Gene expression profiling in the silkworm, Bombyx mori, during early embryonic development

We prepared a cDNA library for a microarray from eggs of the silkworm, Bombyx mori, at the germ-band formation (24 hours after fertilization) stage. Using a microarray constructed with 2,445 ESTs, we screened gene expression profiles during germ-band formation at six specific time points in the early embryonic stages (from the unfertilized egg to the formation of abdominal leg appendages), and determined 241 of these cDNAs to represent genes that were expressed differentially during the germ-band formation stage. These differentially expressed genes grouped into two clusters. In the early and late clusters, 203 and 38 genes were upregulated, respectively. In the upregulated clusters, we isolated several genes that were associated with development and cell communication, including egalitarian, RAD23b, innexin 2, and senescence-associated protein. Northern blot hybridization revealed that the expression patterns of 14 genes had changed in each of the stages. In this study, we assessed changes in the levels of gene expression in relation to the germ-band formation stages in whole Bombyx embryos.     

Physiological and morphological characterization of olfactory descending interneurons of the male silkworm moth, Bombyx mori

In order to understand the neural mechanisms of pheromone-oriented walking in male silkworm moths, Bombyxmori, we have characterized olfactory responses and three-dimensional structure of two clusters (Group-I, Group-II) of descending interneurons in the brain by intracellular recording and staining with lucifer yellow. Neurons were imaged with laser-scanning confocal microscopy. Group-I and Group-II descending interneurons were classified into three morphological types, respectively. In response to the sex pheromone, bombykol, Type-A Group-I descending interneurons showed characteristic flipflopping activity. The Group-I descending interneurons had dendritic arborizations in the lateral accessory lobe and varicose profiles in the posterior-lateral part of the suboesophageal ganglion where the dendritic arborizations of a neck motor neuron (i.e., cv1 NMN) reside. Other types of Group-I descending interneurons exhibited long-lasting suppression of firing. The pheromonal responses of Group-II descending interneurons fell into two classes: brief excitation and brief inhibition. Type-A Group-II descending interneurons showing brief excitation had blebby processes in the posterior-lateral part of the suboesophageal ganglion. Type-B and Type-C Group-II descending interneurons did not have varicose profiles there. Therefore, the neck motor neuron regulating head turning, which accompanies the pheromone-oriented walking, may be controlled by these two types, flipflop and phasic excitation, of descending activity patterns. Accepted: 2 November 1998     

Coordination of flipflopping neural signals and head turning during pheromone-mediated walking in a male silkworm moth Bombyx mori

Male silkworm moths, Bombyx mori, move their heads side-to-side during zigzag walking toward a source of sex pheromone. High-speed video analysis revealed that changes in walking direction were synchronized with this head turning. Thus the direction of the walking is indicated by the direction of the head turning. Head turning was regulated by neck motor neurons which innervate the cervical ventral muscles and the ventral muscles through the second cervical nerve. To determine the role of the `flipflop' state transition in spike activity carried by descending interneurons from the brain to the thoracic ganglion, we recorded pheromonal responses simultaneously from flipflop descending interneurons and a single cervical ventral 1 neck motor neuron. The activity of the cervical ventral 1 neck motor neuron was synchronized to that of the flipflop descending interneurons. The cervical ventral 1 neck motor neuron was morphologically identified using confocal imaging. Our results demonstrate that the flipflop signals play an important role in instructing turning signals during the pheromone-mediated behavior in a male B. mori. Accepted: 11 June 1998     

