Hormonal effects on carotenoid uptake by the silk gland in the silkworm, Bombyx mori

The carotenoid uptake by the silk gland of the silkworm (Bombyx mori), which occurs only during the middle to late period of the last (fifth) instar in the natural condition, was studied in relation to the hormonal controls. During certain stages of the fourth and last instars, the corpus allatum hormone (JH) was found to inhibit the activation of the absorbing function of the silk gland. The absorbing activity was inactivated, if the activated silk gland was implanted into larva at the late stage of the fourth instar in the presence of the moulting hormone (MH). As more ponasterone-A (ecdysone-analogue) was injected into decapitated larvae, the pigmentation of the silk gland was increased; but injection of a high titre inhibited its activity. It seems that, through serial transplantations, the silk gland inactivated experimentally at the late stage of the fourth instar is reactivated in the presence of MH during the middle to late period of the last instar. The results indicate that MH and JH at each stage control the activity of the carotenoid uptake.     

RNA in the degenerating silk gland of Bombyx mori

Nucleic acids in the degenerating posterior silk gland of Bombyx mori were analysed during the period from larval maturation to early pupal stage, by methylated albumin column chromatography and sucrose density gradient centrifugation. During the first half of the period, the amount of RNA decreased rapidly, but no accumulation of degradation products was detected. The ratio $\text{26}\text{S}\text{17}\text{S}$ rRNA decreased slightly. A decrease of sRNA-like polynucleotide (∼4S RNA) was faster than that of rRNA. During the latter half of the period, rRNA continued to decrease, while ∼4S RNA increased in content. This probably resulted from the degradation of rRNA. There was a significant fall in the ratio of $\text{26}\text{S}\text{17}\text{S}$, suggesting that rRNA, at least in part, was degraded by the scheme of 26 S→17 S→∼ 4S. The possibility that a part of rRNA may be released outside the tissue without complete decomposition is discussed.     

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.     

Effect of carotenoid deficiency on photosensitivities in the silkworm, Bombyx mori

The silkworm, Bombyx mori, was reared aseptically on a synthetic diet with and without β-carotene and the effects of carotenoid and vitamin A deficiency on photosensitivities in larval phototaxis, visual function and adult eclosion were studied.β-Carotene or vitamin A acted as a growth-promoting factor in continuous darkness and under photoperiodic conditions. The deficiency of β-carotene decreased the larval phototactic response as growth proceeded. The offspring larvae from eggs laid by β-carotene-deficient moths also lost the phototactic response, but successive rearing with dietary β-carotene or vitamin A re-established the response. The deficiency of β-carotene caused the loss of the electric response by light stimuli in the ocelli of fifth instar larvae and the compound eyes of adult moths. These results indicate that vitamin A is essential for visual function in the silkworm, as reported in other insects. The lack of carotenoid did not affect the development of the pupae or the specific time of eclosion which is regulated by a photoperiodic condition of pupal stage. This observation suggests that the carotenoid and its derivative are not involved in photoreception for the entrainment of the adult eclosion of the silkworm.     

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.     

Biochemical evidence of DNA transport from the silk gland to the fat body of the silkworm, Bombyx mori

β-DNA, a component of DNA found in the pupal fat body of the silkworm, Bombyx mori, has the same GC content but a smaller molecular weight than typical silkworm DNA (α-DNA). Its origin and time of synthesis were studied by MAK column chromatography of phenol extracts after labelling with radioactive precursors.The DNA components of the fat body changed greatly during the early pupal stage, the β-DNA showing a striking increase relative to α-DNA. Thymidine-6-³H and phosphoric acid-³²P injected into the animals 1 day before analysis caused labelling of α-DNA, but not of β-DNA of the fat body, indicating that β-DNA was not synthesized during the stage of its appearance in the fat body.On the other hand, injection of thymidine-6-³H into 2-day-old fifth instar larvae, when DNA of the silk gland was being actively synthesized, gave high incorporation of the isotope into β-DNA of the pupal fat body. The sudden appearance of highly labelled β-DNA in the fat body during the early pupal stage as well as the occurrence of β-DNA in both the silk gland and fat body suggested that DNA might move from the silk gland to the fat body.It is possible that the fat body stores DNA as a nutrient from the degenerating silk gland.     

