Localization, expression, and secretion pathway of diapause hormone in embryo and larva of the silkworm, Bombyx mori

In this paper, the distribution and expression of diapause hormone (DH) at mRNA and protein levels in the central nervous system of Bombyx mori embryo and larva were studied using whole-mount in situ hybridization and immunocytochemistry. Whole-mount immunocytochemistry revealed that the distribution of the DH-like immunoreactivity was throughout the central nervous system including the brain, suboesophageal ganglion (SG) and thoracic ganglia(TG); and that the corpus cardiacum and terminal abdominal ganglion may be the site for DH release due to the presence of strong immunoreactivity. In situ hybridization with the probe labeled by digoxigenin shows that the BomDHmRNA was also localized in the mandibular, maxillary, labial cell clusters. In addition, a pair of lateral neurons in the SG and a pair of ventral midline neurons in each TG expressing the Bom-DH transcript were also identified. Thesere sults were consistent with the localization of Bom-DH mRNA in larva by in situ hybridization and the distribution of the gene by RT-PCR, which is some different from the results reported previously.

Prevalence of Muscardine Disease in Different Silkworm Hybrid, Bombyx Mori L. under Agro-climatic Conditions in Aurangabad （M.S.）, India

A study was carried out in Dr. Babasaheb Ambedkar Marathwada University Campus Aurangabad, Maharashtra State, India for analyzing the prevalence of Muscardine disease during rainy, winter and summer season in bivoltine x bivoltine hybrids viz. CSR2 × CSR4, CSR4 × CSR2 and multi × bivoltine hybrid PM × CSR2. Observation on Muscardine disease was recorded till the onset of spinning. Analysis of results shows that disease prevalence was more in bivoltine x bivoltine hybrids compare to multi × bivoltine hybrids. PM × CSR2 was found to be more resistant towards Muscardine disease compared to other hybrids under agro-climatic conditions of Aurangabad.

Detection of Contamination and Analysis of Vertical Transmission of BmNPV in Eggs and Moths of Bombyx mori

This study reports the molecular detection of Bombyx mori nucleopolyhedrovirus (BmNPV) in silkworm strains of the Universidade Estadual de Maringá Brazilian Germplasm Bank (UBGB). DNA extraction was carried out by using six Bombyx mori female moths of each strain, followed by PCR amplification. A pair of primers was designed based on a specific sequence of the baculovirus genome related to the BmNPV ORF 14. Another pair of primers was used to amplify the silkworm Actin A3 gene segment, which was used as positive control. Twenty gene pools were analyzed, and fifteen revealed a fragment of 443 base pairs (bp), which indicated the presence of the BmNPV. The frequency of contaminated moths was as following: 100% for silkworm strains M18-2, M12-2 and J1;83% for C25, C75 and C24 strains;66% for KR01;50% for M11-A;33% for AS3, B106, M8 and M11 and 16% for C211, E8 and Hindu strains. These are promising results for the identification of contaminated B. mori moths by BmNPV, which may prevent virus proliferation in subsequent generations. We also analyzed DNA samples extracted from B. mori eggs, but the results were not conclusive regarding the detection of the fragments of the expected size (443 bp). The difficulty in detecting BmNPV contamination in B. mori eggs may be due to the low concentration of virus in samples.

Analysis of ESTs and gene expression patterns of the posterior silkgland in the fifth instar larvae of silkworm, Bombyx mori L.

