Polyoxin N,a New Member of the Polyoxin Family

Eclosion hormone was purified 5,000-fold from the extracts of male adult heads of the silkworm, Bombyx mori. The fourteen-step purification procedure consisting of solvent extractions, fractional precipitations and chromatographies afforded a partially purified preparation of eclosion hormone, 1.8 μg of which showed activity in a Bombyx pharate adult. The hormone was inactivated by proteolytic enzymes. The molecular weight of eclosion hormone was estimated to be 8,400 ± 1,000 daltons by gel-filtration on Sephadex G-50.     

Neurohormones in developing embryos of the silkworm,Bombyx mori: The presence and characteristics of prothoracicotropic hormone B

The presence and fluctuation of prothoracicotropic hormone (PTTH) activity have been shown in brainless Bombyx mori pupae and in developing embryos. The embryonic PTTH is only effective in eliciting adult development of brainless pupae. PTTH activity first became detectable in embryos which had developed almost to the stage of differentiation of their neuroendocrine systems. The hormone was partially purified from pharate first-instar larvae (1 day before hatching) and approx a 300-fold purification was achieved. 0.2 μg of the partial purified hormone can evoke adult development in brainless pupae. The molecular weight of the hormone was estimated to be 20,000–30,000 daltons by gel-filtration on a Sephadex G-50. Two distinct forms of PTTH are present in developing embryos of B. mori.     

Eclosion hormone activity in haemolymph of eclosing silkmoth, Bombyx mori

The presence of eclosion hormone in the haemolymph of eclosing adults of the silkworm, Bombyx mori, was demonstrated to provide evidence that the hormone is involved in the induction of eclosion of this insect.The eclosion of the adult occurred within 55–75 min after light-on, when the pupae had been kept under conditions of 16 h light and 8 h dark during adult development. The hormone became detectable in the haemolymph 10–20 min after light-on, followed by eclosion 40 min later. The hormonal activity increased sharply to a maximal level (30 units/ml haemolymph) at 30 min prior to the eclosion and then fell rapidly: detectable eclosion hormone was present in the haemolymph for only 15–20 min. The hormone was partially purified from the haemolymph of eclosing animals and a 400-fold purification was achieved. The preparation was rather stable against heat treatment, and the molecular weight was estimated to be 5,000–10,000 by gel-filtration on a Sephadex G-50 (superfine). The origin of eclosion hormone in the haemolymph is discussed.     

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.     

Effect of the Molecular Chain Length on Biological Activity of Juvenile Hormone Analogs

Twenty six juvenile hormone analogs with various molecular chain length were prepared and bioassayed using allatectomized 4th instar larvae of Bombyx mori L. Among methyl or ethyl esters, the chain length of 17 atoms is the optimal for the high juvenile hormone activity on the silkworm.     

Relationship between the expression of Rab family GTPases and neuropeptide hormones in the brain of Bombyx mori

Rab proteins are small GTPases that play essential roles in vesicle transport. In this study, we examined the expression of Rab proteins and neuropeptide hormones in the brain of the silkworm, Bombyx mori. We produced antibodies against B. mori Rab1 and Rab14 in rabbits. Immunoblotting of samples of brain tissue from B. mori revealed a single band for each antibody. Rab1 and Rab14 immunohistochemical labeling in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Rab1, Rab7 and Rab14 co-localized with bombyxin. Rab1 and Rab7 co-localized with eclosion hormone. Rab1 co-localized with prothoracicotropic hormone. These results suggest that Rab1, Rab7 and Rab14 may be involved in neuropeptide transport in the brain of B. mori. This is the first report on the specificity of Rab proteins for the secretion of different neuropeptides in insects.     

