Toshinobu Yaginuma

Cell cycles in embryos of the silkworm, Bombyx mori: G2-arrest at diapause stage

SummaryIn the silkworm, Bombyx mori, diapause occurs at a specific embryonic stage, i.e. after formation of the germ band with cephalic lobes and telson and sequential mesoderm segmentation. As long as the eggs are incubated at 25° C, cell divisions and morphological development of the embryos cease. To examine changes in percentage of embryonic cells in the G1, S and G2 phases during embryogenesis, nuclear fractions were isolated from embryos, stained with propidium iodide and then subjected to flow cytometric analysis. The percentages of embryonic cells in G1, S and G2 were 10, 35 and 55%, respectively, at the stage of formation of cephalic lobes, whilst 98% of cells were in G2 at diapause stage. After termination of diapause by acclimation at 5° C or by a combination of chilling and HCl, cell division resumed in the embryos. During this period, the cells rapidly entered S phase through G1 from G2, suggesting that their G1 phase was short. In eggs in which diapause was averted by HCl-treatment after incubation at 25° C for 20 h after oviposition, embryonic development proceeded continuously for 9.5 days at 25° C until hatching. Along with this development, the G1 fraction increased to levels of about 90%. These results indicate that embryonic cells are arrested in G2 at diapause and suggest that, concomitant with further embryonic development, cell cycles become slower in proportion to an increasing length of G1. Finally, most of the cells may be arrested in G1, while there is only a small fraction of cells continuously cycling.

Polyol metabolism related to diapause in Bombyx eggs: Different behaviour of sorbitol from glycerol during diapause and post-diapause

Metabolic relationships between glycogen, sorbitol and glycerol were studied through the entire period of diapause in silkworm eggs which had been labelled with 14C-glucose injected during oogenesis. Modified instant thin layer chromatography was suitable for the separation of the carbohydrate components by its rapidity and simplicity and it was confirmed that glycogen, sorbitol and glycerol were then main carbohydrate components of the eggs. At the initiation and termination of diapause an intimate interconversion between glycogen and sorbitol was demonstrated by the coincident and complementary changes and the comparable specific radioactivity. However, the temporal changing pattern of glycerol was different from that of sorbitol: a delayed accumulation occurred through diapause and a prolonged persistence remained after diapause termination. Furthermore, radioactivities per C-atom of glycerol were shown to be constant throughout the entire period of diapause but slightly lower than those of sorbitol. From these results the metabolic correlation and physiological roles of sorbitol and glycerol are discussed in relation to diapause phenomenon in silkworm eggs.

NAD-dependent sorbitol dehydrogenase activity in relation to the termination of diapause in eggs of Bombyx mori

Abstract Some properties of NAD-dependent sorbitol dehydrogenase (NAD-SDH) were determined in crude extracts from chilled, diapause eggs of Bombyx mori. Optimum pH was approx. 8.8 and the apparent K m values for NAD and sorbitol were 0.20 mM and 136 mM, respectively. In the reaction, sorbitol was stoichiometrically converted to fructose, but glycerol and mannitol were not oxidized. During early embryonic development (up to 2 days after oviposition) NAD-SDH activity was almost undetectable in both non-diapause and diapause types of eggs. The activity remained at this level in the diapausing eggs while it increased with age in the non-diapausing ones. After chilling the diapause eggs for 3 months, NAD-SDH activity began to increase gradually and then rose markedly. Also, a rapid increase in NAD-SDH activity was observed in eggs which were treated with HCl to break diapause quickly. These results together with the changing patterns in sorbitol concentrations in the eggs suggest that NAD-SDH is a key enzyme for sorbitol degradation at the termination of diapause in eggs of B. mori. The biochemical significance of NAD-SDH is also discussed in relation to the pathway of glycogen synthesis from sorbitol.

