteration of the fibroin mRNA molecule was apparent from the high association constant of hybridization. An exhaustive hybridization experiment showed that such repetitions comprise at least 90% of each mRNA molecule. The saturation value, in conjunction with the genome DNA content and the mRNA size, indicated the presence of only one fibroin gene per haploid B. mori genome.Hybridization of actinomycin $\text{D}\text{CsCl}$ fractionated DNA indicated that fibroin mRNA can form hybrids with DNA that bands with bulk B. mori DNA. These hybrids appear to involve DNA which is related to, but distinguishable from, true fibroin gene sequences. The fibroin gene-related sequences form mismatched hybrids with the mRNA, are much shorter than the fibroin gene and are dispersed in B. mori DNA of much lower G + C content, and there are many copies of these sequences per B. mori genome.     

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.     

Tissue- and Stage-Specific Expression of Sericin Genes in the Middle Silk Gland of Bombyx mori

Sericin gene expression in the middle silk glands during Bombyx mori larval development was analyzed using probes from a genomic DNA clone for 10.5 kb sericin mRNA. The 10.5 kb mRNA, the most abundant in the fifth instar, is not detected in the third feeding, fourth feeding and fourth moulting stages. It becomes detectable at 2 days of the fifth instar, and accumulates rapidly. The second major mRNA in the late fifth instar, a 9.0 kb component having a similar sequence to the 10.5 kb mRNA, becomes detectable only at 6 and 7 days of the instar by use of the repetitious coding sequence probe of the sericin clone. Using the same probe about 20 kb RNAs with a fainter intensity than that of the major mRNAs are detected. They are present extremely faintly in the third and fourth feeding stages, disappear in the fourth moulting stage, and increase in the fifth instar. Two other faint poly(A)⁺ RNA components are detected by a DNA probe containing the 5' end sequence of the sericin clone. One is 4.3 kb, and appears in the third, fourth and fifth feeding stages but not in the fourth moulting stage. The other is 3.0 kb, and it becomes detectable after 1 day of the fifth instar.     

Isolation and identification of the messenger RNA for silk fibroin from Bombyx mori

The messenger RNA for the protein silk fibroin has been isolated from the posterior silk gland of Bombyx mori and identified by partial sequence analysis. The sequence of mRNA could be predicted because the protein has a simple repetitious primary structure in which glycine residues comprise 45% of all residues and alternate predominantly with alanine and serine.     

Tissue-/stage-dependent expression of a cloned Bombyx mandarina QM homologue

QM, a novel gene that was firstly isolated as a putative tumor suppressor gene from Wilms’ tumor cell line. Although it is well known that the QM gene product plays an important role within the tumor cells, the precise role of QM in the non-tumor cells has remained elusive. With in this mind we isolated a cDNA encoding QM homologue from Bombyx mandarina to understand the function of QM. The 596 bp cDNA has an open reading frame of 219 amino acids and a predicted mol. wt. of 25 kDa. The protein has more than 88% amino acid sequence identity to the QM protein from Drosophila melanogaster. mRNA expression gradually increased from 1–2 days after egg laying to 2 days of finial instar, while very low expressions were detected for either the pupae and the moth stages. The organs, posterior/middle division of silkgland, midgut, fat body and malpighian tubes, also show relatively high mRNA expression levels, respectively. The high degree of conservation and expression of the B. mandarina QM homologous suggest that it has a selectively conserved amino acid sequence due, presumably, to an important biological role which is associated with pupae formation.     

Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins

The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography–tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49 %) were predicted to function as binding proteins and 46 % were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.     