The fibroin gene expression pattern and regulation of the posterior silkgland were studied by means of expressed sequence tags (ESTs) using the first and fifth day larvae of the fifth instar of silkworm, Bombyx mori L (strain: C 108). The results showed that there were 911 repetitive ESTs and 1950 single sequences (Singlets) among total 2861 consentient sequences, which were spliced. 1335 sequences were identified and the other 1526 were unknown. 5560 sequences (55.89%) in the posterior silkgland cell of the silkworm were new ESTs without homology with EST data published by Mita et al. The number of repetitive ESTs and single sequences from the first day larvae of the fifth instar was double more than that of the fifth day of the same instar in the silkworms. The unigenes which were more than 50 in repetitive EST size (contig size) came to only about 0.5% in total consentient sequences. There were significant differences between gene expression frequencies, and expressed genes were related to fibroin synthesis and its secretion and fibroin composition. Comparing the fifth day with the first day of the fifth instar, the genes-expressed quantity of fibroin heavy-chain gene was 18 fold higher, fibroin light-chain gene 9 fold and fibroin P52 gene 8 fold. 508 genes functioned for cellular component and 315 for enzyme after function tracing. These results implied that the gene expression of the first day was mainly for preparation for fibroin synthesis except for the growth of silkgland cells, and the gene expression of the fifth day of the fifth instar was mainly for synthesizing and excreting fibroin. Because the ratio of heavy chain, light chain and p25 of fibroin was not 6:6:1 as theoretically expected, or its special H-chain structure, the H-chain gene was not easy to detect through EST technique. Most of genes among total 2861 consentient sequences functioned for fibroin synthesis and secretion. This suggested the fibroin synthesis and secretion procedure of the posterior silkgland was more complex than the knowledge we have.

Study on fibroin heavy chain of the silkworm Bombyx mori by fluorescence in situ hybridization (FISH)

The sericulture industry plays a very important role in our national economy. Silkworm (Bombyx mori) is always regarded as a model animal and biological reactor. There have been detailed studies on the structure, expression and control and molecular evolution of silk genes. However, few, if any, reports are available on the localization of structural genes in silkworm by molecular cytogenetics. The present experiment has tentatively localized the Fib-H gene at the distal end of the 25th linkage group, namely at the 25-0.0 position, and verified that Fib-H has only one locus, thus providing a temporary solution to the problem about its localization.

Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bombyx mori) on Day 1 and Day 4 in the 5th Instar Stage

The posterior silk gland （PSG） of silkworm is an important organ where fibroin is synthesized and secreted exclusively. Because fibroin constitutes 75-80% of the silk filament, the mechanism governing fibroin secretion, quality and yield of cocoon can be elucidated by the study on the PSG. Using two-dimensional gel electrophoresis （2-DE） and image analysis system, the changes in the protein composition in the PSG cell were investigated on the day 1 （D1） and day 4 （D4） in the 5th instar stage from five different strains of silkworm （Bombyx mori）. While differences at protein level between days and strains were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse among the different strains. The results suggest that the secretion of fibroin is regulated by multiple proteins. The site of regulation and the proteins responsible for the regulation vary with the strain, which leads to differences between strains in the capacity of fibroin secretion in the PSG cell.

Protease inhibitors in Bombyx mori silk might participate in protecting the pupating larva from microbial infection

Pupae inside cocoons rarely suffer from disease. It is apparent that some factors in the cocoon exert antimicrobial effects whereby the pupae inside can be protected from microbial infection. In the present study, we investigated the expression of cocoon protease inhibitors using immunoblotting and activity staining. Enzymatic hydrolysis of cocoon proteins in vitro was performed to characterize their roles in protecting the cocoon from microbial proteases. We found that some protease inhibitors, particularly trypsin inhibitor-like （TIL）-type protease inhibitors, can be secreted into the cocoon layer during the spinning process, thereby providing effective protection to the cocoon and pupa by inhibiting the extracellular proteases that can be secreted by pathogens.

Sex Control of Silkworm Bombyx mori by UsingSensitive Character to IncubatingTemperature and Humidity

Because the male silkworm is economically superior to the female in sericulture,male-silkworm rearing through sex control is an interesting question. In order to rearmale silkworms only, a balanced lethal system was established in Japan and former U.S.S.R.by utilizing the recessive female sex-linked gene. Tajima et al. eliminated femaleindividuals by means of mechanical or manual methods according to the sex-limitedegg color or sex-limited larva marking. In addition, Xu An-ying et al. tried to ob-tain only male individuals by inducing androgenesis. However, the above methodscannot yet be utilized in sericulture production because of their harmful influenceson the vitality of silkworm larvas or the hatching rate.