Carbohydrate metabolism during starvation in the silkworm Bombyx mori

The effect of starvation on carbohydrate metabolism in the last instar larvae of the silkworm Bombyx mori was examined. Trehalose concentration in the hemolymph increased slightly during the first 6 h of starvation and decreased thereafter, whereas glucose concentration decreased rapidly immediately after diet deprivation. Starvation-induced hypertrehalosemia was completely inhibited by neck ligation, suggesting that starvation stimulates the release of a hypertrehalosemic factor(s) from the head. The percentage of active glycogen phosphorylase in the fat body increased within 3 h of starvation and its glycogen content decreased gradually. These observations suggest that production of trehalose from glycogen is enhanced in starved larvae. However, hypertrehalosemia during starvation cannot be explained by the increased supply of trehalose into hemolymph alone, as similar changes in phosphorylase activity and glycogen content in the fat body were observed in neck-ligated larvae, in which hemolymph trehalose concentration did not increase but decreased gradually. When injected into larvae, trehalose disappeared from hemolymph at a rate about 40% lower in starved larvae than neck-ligated larvae. The hemolymph lipid concentration increased during starvation, suggesting that an increased supply of lipids to tissues suppresses the consumption of hemolymph trehalose and this is an important factor in hypertrehalosemia.     

Storage proteins in the silkworm,Bombyx mori

In the silkworm, Bombyx mori, two storage proteins named SP-1 and SP-2 were shown to decline in concentration in the haemolymph and increase in the fat body during the larval-pupal transformation, when protein granules are formed in the fat body at the same time as the degeneration of mitochondria and endoplasmic reticulum. At the larval-pupal ecdysis, in females the two proteins account for 60% of total fat body protein (80% of the soluble protein), while males have very little SP-1 and SP-2 comprises only 20% of the total fat body protein. The concentration of protein granules in the fat body cytoplasm is much greater in females than in males, and the granules in females have partially crystalline inner zones. This is different from males where granules with non-crystalline structure are most numerous.The properties of these proteins purified from pupal fat body are similar to those of Cecropia storage proteins and calliphorin, all of which have molecular weights of around 500,000 and are composed of subunits of mol. wt about 85,000. SP-1 differs from SP-2 by having an exceptionally high content of methionine, but much less glutamate, phenylalanine and tyrosine. SP-1 resembles another female-specific protein, vitellogenin and SP-2 resembles calliphorin in amino acid composition.From these results, it is concluded that SP-1 and SP-2 have storage roles and are deposited in protein granules.     

Immunocytochemical location of vitellin in the egg of the silkworm,Bombyx mori,during early developmental stages

Summary Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.     

Ecdysteroids during early embryonic development in silkworm Bombyx mori: metabolism and functions

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various molecular species of ecdysteroids in free and conjugated forms. In B. mori eggs, 20-hydroxyecdysone (20E) is a physiologically active molecule. In nondiapause eggs, 20E is produced by the conversion of maternal conjugated ecdysteroids (ecdysteroid-phosphates) and by de novo biosynthesis. In contrast, in diapause eggs, neither of these metabolic processes occurs. In de novo biosynthesis of 20E in B. mori eggs, hydroxylation at the C-20 position of ecdysone, which is catalyzed by ecdysone 20-hydroxylase, is a rate-limiting step. Furthermore, we found that a novel enzyme, called ecdysteroid-phosphate phosphatase (EPPase), specifically catalyzes the conversion of ecdysteroid-phosphates to free ecdysteroids. The developmental changes in the expression pattern of EPPase mRNA correspond closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs. EPPase is localized in the cytosol of yolk cells, and the bulk of maternal ecdysteroid-phosphates is bound to vitellin and stored in yolk granules. The vitellin-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase. Therefore, to examine how ecdysteroid-phosphates are hydrolyzed by EPPase during embryonic development further investigations were focused on yolk granules. Recent data indicate that acidification in yolk granules, induced by vacuolar H(+)-ATPase, triggers the dissociation of ecdysteroid-phosphates from the vitellin-ecdysteroid-phosphates complex and the dissociated ecdysteroid-phosphates are released from yolk granules to the cytosol. To explain the process of the increase in the level of 20E during embryonic development in B. mori eggs, a possible model is proposed.     