Silkworm Eggs at Low Temperatures: Implications for Sericulture

Sericulture and apiculture are unique among the field of general entomology, because they contribute to the development of technical bases for the use of insects as a bioresource for human life. Sericulture usually includes all phases of raw silk production and has a long history of developing industrial techniques. Rapid development has been achieved by the improvement of silkworm strains using hybrid breeding and the expansion of the rearing season by controlling embryogenesis (Yokoyama, 1973). Artificial hatching of eggs was less important when larvae were reared only in the spring, for under natural conditions larval hatching takes place in the spring in conceit with the growth of mulberry leaves. In nature, diapause eggs laid in spring never hatch before a long period of cold exposure during hibernation. Thus, no larvae appear in summer or autumn even when mulberry leaves are available. To elicit egg hatch for summer- and autumn-rearing, intense efforts have been made in studies of diapause, cold storage, and artificial hatching of silkworm eggs. This research has not only clarified the physiological mechanisms of diapause, but it has also provided appropriate techniques for obtaining hatchable eggs at any time of the year.

FXPRL-amide peptides induce ecdysteroidogenesis through a G-protein coupled receptor expressed in the prothoracic gland of Bombyx mori

The FXPRL-amide peptide family (pyrokinin/PBAN family) consists of insect peptides that function broadly in insect life processes and are characterized by a conserved C-terminal motif. In the silkworm, Bombyx mori, sex pheromone biosynthesis and induction of embryonic diapause are regulated by peptides from this family. To elucidate other functions of Bombyx FXPRL-amide peptides, we analyzed the tissue expression patterns of two known Bombyx G-protein coupled receptors for these peptides. We found that the Bombyx diapause hormone receptor (BmDHR), is expressed in the prothoracic gland (PG), the organ which synthesizes and releases the insect molting hormones, ecdysteroids. Furthermore, diapause hormone (DH), a member of the Bombyx FXPRL-amide peptides, increases both intracellular Ca(2+) and cAMP concentrations and induces ecdysteroidogenesis in late fifth instar PGs coincident with BmDHR expression in the PGs. DH also has the highest prothoracicotropic activity among the FXPRL-amide peptides, which corresponds well to the ligand specificity of heterologously expressed BmDHR. These results demonstrate that FXPRL-amide peptides can function as prothoracicotropic factors through the activation of BmDHR and may play an important role in controlling molting and metamorphosis.

Distinct effects of different low temperatures on the induction of NAD-sorbitol dehydrogenase activity in diapause eggs of the silkworm, Bombyx mori

SummaryDiapause eggs of the silkworm, Bombyx mori, exposed to 5°C and 0.5°C from 2 or 30 days after oviposition, were examined for changes in contents of glycogen, sorbitol and glycerol. Cold acclimation did not alter the profile of accumulation of sorbitol from that in eggs kept continuously at 25°C. However, acclimation at 5°C resulted in conversion of sorbitol to glycogen, while acclimation at 0.5°C was not accompanied by the utilization of sorbitol. NAD-sorbitol dehydrogenase (NAD-SDH; EC 1.1.1.14) activity was examined in the cold-acclimated eggs. The activity was induced by acclimation at 5°C but not at 0.5°C. Incubation at 0.5°C suppressed any further increase in the activity that had been induced. Temperature-directed changes in NAD-SDH activity paralleled those in sorbitol content. Hatching of the diapause eggs was monitored after cold acclimation for various periods of time and subsequent transfer to 25°C. Incubation at 0.5°C was less effective than 5°C at breaking diapause. The time required for the eggs to hatch in synchrony after acclimation at 5°C coincided with that required for the induction of NAD-SDH activity. These results show that different effects result from acclimation at 5°C and near 0°C with respect to the control of NAD-SDH activity, that utilization of sorbitol is controlled by NAD-SDH activity, and that induction of this activity is temperature-dependent. Furthermore, induction of NAD-SDH activity is involved in the termination of diapause in B. mori.

cDNA cloning, sequencing and temporal expression of the protease responsible for vitellin degradation in the silkworm, Bombyx mori.

