A new method of pebrine inspection of silkworm egg using multiprimer PCR

Using a mixture of several PCR primers, we evaluated whether multiprimer PCR is practically useful for the early and simultaneous detection of several kinds of microsporidia that cause silkworm pebrine. When genomic DNA extracted from silkworm eggs infected with Nosema bombycis was used as the DNA template, the specific DNA sequences were amplified by multiprimer PCR. In addition, similar results were obtained even when genomic DNA extracted from silkworms infected with N. bombycis was used as the DNA template. These findings suggest that multiprimer PCR using several primers designed for this study is practically useful for pebrine inspection of silkworm eggs.     

Molecular basis of the silkworm mutant rel causing red egg color and embryonic death

The coloration and hatchability of insect eggs can affect individual and population survival. However, few genetic loci have been documented to affect both traits, and the genes involved in regulating these two traits are unclear. The silkworm recessive mutant re^l shows both red egg color and embryo mortality. We studied the molecular basis of the re^l phenotype formation. Through genetic analysis, gene screening and sequencing, we found that two closely linked genes, BGIBMGA003497 (Bm-re) and BGIBMGA003697 (BmSema1a), control egg color and embryo mortality, respectively. Six base pairs of the Bm-re gene are deleted in its open reading frame, and BmSema1a is expressed at abnormally low levels in mutant re^l. BmSema1a gene function verification was performed using RNA interference and clustered randomly interspersed palindromic repeats (CRISPR)/CRISPR-associate protein 9. Deficiency of the BmSema1a gene can cause the death of silkworm embryos. This study revealed the molecular basis of silkworm re^l mutant formation and indicated that the Sema1a gene is essential for insect embryo development.     

A defective prostaglandin E synthase could affect egg formation in the silkworm Bombyx mori

We had previously reported a prostaglandin E synthase (bmPGES) in the silkworm Bombyx mori that catalyzes the isomerization of PGH₂ to PGE₂. The present study aimed to provide a genome-editing characterization of bmPGES in B. mori. Results showed bmPGES gene disruption to result in a reduced content of PGE₂. The change affected the expression of chorion genes and egg formation in silkworms. Collectively, the results indicated that bmPGES could be involved in reproduction of B. mori. Therefore, this study provides insights into the physiological role of bmPGES and PGE₂ in silkworms.     

Cytology of egg maturation in the silkworm (Bombyx mori L.) in artificial parthenogenesis

The orientation of bivalentss in the first meiotic metaphase was studied on the squash preparations and sections of eggs in Bombyx mori L., Antheraea pernyi Guèr and Carpocapsa pomonella L. The reduction fissure of bivalents separating the homologous chromosomes lies in the plane of spindle equator; the equation fissure separating the sister chromatids is parallel to the long spindle axis. Complicated rearrangements are observed in the nuclei of the silkworm eggs stimulated to ameiotic parthenogenesis by the high temperature dissociation of bivalents, full destruction of spindle, formation of a new spindle and metaphase plate. The latter includes univalent chromosomes involved in the equation division. As a result, two genetically identical pronuclei with the somatic set of chromosomes are formed.     

家蚕保存系统红色卵的遗传分析

用遗传背景清楚的家蚕Bombyx mori红卵（re）、白卵(w-2、pe)、第4褐卵（b-4）的标志基因系统和正常型黑卵系统与我国家蚕基因库保存的20个红色卵系统杂交,进行顺反测验,分析了它们的卵色支配基因及遗传规律。结果发现：①在03-310系统中存在家蚕卵色新突变pink egg,与红卵re 等位,基因符号为re^p,表型特征为：卵淡红色,成虫蛾眼也为淡红色;②6个系统为红卵（re）的纯合系统,还有５个系统除具有re／re基因型外,还具有支配白色卵或浅红色或橙红色卵的突变基因;③２个系统为第4褐卵（b4）的纯合系统; ④6个系统的红褐色卵为母性影响遗传;⑤发现家蚕卵色基因b-4和r-e的互补关系,b-4/b-4 re/re基因型表现为新的卵色——橙黄色。     

Dopamine is a key factor for the induction of egg diapause of the silkworm, Bombyx mori.