Relationship between photoperiod and secretion of the diapause hormone during larval stages of the silkworm, Bombyx mori L., reared on an artificial diet

The suboesophageal ganglion of the silkworm, Bombyx mori synthesizes sufficient diapause hormone to produce diapause eggs, regardless of the photoperiodic conditions experienced during the larval stages. When larvae destined to produce non-diapause eggs are implanted with the brain-suboesophageal ganglion complex from larvae which have been reared under short-day conditions, the resulting adults lay diapause eggs. The larvae receiving the complex from larvae reared under long-day conditions gave rise to adults which did not produce any diapause eggs. The brains from pupae which have been reared under long-day conditions show an activity inhibiting the secretion of diapause hormone by the suboesophageal ganglion. The mechanism through which the brain controls the secretion of diapause hormone from the suboesophageal ganglion can be modified by photoperiodic conditions during the larval stages.     

Hormonal basis of adult eclosion in the gypsy moth (Lepidoptera: Lymantriidae)

Eclosion hormone was found to control the stereotypic adult eclosion behaviour of Lymantria dispar, the gypsy moth. A bioassay for hormonal activity was developed utilizing pharate adult females, and comparisons were made with the Manduca wing assay. The distribution of eclosion hormone activity was confined to the central nervous system tissues including the protocerebrum, corpora allata/corpora cardiaca complex, thoracic and the last abdominal ganglion. Haemolymph ecdysteroid titres were determined daily throughout pupal-adult development, and the peak activity period was found in 3–4 day pupae. Eclosion hormone activity in the brain and corpora allata/corpora cardiaca complex started to increase when the ecdysteroid titre dropped to background levels. Eclosion hormone in the brain peaked in the pharate adult stage, was released in the haemolymph 1 h prior to eclosion, which coincides with the depletion of activity in the retrocerebral complex, and fell to undetectable levels after the adult emerged.     

Effects of ingested phytoecdysteroids on the growth and development of two Lepidopterous larvae

The effects of ingested or injected 20-hydroxyecdysone on silkworm larvae (Bombyx mori) including death without moulting, death following completion of promoted moulting, death during promoted moulting (ecdysis inhibition) and inhibition in growth with and without effects on moulting, are dependent upon the concentration of exogenous hormone, the precise developmental stage of the treated larvae, and the duration of exposure to the exogenous ecdysteroid. Comparisons of 20-hydroxyecdysone with other phytoecdysteroids in the silkworm and pink bollworm, Pectinophora gossypiella, show a similar but more potent effect induced by ponasterone A, while cyasterone causes an ‘antiecdysone’ effect.     

Consumption of Bt Rice Pollen Containing Cry1C or Cry2A Protein Poses a Low to Negligible Risk to the Silkworm Bombyx mori (Lepidoptera: Bombyxidae)

By consuming mulberry leaves covered with pollen from nearby genetically engineered, insect-resistant rice lines producing Cry proteins derived from Bacillus thuringiensis (Bt), larvae of the domestic silkworm, Bombyx mori (Linnaeus) (Lepidoptera: Bombyxidae), could be exposed to insecticidal proteins. Laboratory experiments were conducted to assess the potential effects of Cry1C- or Cry2A-producing transgenic rice (T1C-19, T2A-1) pollen on B. mori fitness. In a short-term assay, B. mori larvae were fed mulberry leaves covered with different densities of pollen from Bt rice lines or their corresponding near isoline (control) for the first 3 d and then were fed mulberry leaves without pollen. No effect was detected on any life table parameter, even at 1800 pollen grains/cm² leaf, which is much higher than the mean natural density of rice pollen on leaves of mulberry trees near paddy fields. In a long-term assay, the larvae were fed Bt and control pollen in the same way but for their entire larval stage (approximately 27 d). Bt pollen densities ≥150 grains/cm² leaf reduced 14-d larval weight, increased larval development time, and reduced adult eclosion rate. ELISA analyses showed that 72.6% of the Cry protein was still detected in the pollen grains excreted with the feces. The low exposure of silkworm larvae to Cry proteins when feeding Bt rice pollen may be the explanation for the relatively low toxicity detected in the current study. Although the results demonstrate that B. mori larvae are sensitive to Cry1C and Cry2A proteins, the exposure levels that harmed the larvae in the current study are far greater than natural exposure levels. We therefore conclude that consumption of Bt rice pollen will pose a low to negligible risk to B. mori.     