In vitro diapause substance in the silkworm, Bombyx mori

By means of in vitro studies, in which isolated suboesophageal ganglions of the Bombyx silkworm were cultured, it was shown that at least two kinds of substances are biosynthesized and exert independent effects on determination of diapause or non-diapause in silkworm eggs. They are referred to as the diapause and non-diapause substance, respectively. Whether diapause or non-diapause eggs are laid may depend upon the different quality of these substances.     

Properties of flacherie virus of the silkworm, Bombyx mori

The flacherie virus of the silkworm, Bombyx mori, was isolated from infected larvae reared under aseptic conditions. Two types of infectious particles, tentatively designated FVS I and FVS II, were separated by density gradient centrifugation. Some properties of the separated particles were investigated. Electron micrographs showed that FVS I and FVS II were spherical particles with diameters of 27 ± 2 nm and 22 ± 2 nm, respectively. The sedimentation coefficients of FVS I and FVS II were 180 S and 134 S, respectively. It was concluded from experiments of incorporation of ³H-uracil inoculated into diseased larvae at late stage of flacherie disease that the nucleic acid of FVS II was RNA. The two types of particles were present in Sakaki and Wadayama strains of flacherie virus.     

Characterization and identification of the integrin family in silkworm, Bombyx mori

As an important economic insect, Bombyx mori is also a useful model organism for lepidopteran insect. Integrins are evolutionarily conserved from sponges to humans, and play vital roles in many physiological and pathological processes. To explore their diverse functions of integrins in insect, eleven integrins including six α and five β subunits were cloned and characterized from silkworm. Our results showed that integrins from silkworm own more family members compared to other invertebrates. Among those α subunits, integrins α1, α2, and the other four subunits belong to PS1, PS2, and PS3 groups, respectively. The β subunits mainly gather in the insect βν group except the β1 subunit which belongs to the insect β group. Expression profiles demonstrated that the integrins exhibited distinct patterns, but were mainly expressed in hemocytes. α1 and β2 subunits are the predominant ones either in the embryogenesis or larva stages. Interestingly, integrins were significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo. These results indicate that integrins perform diverse functions in hemocytes of silkworm. Overall, our results provide a new insight into the functional and evolutionary features of integrins.     

Involvement of glycine transfer ribonucleic acids in development of the posterior silk gland of Bombyx mori

Glycine transfer RNAs from the two physiological phases, V-2, the stage of maximum growth, and V-5, the stage of maximum fibroin production, during the development of the posterior silk gland of Bombyx mori were examined. The tRNAs from both phases could be fractionated into two major isoaccepting species on a benzoylated DEAE-cellulose column. No significant qualitative differences were observed among the tRNAs, but the total amount of the isoaccepting species of tRNA^Gly in each gland of V-5 stage was 6-fold higher than the amount of tRNA^Gly in the V-2 gland. The codon recognition properties of the tRNA^Gly species were examined. It was found that tRNA^Gly₁ responded to the copolymer (G:U) preferentially while tRNA^Gly_II recognized the copolymer (A:G). The ratio between the extent of incorporation of labeled glycine from glycyl-tRNA^Gly₁ and glycyl-tRNA^Gly_II into protein in a cell-free system utilizing polysomes from the V-5 glands was similar to the relative abundance of the isoaccepting species present in the glands at that time. It also reflected the ratio between the corresponding codons assigned for glycine based on the sequence analysis of fibroin-mRNA [Suzuki, Y., and Brown, D. D. (1972) J. Mol. Biol.63: 409]. These results suggest that the abundance of tRNA^Gly in the posterior silk gland and the changes in the relative amounts of the isoaccepting species are quite specific for the development of the gland.     

Expression pattern of immunoglobulin superfamily members in the silkworm, Bombyx mori

Immunoglobulin superfamily (IgSF) proteins are involved in cell adhesion, cell communication and immune functions. In this study, 152 IgSF genes containing at least one immunoglobulin (Ig) domain were predicted in the Bombyx mori silkworm genome. Of these, 145 were distributed on 25 chromosomes with no genes on chromosomes 16, 18 and 26. Multiple sequence alignments and phylogenetic evolution analysis indicated that IgSFs evolved rapidly. Gene ontology (GO) annotation indicated that IgSF members functioned as cellular components and in molecular functions and biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis suggested that IgSF proteins were involved in signal transduction, signaling molecules and interaction, and cell communication. Microarray-based expression data showed tissue expression for 136 genes in anterior silkgland, middle silkgland, posterior silkgland, testis, ovary, fat body, midgut, integument, hemocyte, malpighian tubule and head. Expression pattern of IgSF genes in the silkworm ovary and midgut was analyzed by RNA-Seq. Expression of 105 genes was detected in the ovary in strain Dazao. Expression in the midgut was detected for 74 genes in strain Lan5 and 75 genes in strain Ou17. Expression of 34 IgSF genes in the midgut relative to the actin A3 gene was significantly different between strains Lan5 and Ou17. Furthermore, 1 IgSF gene was upregulated and 1 IgSF gene was downregulated in strain Lan5, and 4 IgSF genes were upregulated and 2 IgSF genes were downregulated in strain Ou17 after silkworms were challenged with B. mori cypovirus (BmCPV), indicating potential involvement in the response to BmCPV-infection. These results provide an overview of IgSF family members in silkworms, and lay the foundation for further functional studies.     