The silkworm, Bombyx mori, enters diapause in the early embryonic stage. Embryonic diapause is induced by incubating eggs of the maternal generation at high temperature (diapause type), whereas incubation at low temperature results in non-diapausing progeny (non-diapause type). Measurement of catecholamine concentrations in haemolymph and brain-subesophageal ganglia (Br-SGs) showed that only dopamine concentrations in both tissues are consistently higher in diapause-type than non-diapause-type larvae and pupae. In particular, the difference in dopamine concentrations in both tissues increases around pupal ecdysis. During the early pupal stage, Dopa decarboxylase activities and mRNA concentrations in Br-SGs were also much higher in diapause-type than non-diapause-type insects. Elevation of dopamine levels induced by feeding Dopa to penultimate-instar and last-instar larvae, and by injecting Dopa or dopamine into pupae 2 days after pupation made the non-diapause-destined insects lay diapause-destined eggs at 59% and approximately 70% frequencies, respectively. Furthermore, injection of Dopa or dopamine elevated mRNA levels of the diapause hormone in the Br-SGs of non-diapause-type pupae 1 day after injection. Incubation of Br-SGs isolated from non-diapause-type day-2 pupae with Dopa or dopamine also stimulated the expression of diapause hormone mRNA. These data indicate that environmental stimuli during embryonic development increase dopamine levels in both hemolymph and Br-SGs from the larval stage to early pupal stage, which results in laying of diapause-destined eggs by female adults through enhanced expression of the diapause hormone gene.     

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.     

Is the egg size determining gene,Esd, on the W chromosome identical with the sex-linked giant egg gene,Ge, in the silkworm?

The presence of the egg size-determining (Esd) gene, which acts as a quantitative gene, on the W chromosome of the silkworm was revealed in a previous study by using two types of triploid females, ZZW and ZWW, (Kawamura, Genetica 76: 195–201). The females with the sex-linked giant egg (Ge) gene deposit eggs as large as those laid by tetraploids. If the Ge mutant is induced by translocation of a fragment of the W carrying Esd onto the Z by chance, the egg size increase in the Ge strain and in tetraploids may be easily explained by the double dose of Esd. The measurement of the length of the Z-W bivalent in oocytes showed that the Z of the Ge strain was much longer than that of the other strains which do not carry the Ge gene. The result suggests that the Ge gene is identical with the Esd on the W chromosome of the silkworm.     

Molecular characterization of DnaJ 5 homologs in silkworm Bombyx mori and its expression during egg diapause

A comparison of the cDNA sequences （1 056 bp） of Bombyx mori DnaJ 5 homolog with B. mori genome revealed that unlike in other Hsps, it has an intron of 234 bp. The DnaJ 5 homolog contains 351 amino acids, of which 70 contain the conserved DnaJ domain at the N-terminal end. This homolog orB. mori has all desirable functional domains similar to other insects, and the 13 different DnaJ homologs identified in B. mori genome were distributed on different chromosomes. The expressed sequence tag database analysis of Hsp40 gene expression revealed higher expression in wing disc followed by diapause-induced eggs. Microarray analysis revealed higher expression of DnaJ 5 homolog at 18th h after oviposition in diapause-induced eggs. Further validation of DnaJ 5 expression through qPCR in diapause-induced and nondiapause eggs at different time intervals revealed higher expression in diapause eggs at 18 and 24 h after oviposition, which coincided with the expression of Hsp70 as the Hsp 40 is its co-chaperone. This study thus provides an outline of the genome organization of lisp40 gene, and its role in egg diapause induction in B. mori.     

家蚕不同品系卵巢管数目变异及其与造卵数的关系

选取西南大学家蚕Bombyx mori基因库中157个品系及杂交材料的雌蛾进行解剖和观察,调查了卵巢管数目的多样性,并初步研究了其生物学特性。结果发现,家蚕资源品系大范围存在雌蛾卵巢管数目变异现象。157个品系及其杂交材料中有87个发生了卵巢管数目变异现象,占55.41%,变异类型有11种。不同类品系间卵巢管数目变异率差别显著,其中突变品系的变异率最高,达60.19%;突变系杂交材料的变异率最低,有37.50%。在发生卵巢管数目变异的突变品系中,个体变异率最高达50%,最低为2%。调查部分品系发现,品系内卵巢管数目增加并不见总造卵数一定增加,而单根卵巢管平均造卵数均低于（4,4）正常型。通过对实用型品系21-871和突变系06-051上下世代雌蛾的解剖观察,发现这两个品系的上下代雌蛾卵巢管数目均有相近的变异趋势,提示卵巢管数目变异有遗传性。     

家蚕卵显微注射操作对蚕卵孵化及畸形蚕发生的影响

早期胚胎显微注射是目前获得转基因家蚕Bombyx mori的主要途径。显微注射操作对蚕卵的损伤导致注射后的蚕卵孵化率降低, 是家蚕转基因工作的主要障碍之一。本研究对不同卵龄的蚕卵进行了开孔或注射实验, 并对产后5 h的蚕卵上背侧、 腹侧、 前极、 后极和中央等5个不同的位置进行了开孔实验, 调查了卵孵化率和体形异常蚕的产生情况。结果表明： 较早卵龄期的注射或从蚕卵背侧的注射可以获得高的孵化率。腹侧注射产生大量的体形异常蚕而背侧注射的蚕完全正常。通过调整注射时期和注射位置避开上述影响可以减少死卵和畸形蚕, 提高孵化率。本研究为改进家蚕转基因操作技术提供了有效的参考。     