Cyclic AMP phosphodiesterase activity during the development of the silkworm, Bombyx mori

Cyclic AMP phosphodiesterase activity has been demonstrated in homogenates of the silkworm, Bombyx mori, throughout larval, pupal, and pharate adult life. The enzyme exhibits a pH optimum at pH 8·0, and the enzyme activity was inhibited in the presence of caffeine and theophylline. The enzyme activity increases after hatching, and a relatively higher level of the activity is maintained through the larval stage. The activity, however, falls markedly before pupation and increases sharply once again before emergence.     

Eclosion and hatching in cockroach first instar larvae: A stereotyped pattern of behaviour

Cockroach pharate first instar larvae (Periplaneta americana) spontaneously initiate eclosion and hatch from the oötheca after 30 days' incubation at 29°C. The caudal-to-rostral peristaltic movements involved in both eclosion and hatching are initiated even when the first instar larvae have been removed from the oötheca and incubated separately in organ culture dishes. Therefore, environmental stimuli and conditions in situ are not necessary for the onset of eclosion. However, environmental factors associated with the cuticle control the termination and/or duration of the eclosion behaviour sequence. Larvae which had cuticles glued to their bodies had longer than normal eclosion episodes while larvae which experienced premature cuticle removal immediately ceased the movements. Cuticle removal immediately ‘switched’ behaviour from that of the larva to that of the adult with the characteristic walking gait. Eclosion in larvae removed from the oötheca could be initiated by tactile stimulation. A rôle for this response in synchronizing the eclosion-hatching movements of the many larvae within an oötheca is suggested.     

Occurrence of α-ecdysone in the developing embryos of the silkworm, Bombyx mori

From an acetone-ethanol extract of the developing embryos of the silkworm, Bombyx mori, the presence of α-ecdysone, but not of β-ecdysone, was shown by high pressure liquid chromatography and bioassay. The amount of α-ecdysone was estimated to be 0.74 μg per gram of eggs. The absence of a hydroxylating system at C-20 in the embryos is suggested.     

Molecular characterization and expression analysis of BmNOX in two strains of Bombyx mori with contrasting viral resistance phenotype

We recently documented the identification of a 26.5 kDa protein named BmNox in the gut fluid of Nistari strain of Bombyx mori, which possessed antiviral activity against BmNPV in vitro. In this report, we report the characterization of the full‐length gene encoding BmNOX and the levels of expression of this gene in select tissues of silkworm larvae from a BmNPV‐susceptible and a BmNPV‐resistant strain to the defense capability in Bombyx mori larvae challenged with BmNPV. We also evaluated the BmNox expression in various stages of larval life of a resistant and a susceptible strain of Bombyx mori selected from among a panel of strains of silkworm. Nistari, a multivoltine strain of silkworm, expressed BmNOX during all five larval stages, and were highly resistant to BmNPV infection. In sharp contrast, CSR₂, a bivoltine strain, showed weaker expression of BmNOX in the anterior midgut in larval life and was highly susceptible to BmNPV infection. BmNOX is a secretory protein with dual expression in gut fluid and mid gut tissue. BmNOX is expressed heavily in the posterior mid gut, with weaker expression in the fore‐ and mid‐gut regions. © 2010 Wiley Periodicals, Inc.     

Synthesis and Anti-juvenile Hormone Activity of 1-Citronellyl-5-substituted Imidazoles

A number of 1-citronellyl-5-substituted imidazoles were synthesized and bioassayed on the silkworm, Bombyx mori, in order to assess their anti-juvenile hormone activity. Most of the 1-citronellyl-5-substituted imidazoles induced precocious metamorphosis in the 3rd and 4th instar larvae of B. mori by topical application. The percentage of precocious metamorphosis correlated well with the dosage. Among the compounds tested, l-citronellyl-5-(2-chlorophenyl)imidazole (8) and the 2-methylphenyl analog 10 showed the highest activity. When compounds 8 and 10 were applied to the 4th instar larvae at 10 μg/larva, precocious pupation was induced in 100% without any lethal effects.     