1. We have cloned the cDNA encoding the vitellin (Vtn)-degrading protease (30 k Vtn protease and 24 k Vtn protease) of the silkworm, Bombyx mori, and determined the primary structure by sequencing the cDNA along with mRNA. 2. The deduced amino acid sequence comprised 264 amino acid residues and had high homology to the trypsin-like proteases of vertebrates and invertebrates. 3. Northern blot analysis using the cDNA as a probe revealed that the transcription of the Vtn protease gene occurred at the restricted stage of embryogenesis when the protease activity appeared. 4. The in vitro translation experiment demonstrated that a 32 kDa polypeptide was the primary translation product and the translation activity changed according to transcriptional activity. 5. By Western blotting using the antiserum against each Vtn protease, two enzymes were shown to share the common antigenicity, and the titer of both enzyme proteins changed closely related with activity of proteases. 6. These results led us to conclude that the biosynthesis of Vtn protease is regulated at the level of transcription.

Functional analysis of Ultrabithorax in the silkworm, Bombyx mori, using RNAi

The formation of abdominal appendages in insects is suppressed by the Hox genes Ultrabithorax (Ubx) and abdominal-A (abd-A), but mechanisms of the suppression can differ among species. As the function of Ubx and abd-A has been described in only a few species, more data from various insects are necessary to elucidate the evolutionary transition of regulation on abdominal appendages. We examined the function of Ubx in the silkworm Bombyx mori (Bm-Ubx) by embryonic RNA interference (RNAi). This is the first case in which functional analysis for Ubx is performed in lepidopteran insects. Larvae treated with Bm-Ubx dsRNA displayed an additional pair of thoracic leg-like protuberances in A1, whereas the other abdominal segments had no transformation. Our results suggest that Bm-Ubx is a suppressor of leg development in A1.

Insect Morphological Diversification Through the Modification of Wing Serial Homologs

A Wing by Another Name? Wings and their derivatives on the second and third thoracic segments represent the only known dorsal appendages in modern insects; however, the existence of wing homologs in non-winged segments has been suggested from fossil insects. Ohde et al. (p. 495, published online 14 March) identify part of the body wall on the first thoracic segment and pupal dorsolateral outgrowths as modified pairs of wings in the mealworm beetle. These organs could be transformed into wings and thus share the central wing developmental mechanism. Modification rather than loss of dorsal appendages has provided a diversifying mechanism for the insect body plan. Fossil insects living some 300 million years ago show winglike pads on all thoracic and abdominal segments, which suggests their serial homology. It remains unclear whether winglike structures in nonwinged segments have been lost or modified through evolution. Here, we identified a ventral lateral part of the body wall on the first thoracic segment, the hypomeron, and pupal dorsolateral denticular outgrowths as wing serial homologs in the mealworm beetle Tenebrio molitor. Both domains transform into winglike structures under Hox RNA interference conditions. Gene expression and functional analyses revealed central roles for the key wing selector genes, vestigial and scalloped, in the hypomeron and the denticular outgrowth formation. We propose that modification, rather than loss, of dorsal appendages has provided an additional diversifying mechanism of insect body plan.

Changes in the Expression of Soluble and Integral-Membrane Trehalases in the Midgut During Metamorphosis in Bombyx mori

Abstract To elucidate the relationship between soluble trehalase (Treh1) and integral-membrane trehalase (Treh2) in the Bombyx mori midgut, expression profiles for both proteins and mRNAs were examined during metamorphosis by using Western-blotting and quantitative real-time PCR analyses. Two bands of Treh2 (about 74 kDa) were detected in the midgut of 0-day-old 5th (last) instar larvae. Levels of Treh2 decreased as the developing larvae approached spinning (8 days old). In contrast, towards the onset of the spinning stage, Treh1 (68 kDa) was clearly observed, and levels increased until the middle of the pupal stage. Treh2 mRNA expression relative to Bmrp49 mRNA expression was almost constant, although fluctuations were detected. Treh1 mRNA expression relative to Bmrp49 mRNA increased sharply just after spinning. To further examine the expression mechanism of the Treh1 gene in midgut, actively feeding larvae (4 days old) were starved or ligated between the 4th and 5th segments. Injection of a molting hormone into the larval-isolated abdomen led to activation of Treh1, demonstrating that molting hormone acts on the midgut and activates this gene.