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.     

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.     

Ecdysteroid levels and developmental events during larval moulting in the silkworm, Bombyx mori

In the silkworm, Bombyx mori, apolysis of the spiracle is the first visible sign of the initiation of a larval moult. After spiracular apolysis, the characteristic sequence of new spiracle formation can be recognized easily through the cuticle around the old spiracle. This sequence, called the spiracle index, was used as the basis for a precise developmental chronology of the larval moulting period. At certain spiracle-index stages, histological changes in the larval ventral abdominal integument were also examined. By allatectomy and abdominal ligations, the critical periods for the secretion of juvenile hormone and ecdysone respectively were found to be at the initiation of spiracular apolysis and at the time when general apolysis was complete. The haemolymph-ecdysteroid level as measured by radioimmunoassay was about 30–50 ng/ml during the first 2 days of the 4th instar, 60–70 ng/ml at the time of initiation of spiracular apolysis, and 200 ng/ml at the time of completion of apolysis of the general body surface. The maximal level of the hormone was about 290 ng/ml at the mid-moulting period. The relationship between the ecdysteroid titre and morphological events occurring during the larval moult was 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.     

Temporal organization of endocrine events underlying larval-pupal metamorphosis in the silkworm, Bombyx mori

The temporal organization of endocrine events underlying larval-pupal moult was examined. After the phagoperiod of 6–7 days, the last instar larvae of Bombyx mori purged the gut contents and then pupated 4–5 days thereafter. Juvenile hormone titer was considerably high on the first day of the fifth instar, but declined to an undetectable level on day 3. Release of prothoracicotropic hormone (PTTH) began after the decline in the junvile hormone titer and completed during the night of day 4. Prothoracic glands began to secrete ecdysone on day 5. Larvae could pupate on schedule if their brains were removed after the PTTH release. One time of PTTH release during the feeding stage could satisfy the requirement for pupation occuring on schedule. The phagoperiod could be shortened by allatectomy early in the fifth instar and prolonged by the injection of a certain dose of juvenile hormone analogue before the gated PTTH release, accounting for the role of juvenile hormone in the timing of PTTH release.     

Temporal organization of endocrine events underlying larval-larval ecdysis in the silkworm, Bombyx mori

The timing of ecdysis in the penultimate instar of Bombyx mori was demonstrated to be under the control of a circadian clock. The temporal organization of secretion of prothoracicotropic hormone (PTTH), ecdysone and juvenile hormone was studied with particular attention to the circadian control of the timing of hormone release. PTTH release occurs, at least, in the second and third night. The latter is responsible for evoking the larval ecdysis. Prothoracic gland initiates ecdysone secretion abruptly with a very short span after the second PTTH release and secrete enough amount of ecdysone for larval moulting, which takes place 11 h later. Juvenile hormone titer is relatively high before the second PTTH release and corpus allatum becomes dispensable for ensuring the larval moulting in 1.5 h. Based on these findings, interpretations for the endocrine system underlying precocious pupation and formation of intermediates, which are produced by neck ligation, are presented.     

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.     

Prothoracotropic action of ecdysone analogues in Bombyx mori

The prothoracotropic action of ecdysone analogues was examined, using the brainless, diapausing pupae of Bombyx mori with or without inclusion of prothoracic glands. The most effective of those hormones to stimulate prothoracic glands to secrete the moulting hormone was found to be cyasterone. The other analogues such as ponasterone A, ecdysterone, and inokosterone showed a lower activity with regard to prothoracotropic action. The female prothoracic glands were found to be more sensitive to the ecdysones than the male ones. The time lag from hormone injection to emergence indicated the dual actions of the injected ecdysones, directly on the target organs and indirectly on the prothoracic glands subsequent to secreting the moulting hormone.     

Releasing signals of oviposition behaviour in Bombyx mori

The oviposition behaviour of Bombyx mori was studied by eliminating the sensory input received by the tactile setae of the anal papillae.It is shown that the removal of mechanical stimuli of the setae affects oviposition and its time course to some extent. In comparing this experimental result with the theory of Lorenz, summarized in the ‘hydromechanic model’, it is postulated that the sensory information from the mechanoreceptive setae surrounding the ovipositor is a key stimulus which releases the accumulated action-specific energy (‘drive’) and triggers and controls the organs involved in the behaviour.     

Degenerative changes and carotenoid uptake in the silk gland of the silkworm Bombyx mori

In the middle silk gland of the silkworm Bombyx mori, especially in the middle region, structural changes were studied in relation to absorbing activity, using a transplantation method. The physiologically active gland, which was prepared by the decapitation at the feeding stage of the fourth larval instar, maintained a normal structure when placed in the larval body cavity during the middle stage of the fourth instar or during the early stage of the fifth (last) larval instar. But, if the gland was placed there during the fourth larval-larval pharate stage, histolytic changes, e.g. invagination of tunica propria, its separation from the cell and contraction of the cell, took place in the tissue. These results suggest that, once activated, cells in the middle region of the middle silk gland undergo degenerative changes even in the presence of the corpus allatum hormone during the larval-larval pharate period.     

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.     

Changes in vitellin and other yolk proteins during embryonic development in the silkworm, Bombyx mori

Changes in the amounts of vitellin and other yolk proteins of the eggs of the silkworm, Bombyx mori were investigated during embryonic development using polyacrylamide gel electrophoresis and immunotitration techniques. In the newly laid eggs, soluble proteins were separated into at least nine bands after electrophoresis. The major band was identified as vitellin, accounting for about 40% of the total proteins. The four predominant bands including vitellin exhibited the same mobility as the proteins of haemolymph, but one other major band was specific to the eggs, accounting for about 20% of the proteins.During embryonic differentiation 6–7 days after oviposition, the total protein content did not decrease and the banding patterns and their relative concentrations remained unchanged as a whole. However the concentration of the egg specific protein steadily decreased. During subsequent larval differentiation until hatching, the total proteins were utilized to about 50% of the initial levels: the rapid degradation was observed in almost every species of proteins.An immunotitration experiment further demonstrated that vitellin was not utlilized during embryonic differentiation but was consumed markedly during larval differentiation. However, about 30% of initial level was reserved in the newly hatched larvae. Such a prolonged persistence of vitellin is discussed in relation to protein metabolism during embryonic development in silkworms.     

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.     

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.     

Primary structure of Bombyx mori posterior silkgland tRNAPhe

tRNA^Phe was isolated from posterior silkgland from Bombyx mori and hydrolysed to mixtures of oligonucleotides. |³²P|5′ end labelling of the oligonucleotides and sequence study indicates that the major component of Bombyx mori tRNA^Phe is similar to mammalian tRNA^Phe, the minor component differing from the major one by one nucleotide only.     

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.     

Gene-controlled incorporation of haemolymph protein into the ovaries of Bombyx mori

The haemolymph protein concentration in Bombyx mori decreases normally by about one-fourth during pharate adult development. In females homozygous for the small egg gene, the concentration of haemolymph protein remained constant throughout the pupal and pharate adult stages. The sm gene does not influence the synthesis of vitellogenic female protein of pupal and pharate adult haemolymph (FP). Normal ovaries transferred to the haemocoele of sm females undergo normal vitellogenesis. In the absence of normal alleles of sm, the ovaries encounter difficulties in the incorporation of FP into their oöcytes from pharate adult haemolymph. These results suggest that an active translocation mechanism is involved in the transfer of haemolymph protein into the ovaries.     

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.