INHIBITORY EFFECT OF POLY I:C AND 2′,5′-OLIGO (A) ON CYTOPLASMIC POLYHEDROSIS VIRUS OF SILKWORM, BOMBYX MORI L.

One of the major diseases in sericulture is the mid gut polyhedrosis, in which the pathogen is the cytoplasmic polyhedrosis virus （CPV）. After the silkworm larvae had been fed or injected with Poly Ⅰ: C or 2′, 5′-oligo （A） （2′, 5′-P3A3）, silkworms increased their ability to inhibit CPV, eventually, the attack rate of the silkworm decreased by about 40—50％. Moreover, it was found that Poly Ⅰ: C was able to induce some new proteins which seemed to be connected with the antiviral activity of the silkworm larva.

关于桑蚕(Bombyx mori L.)对人工饮料摄食性的研究──Ⅰ.不同收蚁时间和饲料含水率对小蚕发育的影响

为解决发育不齐的问题，就家蚕不同收蚁时间、不同饲料含水率对家蚕幼虫发育的影响进行了研究。结果发现：①发育整齐度，孵化当天收蚁＞二夜包＞三夜包＞四夜包；②饲料含水率以73％左右为适当．过高或过低都会延缓蚕的发育，影响发育整齐度。

桑蚕(Bombyx mori L.)1～2龄人工饲料1回育3～5龄桑叶育及其成本分析

通过春、夏２期桑蚕１～２龄人工饲料１回育３～５龄桑叶育的农村试养，结果表明，试验区的茧丝质成绩春季基本接近全龄桑叶育，夏季优于全龄桑叶育；通过小蚕人工饲料育的成本分析，采用１～２龄人工饲料１回育每盒蚕种可节省７．５３元。

Bombyx mori Pyridoxal Kinase cDNA Cloning and Enzymatic Characterization

Pyridoxal kinase （PLK） （EC 2.7.1.35） catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal-5＇-phosphate （PLP）, an important cofactor for many enzymatic reactions. Bombyx mori, similar to mammals, relies on a nutritional source of vitamin B6 to synthesize PLP. This article describes how a cDNA encoding PLK was cloned from Bombyx mori using the PCR method （GenBank accession number： DQ452397）. The cDNA has an 894 bp open reading frame and encodes a protein of 298 amino acid residues with a molecular mass of 33.1 k.Da. The amino acid sequence shares 48.6% identity with that of human PLK, and it also contains signature conserved motifs of the PLK family. However, the protein is 10 or more amino acids shorter than the PLK from mammals and plants, and several amino acid residues conserved in the PLK from mammals and plants are changed in the protein. The cDNA cloned was expressed successfully in Escherichia coli using the T7 promoter/T7 RNA polymerase expression system, and the crude extracts containing the expressed product were found to have strong PLK enzymatic activity with a value of 30 nmol/min/mg, confirming that the cDNA encodes the functional PLK of Bombyx mori. This is the first identification of a gene encoding PLK in insects.

1-Deoxynojirimycin Content and Alfa-Glucosidase Inhibitory Activity and Heat Stability of 1-Deoxynojirimycin in Silkworm Powder

Silkworm powder containing 1-deoxynojirimycin (DNJ) has α-glucosidase inhibitory activity and is promising as a complementary and alternative medicine (CAM) agent in Japan. Silkworm powder produced in Korea was extracted with 75% ethanol. The extract was derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl), and DNJ-FMOC content was measured by HPLC. Then, alfa-glucosidase inhibition by silkworm and mulberry powder in pig liver crude enzyme was assayed using 4-nitrophenyl-alfa-D-glucopyranoside as a substrate. Silkworm powder DNJ content (0.39 to 0.58%) was higher than that in mulberry powder (0.08 to 0.12%). The alfa-glucosidase inhibitory activity of silkworm powder was more potent than that of mulberry leaves and green tea. Silkworm powder DNJ was stable upon heating to 121?C for up to 15 min.