Eclosion hormone activity in developing embryos of the silkworm, Bombyx mori

The ultimate timing of hatching in the silkworm, Bombyx mori, is controlled by a circadian oscillator. The presence of eclosion hormone in developing embryos of the silkworm is demonstrated. Eclosion hormone activity first becomes detectable in embryos which have developed almost to the stage of the differentiation of the neuroendocrine system. Hormonal activity increases sharply to a maximum level 1 day before hatching and falls by about a half in the newly hatched larvae. Eclosion hormone was partially purified from the pharate first-instar larvae and approx, a 2100-fold purification was achieved. The molecular weight of the embryo eclosion hormone is estimated to be 7000 ～ 9000 Daltons by gel-filtration on Sephadex G-50 (superfine). The role of eclosion hormone on hatching behaviour of the silkworm, Bombyx mori, is discussed.     

Nucleic acid of flacherie viruses of the silkworm, Bombyx mori

It was reported previously that two spherical flacherie viruses of silkworm, FVS I and FVS II, had been isolated from flacherie silkworm larvae and the nucleic acid of FVS II was RNA as suggested by the experiments of incorporation of [³H]-uracil. In this paper, it has been confirmed by biochemical methods that the nucleic acid of FVS I and FVS II is RNA. FVS I and FVS II were labeled with ³²P in flacherie silkworms, and the viruses were analyzed by sucrose density gradient centrifugation. When the ³²P-labeled compound in the viruses was treated with 0.5 n KOH, the acid-insoluble ³²P-labeled compound changed to acid-soluble compounds. It was determined by paper chromatography and ion-exchange column chromatography that the alkali-decomposed compounds included four ribonucleotides. Therefore, the viral nucleic acid of FVS I and FVS II was determined to be RNA. The correlations between FVS I and FVS II particles were discussed, and it was suggested that FVS I and FVS II might be closely related or were the same viral species.     

Hormonal control of storage protein synthesis and uptake by the fat body in the silkworm, Bombyx mori

The female silkworm, Bombyx mori, rapidly accumulates two storage proteins, that are synthesized by the fat body, in the haemolymph during the feeding stage of the last-larval instar, and then sequesters them from the haemolymph into fat body during the larval-pupal transformation.The rapid synthesis and uptake of storage proteins by the fat body are shown to be induced by allatectomy in the early-penultimate larval instar. A juvenile hormone analogue, methoprene, is highly effective in inhibiting the allatectomy-induced synthesis, and, in a higher dosage, further blocks the uptake. Allatectomy in the late-penultimate larval instar shortly before moulting does not enhance the storage protein synthesis, but causes the uptake to occur two days earlier in the last-larval instar. Injection of 20-hydroxyecdysone is not stimulatory for synthesis of the proteins, but is effective to induce their uptake. Starvation during the early last-larval instar completely blocks the synthesis.From these results, it is suggested that storage protein synthesis is induced in the absence of juvenile hormone by some supplementary stimulus, possibly the supply of nutrient after feeding, and uptake is induced by ecdysteroids after a decline in the juvenile hormone level.     

Characterization of a novel chromodomain-containing gene from the silkworm, Bombyx mori

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein across different eukaryotic species, and is crucial in the establishment and maintenance of heterochromatin. HP1 proteins have two distinct functional domains, an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD), which are required for the selective binding of HP1 proteins to modified histones. During our screen for HP1-like proteins in the Bombyx mori genome, we found a novel silkworm gene, Bombyx mori chromodomain protein 1 (BmCdp1), encoding a putative chromobox protein with only two CDs. The BmCdp1 family proteins are closely related to the HP1 proteins, and most of them belong to insect lineages. qRT-PCR analysis indicated that BmCdp1 mRNA was most abundantly expressed in early embryos, and relatively higher expression was observed in larval testes, hemocytes, and pupal ovaries. Western blot and immunostaining experiments showed that BmCdp1 was localized mainly in the nucleus of BmN4 cells. We searched BmCdp1-bound loci in the Bombyx genome by ChIP-seq analysis using Flag-tagged BmCdp1-expressing BmN4 cells. Combined with ChIP-qPCR experiments, we identified two reliable BmCdp1-bound loci in the genome. siRNA-mediated knockdown of BmCdp1 in BmN4 cells and early embryos did not affect the expression of the gene located close to the BmCdp1-bound locus.     