Effects of anti-juvenile hormone “ETB” on the development and metamorphosis of the silkworm, Bombyx mori

As in the tobacco hornworm Manduca sexta, the synthetic juvenile hormone analogue ETB (ethyl 4-[2-(tert-buthylcarbonyloxy)butoxy]benzoate) showed both juvenile hormone-like and anti-juvenile hormone activities in the silkworm, Bombyx mori. When ETB was topically applied to allatectomized 4th-instar larvae, the compound counteracted the effects of allatectomy, such as induction of precocious metamorphosis and black pigmentation in the larval markings. Therefore, ETB had juvenile hormone activity, but it could neither induce brown pigmentation in the markings nor induce an extra-larval moult as can juvenile hormone.When intact 3rd-instar larvae were treated with the compound, the majority underwent precocious metamorphosis in the 4th-instar, and later formed fertile miniature adults. Some moulted into larval-pupal intermediates or 5th-instar larvae with darkened larval markings and/or with abnormality of specific regions of the silk-gland. The optimal dose for such anti-juvenile effects was about 1–10 μg/larva, and higher doses showed less activity. Such anti-juvenile hormone effects of ETB were counteracted by administration of the juvenile hormone analogue, methoprene, before a certain critical time in the 4th-instar. The corpora allata of treated larvae appeared cytologically normal, and the corpora allata from ETB-induced miniature moths secreted juvenile hormone when implanted into allatectomized 4th-instar larvae.     

Maturation and hatching of eggs from silkworm ovaries preserved in liquid nitrogen

Ovarian imaginal discs prepared from fifth-instar larvae of the silkworm, Bombyx mori were treated with graded concentrations of glycerol, cooled at a rate of 1°C/min to ?35°C and preserved in liquid nitrogen for 2 days or more and then rapidly thawed (500°C/min). The frozen and thawed ovaries were transplanted into fifth-instar female larvae, in which more than 20% of the ovaries developed to produce mature eggs with a chorion according to the state of host development. By parthenogenetic activation, the mature eggs started embryogenesis and hatched to produce larvae. About 50% hatching occurred in the eggs developed in a C 108 × Cambodge host, and about 10% in a C 108 × Aojuku host. The hatched larvae completed post-embryonic development as did the normal larvae.     

A virus-inactivating protein isolated from the digestive juice of the silkworm, Bombyx mori

A protein that can precipitate nuclear polyhedrosis virus (NPV) in vitro was isolated from the digestive juice of silkworm larvae (Bombyx mori) by the procedures of gel filtration and ion-exchange and hydroxylapatite column chromatography. The SDS-polyacrylamide gel electrophoretic and the ultracentrifugal analyses showed that the purified substance was a homogenous simple protein. The molecular weight of the purified protein was 27,000–28,000 and the sedimentation coefficient was 2.61 S. This protein had an additional activity to inactivate NPV of B. mori in vitro, somewhat analogous to serological neutralization by serum proteins. Electron microscope observations showed that amorphous materials could be found on the surface of envelopes and that the nucleocapsids disappeared.     

The delta-endotoxin of Bacillus thuringiensis. V. On the occurrence of endotoxin fragments in hemolymph

Leucine-³H labeled crystals of Bacillus thuringiensis δ-endotoxin were fed to last-instar larvae of spruce budworm, Choristoneura fumiferana, eastern forest tent caterpillar, Malacosoma disstria, and silkworm, Bombyx mori. Radioactivity was detected in hemolymph 1 min after feeding in the first two species, but not until 3–5 min after feeding in silkworm larvae. Most of the radioactivity from hemolymph of all three species eluted from gel filtration columns at the same elution volume indicating similar moleculear weights (<1800 daltons).     

Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus

The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production.