Analysis of SSR Fingerprints in Introduced Silkworm Germplasm Resources

Thirty-five SSR markers were used to construct 96 silkworm races fingerprint. All the SSR markers were polymorphic and unambiguously separated silkworm strains from each other. A total of 467 alleles were detected with a mean value of 13.34 alleles/locus （range 3-28）. The mean polymorphism index content （PIC） was 0.71 （range 0.299-0.919）. UPGMA cluster analysis of Nei＇s genetic distance grouped silkworm strains on the basis of their origin. The results indicated that SSR markers are efficient tools for fingerprinting cultivars and conducting genetic diversity studies in the silkworm.

Comprehensive Investigation to Analyze the Amino Acid Constituents of the Spent Silk Worm Pupae of Anthereaea assama

Spent silk worm pupae ofAntheraea assama were analyzed for its amino acid constituents and compared with the amino acid composition of fiber from A. assama, A. pyrni, and Bombyx mori. Amino acid percent was determined by UV Spectrophotometer at 570 nm. Infra Red spectra recorded the presence of different amino acids in the spent silk worm pupae powder which corroborate with the amino acids present in the fiber of different silk species.

家蚕30K蛋白BmLP1的表达模式及其在胚胎中的作用

家蚕30K蛋白是家蚕血液和卵中的高丰度蛋白,参与营养、免疫、抑制细胞凋亡等功能.探究30K蛋白在家蚕体内的表达模式有助于更清楚地了解30K蛋白的功能.利用生物信息学方法发现4种30K蛋白基因Bmlp1,Bmlp2,Bmlp3和Bmlp7都含有信号肽.其中,Bmlp1与其他30K蛋白的序列相似性小于50%.半定量RT-PCR结果表明,Bmlp1从5龄第3 d到化蛹第1 d在脂肪体中表达量都比较高,之后表达量降低.Western blotting结果表明,BmLP1从5龄第5 d到化蛹第4 d在血淋巴中的含量都比较高,之后含量降低.与血淋巴中的变化趋势不同,卵巢中的BmLP1在上蔟第2 d被检测到,之后含量一直增加,化蛾时含量达到最高.这是由于上蔟第2 d卵巢管迅速长大,血液中的BmLP1等营养蛋白逐渐转运至卵巢管的未受精卵并开始积累.免疫组织化学定位结果表明,在胚胎发育前期,BmLP1分布于蚕卵的卵黄颗粒中,在孵化前期,BmLP1被吞入胚胎的肠道内.体外孵育结果表明,蚁蚕粗提物可以降解BmLP1,暗示蚁蚕肠道内可能存在某种蛋白酶可以将大部分BmLP1水解,最终为蚁蚕的孵化提供能量或者氨基酸.系统分析了BmLP1在不同发育时期脂肪体、血液、卵巢和胚胎中的表达特征,揭示了其在胚胎发育过程中的重要作用.