Evaluation of Staphylococcus aureus virulence factors using a silkworm model

Previous studies have indicated that the silkworm model is useful for identifying virulence genes of Staphylococcus aureus, a human pathogenic bacterium. Here we examined the scope of S.?aureus virulence factors that can be evaluated using the silkworm model. Gene-disrupted mutants of the agr locus, arlS gene and saeS gene, which regulate the expression of cell surface adhesins and hemolysins, exhibited attenuated virulence in silkworms. Mutants of the hla gene encoding α-hemolysin, the hlb gene encoding β-hemolysin, and the psmα and psmβ operons encoding cytolysins, however, showed virulence in silkworms indistinguishable from that of the parent strain. Thus, these S.?aureus cytolysins are not required for virulence in silkworms. In contrast, the gene-disrupted mutants of clfB, fnbB and sdrC, which encode cell-wall-anchored proteins, attenuated S.?aureus virulence in silkworms. In addition, the mutant of the srtA gene encoding sortase A, which anchors cell-wall proteins, showed attenuated virulence in silkworms. These findings suggest that the silkworm model can be used to evaluate S.?aureus cell-wall proteins and regulatory proteins as virulence factors.     

Cleavage in a saponin model of the sea urchin egg

A cell model, in which cleavage could be induced, was obtained from fertilized sea urchin eggs by putting eggs that were in the first cleavage into a solution containing 3 X 10(-5) g/ml saponin and suitable amounts of ATP and Ca2+. The cell membrane became freely permeable to ATP and Ca2+ within 1 minute. The respective optimal concentrations of ATP and Ca2+ that advanced the cleavage furrow in this model were 2 mM and 10(-8) M. With the optimal ATP and Ca2+ concentrations, the cleavage furrow of the model advanced at a rate that differed little from that in living eggs. The cleavage furrow soon receded, however, when the concentration of ATP was decreased to less than 1 mM or increased to more than 3 mM, as well as when the concentration of Ca2+ was increased to more than 10(-7) M.     

RH-5992--an ecdysone agonist on model system of the silkworm Bombyx mori

RH-5992 is a novel synthetic non-steroidal ecdysteroid agonist with a high selectivity towards Lepidopteran species. The effect of ecdysone agonist RH-5992 on larval period, larval weight, silk gland weight and haemolymph protein profile were examined in the model organism, the larvae of silkworm, Bombyx mori. The LD50 values were found to be 16.21 and 12.01 micrograms/larva for 72 and 96 hr respectively. In the present study, three sublethal concentrations of 1/5th, 1/10th and 1/20th of LD50 at 72 hr were selected and applied on the mid-dorsal line of the silkworm B. mori. The maximum mortality of 35% was observed in the group which received the highest (3.2 micrograms/larva) concentration of RH-5992. The mortality rate was found to be dose dependent as well as time dependent. Interesting results were observed in haemolymph profile of the RH-5992 treated larvae as staining intensity of 30 kDa protein decreased significantly whereas the effect was not marked on other major proteins like storage proteins and vitellogenin polypeptides. From the results, it is confirmed that RH-5992 causes changes in larval characters and protein profile of silkworm B. mori. It is proposed that RH-5992 may cause negative effect specifically on reproductive characters like development of ovary and egg production due to decrease in 30 kDa protein.     

A novel system for separation and purification of egg pigments from the nondiapause red-egg mutant Re-nd in the silkworm,Bombyx mori

Re-nd, which was induced from the wild-type C108 by the chemical mutagen N-methane-N-methylnitrourea, is a nondiapause red-egg mutant of silkworm Bombyx mori. The special significance of the Re-nd mutant is that it is an independent dominant mutant. The aim of this study was to establish the type of pigment responsible for the red coloration in the Re-nd mutant eggs in silkworm. We compared the eggs of Re-nd mutants with those of the other B. mori egg color strains and confirmed that the Re-nd mutant is the only strain with red color and red pigment granules in nondiapause, showing this mutant belongs to the pigmentation in the serosa. We speculated that the red substance, which contributed to the bright red pigmentation for nondiapause eggs of the Re-nd mutant, could potentially be a novel pigment according to its solubility, optimum absorption peak, and oxidation–reduction reaction. Moreover, we have successfully constituted the system for enrichment, extraction, and purification of the red substance responsible for the Re-nd mutant, providing a new method for the separation and purification of other known and unknown pigments or substances.