Arrest of adult development in debrained pupae of the silkworm, Bombyx mori

The occurrence of developmental arrest after brain removal in pupae of Bombyx mori was examined using a racial hybrid Chinese No. 115 × Japanese No. 122. The results are as follows: (1) Adult development was blocked for a long period in most insects debrained shortly after larval-pupal ecdysis; (2) the earlier the brain removal, the more arrested pupae were obtained; (3) the critical period of brain hormone secretion for adult development was earlier in the female than in the male; (4) the developmental arrest which had been induced in female pupae tended to terminate earlier than that in males; (5) the developmental arrest which was induced by extremely early brain removal terminated earlier than that obtained by later brain removal.     

Transport of sugars by the fat body of the silkworm, Bombyx mori

The transport of glucose and α-methyl glucoside into the fat body of the silkworm, Bombyx mori L., has been studied. Glucose is transported into the tissue by a mechanism similar to facilitated diffusion and α-methyl glucoside by a diffusion process. The uptake of these sugars is neither energy dependent nor coupled to a phosphotransferase system.     

Penetration of phytoecdysones through the pupal cuticle of the silkworm, Bombyx mori

The penetrability of some phytoecdysones, ecdysterone, inokosterone, ponasterone A, and cyasterone, through silkworm pupal cuticle was tested and their effect on pupal-adult development is described. The first three chemicals applied topically to fresh pupae accelerated the pupal-adult development and induced abnormal adults with aberrant legs and antennae, indicating penetration of phytoecdysones through fresh pupal cuticle. Females were more sensitive to the chemicals than males as they showed many more abnormalities. When pupae 1 day after ecdysis were treated topically with phytoecdysones, they transformed into normal adults, suggesting no penetration of ecdysones through old pupal cuticle.     

Rhythmic contractile movements of the larval midgut of the silkworm, Bombyx mori

The raidgut of intact larvae of Bombyx mori exhibited active contractile movements. Contraction waves were generated rhythmically at several regions of the midgut. The waves passed in both oral and aboral directions from their sites of origin. Midgut movements were depressed during moulting. The midgut continued to move normally when tetrodotoxin was injected into the larval haemocoel at doses sufficient to paralyze somatic muscle. Ligation of larvae paralyzed with tetrodotoxin behind the second or third body segment resulted in the abolition of the contraction waves in the midgut portion anterior to the ligature. A ligature applied behind other body segments did not hamper midgut motor activity irrespective of whether or not an abdominal ganglion had been extirpated. The frequency of contraction and the rate of food transport in the midgut were increased when larvae were administered serotonin or when their body temperature was raised.     

Vibration receptive sensilla on the wing margins of the silkworm moth Bombyx mori

Bristles along the wing margins (wm-bristles) of the silkworm moth, Bombyx mori, were studied morphologically and electrophysiologically. The male moth has ca. 50 wm-bristles on each forewing and hindwing. Scanning electron microscopy revealed that these wm-bristles are typical mechanosensilla. Leuco-methylene blue staining demonstrated that each wm-bristle has a single receptor neuron, which is also characteristic of the mechanosensillum. The receptor neuron responded to vibrating air currents but did not respond to a constant air current. The wm-bristles showed clear directional sensitivity to vibrating air currents. The wm-bristles were classified into two types, type I and type II, by their response patterns to sinusoidal movements of the bristle. The neuron in type I discharged bursting spikes immediately following stimulation onset and also discharged a single spike for each sinusoidal cycle for frequencies less than ca. 60 Hz. The neuron in type II only responded to vibrations over 40 Hz and, specifically at 75 Hz, discharged a single spike for each sinusoidal cycle throughout the stimulation period. These results suggest that the two types of wm-bristles are highly tuned in different ways to detect vibrations due to the wing beat. The roles of the wm-bristles in the wing beat are discussed.