Developmental and sex-dependent regulation of storage protein synthesis in the silkworm,Bombyx mori

The mechanism of sex-dependent expression of a major plasma protein, referred to as storage protein 1 (SP-1) was studied during development of the silkworm, Bombyx mori. SP-1 occurred in the hemolymph of the female as well as in the male larvae until the end of the fourth larval instar. In the last instar larvae, the amount of SP-1 in the hemolymph greatly increased in females, but markedly declined in males. The level of fat body mRNA for SP-1 reflected the developmental and sex-dependent changes in the hemolymph concentration of SP-1. The developmental patterns of hemolymph proteins in the third and the fourth instar larvae of sex-mosaic individuals were quite analogous to those observed in normal larvae at the same developmental stages. The hemolymph concentration of SP-1 at the last larval instar of the sex mosaics varied among individuals irrespective of the gonad compositions. In vitro culture of the fat body cells dissected from several locations of a sex-mosaic larva provided evidence that each fat body cell in a common hemolymph milieu synthesizes a high (female type) or a low (male type) level of SP-1 depending on the sex chromosome composition. The amount of vitellogenin in the hemolymph of the sex-mosaic pupae was in proportion to that of SP-1 at the last larval instar. From these results, it is suggested that the sex-dependent expression of SP-1 and vitellogenin in B. mori is genetically determined and developmentally regulated without participation of the reproductive organs or any sex-specific humoral factors.     

Impact of heat shock on heat shock proteins expression, biological and commercial traits of Bombyx mori

We report the thermotolerance of new bivoltine silkworm, Bombyx mori strains NB₄D₂, KSO₁, NP₂, CSR₂ and CSR₄and differential expression of heat shock proteins at different instars. Different instars of silkworm larva were subjected to heat shock at 35°C, 40°C and 45°C for 2 hours followed by 2 hours recovery. Heat shock proteins were analyzed by SDS‐PAGE. The impact of heat shock on commercial traits of cocoons was analyzed by following different strategies in terms of acquired thermotolerance over control. Comparatively NP₂ exhibited better survivability than other strains. Resistance to heat shock was increased as larval development proceeds in the order of first instar > second instar > third instar > fourth instar > fifth instar in all silkworm strains. Expression of heat shock proteins varies in different instars. 90 kDa in the first, second and third instars, 84 kDa in the fourth instar and 84, 62, 60, 47 and 33 kDa heat shock proteins in fifth instar was observed in response to heat shock. Relative influence of heat shock on commercial traits that correspond to different stages was significant in all strains. In NB₄D₂, cocoon and shell weight significantly increased to 17.52% and 19.44% over control respectively. Heat shock proteins as molecular markers for evaluation and evolution of thermotolerant silkworm strains for tropics was discussed.     

Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori‐type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non‐transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.     

The expression analysis of silk gland-enriched intermediate-size non-coding RNAs in silkworm Bombyx mori

Small non-protein coding RNAs （ncRNAs） play important roles in development, stress response and other cellular processes. Silkworm is an important model for studies on insect genetics and control of Lepidopterous pests. We have previously identified 189 novel intermediate-size ncRNAs in silkworm Bombyx mori, including 40 ncRNAs that showed altered expression in different developmental stages. Here we characterized the functions of these 40 ncRNAs by measuring their expressions in six tissues of the fifth instar larvae using Northern blot and real-time polymerase chain reaction assays. We identified nine ncRNAs （four small nucleolar RNAs and five unclassified ncRNAs） that were enriched in silk gland, including four ncRNAs that showed silk gland-specific expression. We further showed that three of nine silk gland-enriched ncRNAs were predominantly expressed in the anterior silk gland, whereas another three ncRNAs were highly accumulated in the posterior silk gland, suggesting that they may play different roles in fibroin synthesis. Furthermore, an unclassified ncRNA, Bm- 152, exhibited converse expression pattem with its antisense host gene gartenzwerg in diverse tissues, and might regulate the expression of gartenzwerg through RNA-protein complex. In addition, two silk gland-enriched ncRNAs Bm-102 and Bm-159 can be found in histone modification complex, which indicated that they might play roles through epigenetic modifications. Taken together, we provided the first expression and preliminary functional analysis of silk gland-enriched ncRNAs, which will help understand the molecular mechanism of silk gland-development and fibroin synthesis.