家蚕体内细菌感染模型建立及不同菌株的协同致病特征分析

【目的】建立家蚕体内细菌感染模型,揭示不同细菌协同感染家蚕的致病力,明确云南蚕区家蚕细菌病的发病原因及其规律,为指导家蚕生产过程中细菌病的防治提供科学依据。【方法】从家蚕龄期、感染浓度和感染方法3个因素出发构建家蚕体内细菌感染模型,然后选用从云南地区自然发病蚕体中分离获得的5株肠道致病菌[Mammaliicoccus sciuri(葡萄球菌属)、粘质沙雷氏菌(Serratia marcescens)、弗氏柠檬酸杆菌(Citrobacter freundii)、Klebsiella variicola(克雷伯菌属)和Pseudomonas soli(假单胞菌属)],开展5种单菌感染及17种组合协同感染试验。【结果】通过建立家蚕体内细菌感染模型发现,家蚕群体添食细菌比单头添食的存活率低;细菌感染浓度选择时建议二龄期家蚕感染选用5.0 OD/mL、四龄期和五龄期家蚕感染选用10.0 OD/mL。选取的5株细菌对家蚕均具有致病力,整体上表现为随着细菌感染时间的延长,家蚕存活率逐渐降低;小蚕期感染细菌对家蚕的影响更明显,至五龄期有大量家蚕发病死亡,与养蚕过程中的细菌病发生规律相同。无论是小龄期感染还是大龄期感染,细菌协同感染的家蚕存活率总体上低于单菌感染,尤其是粘质沙雷氏菌参与的协同感染,至上蔟期的家蚕存活率均较低。此外,无论是二龄期感染还是四龄期感染,夏季家蚕的存活率总体上均高于春季家蚕,说明云南蚕区夏季家蚕的抗病力优于春季家蚕。【结论】5株分离自云南地区自然发病蚕体的病原菌对家蚕产生明显致病性,其致病性与感染龄期、感染浓度、不同细菌组合及感染季节等均有关,总体上具体表现为小蚕期感染较大蚕期感染对家蚕的影响更明显,细菌协同感染比单菌感染的危害更严重,且夏季家蚕感染后的存活率高于春季家蚕。

Differences in nutrient uptake between the fat body and embryonic primary cultures of silkworm （Bombyx mori）

Nutrition utilization and by-product formation in cultured insect cells has been investigated in several insect cells and has been of great interest to cell culturists and physiologists. In this research the biochemical changes in embryonic and fat body primary cultures of silkworm, Bombyx mori, have been compared. TC-100 medium supplemented with 10% and 20% FBS was used in embryonic and fat body primary cultures, respectively. Medium was renewed every week and the amount of glucose, uric acid, urea, total protein and alkaline phosphatase were measured in the samples from medium of primary cultures using spectrophotometeric methods. All biochemical macromolecules except uric acid showed significant changes. Glucose decreased in embryonic tissues, while in fat body culture its amount increased. Urea accumulation in embryonic culture was higher than in the fat body cultures. Since urea is a by-product, this accumulation could be due to higher utilization of amino acids. Total protein showed considerable changes and was consumed by embryonic culture more than the fat body＇ s. Alkaline phosphatase showed stronger activity in embryonic cells.

桑蚕的强制牵伸抽丝及其纤维性能

为提升蚕丝的力学性能,采用一种圆锥斜面牵伸装置对五龄桑蚕进行强制牵伸抽丝以制备高强牵伸丝,研究不同牵伸速度对桑蚕牵伸丝形貌、力学性能的影响,并将牵伸丝与蚕丝进行对比与分析。结果表明:由强制抽丝获得的牵伸丝表面光滑平坦,丝胶均匀附着;随着牵伸速度的增加,牵伸丝截面逐渐向弓形转变;牵伸丝的综合力学性能显著优于蚕丝,且其断裂强度、弹性模量、断裂比功与断裂伸长率随着牵伸速度的增加均有不同程度的增加;其中,以接近桑蚕自然吐丝速度(1 cm/s)牵伸制备的牵伸丝的断裂强度与蚕丝相当,约为3.00 cN/dtex,但其弹性模量(93.10 cN/dtex)远高于蚕丝(76.90 cN/dtex);当牵伸速度增加至4 cm/s时,可获得力学性能最佳的牵伸丝,其断裂伸长率为22.49%,断裂强度为3.39 cN/dtex,弹性模量为95.76 cN/dtex,断裂比功为0.62 cN/dtex,相比于蚕丝的平均值分别提高了7%、13.76%、24.53%、31.91%。