Characterization of termite lipophorin and its involvement in hydrocarbon transport

The transport of lipids constitutes a vital function in insects and requires the plasma lipoprotein lipophorin. In all insects examined to date, cuticular hydrocarbons are also transported through the hemolymph by lipophorin, and in social insects they play important roles not only in water proofing the cuticle but also in nestmate recognition. High-density lipophorin (HDLp), isolated from Reticulitermes flavipes plasma by KBr gradient ultracentrifugation, contains 66.2% protein and 33.8% lipids; hydrocarbons constitute its major neutral lipid (20.4% of total lipids). Anti-lipophorin serum was generated in rabbit and its specific association with lipophorin, and not with any other plasma proteins, was verified with Western blotting. Immunoprecipitation also confirmed that this antibody specifically recognizes lipophorin, because all hemolymph hydrocarbons of the termites R. flavipes and R. lucifugus and the cockroach Supella longipalpa, which associate only with lipophorin, were recovered in the immunoprecipitated protein. Cross-reactivity of the antiserum with lipophorin from related species was investigated by double immunodiffusion with 10 termite species in the genera Reticulitermes, Coptotermes, Zootermopsis, and Kalotermes, and with five cockroach species. Involvement of lipophorin in hydrocarbon transport was shown by injecting HDLp antiserum into Zootermopsis nevadensis and then monitoring the de novo biosynthesis of hydrocarbons and their transport to the cuticular surface; the antiserum significantly disrupted hydrocarbon transport. ELISA revealed a gradual increase in the lipophorin titer in successively larger R. flavipes workers, and differences among castes in lipophorin titers were highest between nymphs and first instar larvae.     

Changes in calmodulin levels during development of the silkworm,Bombyx mori

Calmodulin levels were measured in various tissues during the larval-adult development of the silkworm, Bombyx mori. In the larval period, calmodulin levels in fat body, midgut and testis were in a range of 0.3–1.7 μg/mg protein and remained almost constant during larval growth. The silk gland contained a relatively high (0.2 μg/mg protein) level of calmodulin early in the fifth instar which gradually decreased during maturation of the larva. At pupation, testis calmodulin dropped from 1.5 to 1.7 μg/mg protein to about 1 μg/mg, and remained constant thereafter. The most striking change occurred in fat body calmodulin which fell from 0.5 to 0.6 μg/mg in the larval stage to 0.01–0.03 μg/mg during pupal-adult metamorphosis. Midgut calmodulin levels were unchanged at pupation and remained constant during pupal-adult development.When expressed on per g wet weight basis, calmodulin levels in silkworm tissues were comparable to mammalian tissue levels. However, only 2–4% of the total calmodulin in silkworm tissues was in a membrane-bound form compared to 20–60% for membrane-bound calmodulin in mammals.     

家蚕拟浆细胞具有吞噬功能

目的：通过观察家蚕Bombyx mori吞噬细胞的微细结构,来确定拟绛色细胞是否也具有吞噬功能。方法：用荧光小球微量注射家蚕pnd pS品系的幼虫,经荧光染色剂丫啶橙和碘化丙啶染色循环血细胞后,在荧光显微镜下观察并扫描拍摄。结果：观察发现除颗粒细胞和浆血细胞外,一些原血球细胞（血干细胞）和拟绛色细胞（多酚氧化酶）也能吞噬荧光小球。在拟绛色细胞里还发现许多和颗粒细胞一样的能被丫啶橙染色的颗粒。尽管在小球细胞中没有发现被吞噬的荧光小球,但该类血球有比较多的能被丫啶橙染色的大颗粒,这表明它们可能是已经被吞噬的凋亡小体。结论：除颗粒细胞和浆血细胞外,一些原血球细胞和拟绛色细胞也能吞噬荧光小球。说明拟绛色细胞也具有吞噬功能。     

Dietary sterol preference in the silkworm, Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that beta-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: beta-sitosterol > ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouth part might be different from the sterol recognition mechanism present in sterol metabolism.     

Changes in lipid droplet localization during embryogenesis of the silkworm, Bombyx mori

Lipid droplets are considered one of the most important energy sources in lepidopteran eggs during late embryogenesis, but the process of their incorporation into the embryo is as yet unknown. The present study focused on the process of transition of lipid droplets from the extraembryonic yolk to the embryo of the silkworm Bombyx mori, using morphological and biochemical approaches. The morphological study revealed that the incorporation of lipid droplets from the extraembryonic yolk into the embryo occurs at three points and in three different ways during the development of the embryo. Some lipid droplets were translocated directly from the extraembryonic yolk to the embryo before the blastokinesis stage. However, the majority of lipid droplets together with the other components of the extraembryonic yolk were incorporated in the embryo via both morphogenetic inclusion during dorsal closure and ingestion of the extraembyonic yolk by the developing caterpillar prior to hatching. Similar results were obtained from the biochemical study. Thus, we propose that there are three steps in the incorporation of lipid droplets from the extraembryonic yolk into the embryo. In addition, morphological and biochemical data concerning the total amount of lipid droplets in the egg suggested that lipid droplets were mainly consumed during late embryogenesis, seeming to synchronize with tracheal development.     

Characterization of Tudor-sn-containing granules in the silkworm,Bombyx mori

The Tudor-sn protein, which contains four staphylococcal nuclease domains and a Tudor domain, is a ubiquitous protein found in almost all organisms. It has been reported that Tudor-sn in mammals participates in various cellular pathways involved in gene regulation, cell growth, and development. In insects, we have previously identified a Tudor-sn ortholog in the silkworm, Bombyx mori, and detected its interactions between with Argonaute proteins. The role of Tudor-sn in silkworm, however, still remains largely unknown. In this study, we demonstrated that silkworm Tudor-sn is a stress granule (SG) protein, and determined its interactions with other SG proteins using Bimolecular Fluorescence Complementation assay and Insect Two-Hybrid method. Depletions of Argonaute proteins and SG-marker protein Tia1 by RNAi impaired the involvement of Tudor-sn in the SG formation. Protein domain deletion analysis of Tudor-sn demonstrated that SN2 is the key domain required for the aggregation of Tudor-sn in SGs.     

Sterol composition in larvae of the silkworm, Bombyx mori

Sterols in silkworm larvae were analyzed. Cholesterol was predominantly detected in all tissues examined. Dietary phytosterols and desmosterol, a putative biosynthetic intermediate from phytosterols to cholesterol, were also detected, indicating that imperfect intestinal conversion from phytosterols to cholesterol influences the sterol composition in larval tissues.     

家蚕Hox基因研究进展

Hox基因（homeobox genes）在昆虫躯体模式（body plan）的发育调控机制中扮演着重要角色,其表达具有严格的组织特异性和胚胎发育的程序性。家蚕Bombyx mori作为鳞翅目昆虫的代表,其Hox基因也陆续得到鉴定。在家蚕中存在一个拟复等位基因群--E群基因,其突变表型均与过剩斑纹和过剩附肢有关,这可能与Hox基因有着密切联系。家蚕全基因组测序完成后,发现其Hox基因簇中存在12个特有的homeobox基因（Bmshx1~Bmshx12）, 说明家蚕Hox基因可能具有独特的生物学意义。我们还利用家蚕基因芯片数据分析了Bmlab与Bmpb基因的组织表达特征。通过对家蚕Hox基因的研究,探索家蚕躯体模式建立机制,可望为解析其他鳞翅目昆虫的躯体模式的建立机制提供理论依据。本文就家蚕Hox基因的表达、功能及其与E群突变的关系等方面进行了综述。     

The early embryonic mitosis in normal and cooled eggs of the silkworm,Bombyx mori

Early embryonic mitosis of the silkworm, Bombyx mori, was morphologically studied in the normal eggs and in the eggs treated by low temperature (?10°C). The first embryonic mitosis is observed in the eggs at 120 to 150 minutes after deposition at 26°C. After egg and sperm pronuclei unite, a spindle is formed in each of the pronuclei independently. At metaphase and anaphase paternal and maternal chromosomes are in separate groups on a spindle (gonomeric) and karyogamy takes place at telophase when they reach the poles. The second embryonic mitosis is shown in the eggs at 180 to 210 minutes after deposition. The division of two nuclei is not synchronous in the silkworm, and the mitosis is not gonomeric. In the eggs treated by low temperature, spindle fibers are not observed at all at ?10°C, and chromosomes, which form two deeply stained masses of irregular shape, are seen in the less stained area of spindle shape. When the eggs are returned to 26°C, some eggs go into normal gonomeric division, while some form two small and compact spindles, which seem to be derived from each of the pronuclei. It was observed that these compact spindles are able to continue mitosis.