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.

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。

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.

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.

抗血液型脓病家蚕新品种‘明·湖×春·江’多地农村试验初报

‘明·湖×春·江’是浙江省农业科学院蚕桑与茶叶研究所育成的一对抗血液型脓病家蚕新品种,2022年分别通过浙江省主要农作物品种审定委员会、国家畜禽遗传资源委员会审定。2023年春期,四川、江西、陕西、山东等地进行了‘明·湖×春·江’的引进试验,结果显示,新品种适应性强,在各地均表现孵化眠起齐一,食桑旺盛,强健好养,蚕体和茧形大,产茧量明显高于各地对照品种,健蛹率高,解舒优,茧丝长和解舒丝长长,洁净与万米吊糙优,适于各试验省区推广饲养。

家蚕1-2龄不同人工饲料育对生长发育和饲料效率的影响

本试验以优食一号蚕品种为材料,以全龄桑叶育为对照,探讨了1—2龄颗粒饲料育及1—2龄蒸煮饲料育、3—5龄桑叶育,对全龄生长发育、茧质及5龄期消化吸收和饲料效率的影响。结果表明,该品种对颗粒饲料和粉体蒸煮饲料的摄食性优良,但颗粒饲料育家蚕的发育经过比桑叶育和蒸煮饲料育延长,两种饲料育1—2龄的蚕体重均低于桑叶育,但3龄改为桑叶育之后,饲料育家蚕的生长发育加快,体重增长倍数明显高于全龄桑叶育,熟蚕体重基本达到对照区水平。小蚕饲料育的小区产茧量和全茧量与全龄桑叶育无明显差异,但茧层量略低于全龄桑叶育,茧层率则以1—2龄蒸煮饲料育最高。1—2龄饲料育试验区的5龄食下量、消化量、消化率均低于全龄桑叶育对照区,但5龄体重转化效率、蚕茧生产效率、蚕茧转化效率、茧层生产效率及茧层转化效率等各项饲料效率指标,均是小蚕饲料育试验区高于对照区,其中以1—2龄颗粒饲料育最高。本试验结果说明,小蚕饲料育提高了家蚕的代偿生长能力和饲料效率,主要是由于5龄期食下量减少,而体重转化效率显著提高所致。

不同品种桑蚕牵伸丝的结构与性能

利用自主研制的圆锥斜面强制牵伸抽丝装置对不同品种五龄桑蚕进行强制抽丝,通过拉伸测试、傅里叶红外光谱及X射线衍射对不同品种牵伸丝与相应原茧丝的结构与性能进行表征与分析。结果表明:两种牵伸丝的力学性能均优于其对应茧丝,黄色雄蚕牵伸丝的力学性能更佳,其初始模量、断裂强度、断裂比功与伸长率的平均值分别为117.35、4.55、0.75 cN dtex与26.54%。牵伸作用会导致丝纤维晶粒尺寸减小、结晶度提高,从而赋予丝纤维更为优异的力学性能。

家蚕抗血液型脓病斑纹全限性品种锦绣3号的育成

家蚕品种洞·庭×碧·波曾是长江流域及其他区域的主推品种之一,但存在茧丝质等性状退化和自身抗频发性血液型脓病能力偏弱的现象。以洞、庭、碧、波4个原种及其改良品系作为受体,以家蚕血液型脓病抗性材料HKC作为供体,采用杂交和连续回交方法将抗性基因导入受体品种,同时通过插入杂交方式导入优良血缘以改良丝质。采用系统分离、添毒筛选、活蛹缫丝等技术,育成了对家蚕血液型脓病具有高度抵抗性的家蚕新品系,并组配成优质、高产新组合7521改K·秋丰BK×7522NK·854BNK(华·康×湘·泰)。国家品种试验结果表明,新品种万蚕产茧量18.24 kg、虫蛹率97.69%、茧丝长1186.2 m、解舒率80.41%、洁净95.64分、鲜毛茧出丝率17.37%,各项指标均优于对照品种秋丰×白玉。BmNPV对新品种的LD50≥2.92×10^(9)mL^(-1),新品种的抗病力显著增强。该品种于2022年12月通过国家畜禽遗传资源委员会审定,适合在长江和黄河流域夏秋季饲养,审定名为锦绣3号。

两种不同剂型人工饲料饲养家蚕的比较试验

为了比较不同剂型人工饲料对家蚕的饲养效果,本试验以‘优食一号’为试验品种,采用1~2龄饲料育、3~5龄桑叶育的饲养模式,研究粉体饲料和膨化颗粒饲料对家蚕生长发育的影响。结果表明:两种剂型的人工饲料饲育家蚕的疏毛率基本一致,低于桑叶育;2种剂型的人工饲料饲育家蚕的体重和龄期经过无显著差异,均显著低于桑叶育;颗粒饲料育家蚕的生长发育略低于粉体饲料育和桑叶育家蚕;2种剂型的人工饲料的茧丝长、茧丝纤度和解数率比桑叶组低,茧丝质量无显著性差异。表明2种剂型的人工饲料均可用于小蚕期使用,大蚕转桑后补偿效应显著。

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.

Accumulation of 1-deoxynojirimycin in silkworm,Bombyx mori L.

1-deoxynojirimycin (1-DNJ) contents in the silkworm,Bombyx mori,at different developmental stages and tissues were investigated by using reverse-phase high-performance liquid chromatography. The 1-DNJ contents of silkworm larvae change significantly with their developmental stages. The male larvae showed higher accumulation efficiency of 1-DNJ than the females and also a significant variation was observed among the silkworm strains. The present results show that tissue distribution of 1-DNJ was significantly higher in blood,digestive juice,and alimentary canal,but no 1-DNJ was observed in the silkgland. Moreover,1-DNJ was not found in silkworms fed with artificial diet that does not contain mulberry leaf powder. This proves that silkworms obtain 1-DNJ from mulberry leaves; they could not synthesize 1-DNJ by themselves. The accumulation and excretion of 1-DNJ change periodically during the larval stage. There was no 1-DNJ in the newly-hatched larvae and 1-DNJ was mainly accumulated during the early and middle stages of every instar,while excreted at later stages of larval development. Further,it is possible to extract 1-DNJ from the larval feces and it is optimal to develop the 1-DNJ related products for diabetic auxiliary therapy.

An efficient and safe strategy for germ cell-specific automatic excision of foreign DNA in F1 hybrid transgenic silkworms

The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products.In sericulture,only the first filial generation(F1)hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms,but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs.To address this issue,we developed a safe and efficient strategy using the GAL4/Upstream activating sequence(UAS)system,the FLP/flippase recognition target(FRT)system,and the gonad-specific expression gene promoters(RSHP1p and Nanosp)for the germ cell-specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms.We established 2 types of activator strains,R1p::GAL4-Gr and Nsp::GAL4-Gr,containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp,respectively,and 1 type of effector strain,UAS::FLP-Rg,containing the UAS-linked FLP gene expression cassette.The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silkworms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100%,whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73%to 80.3%.Additionally,we identified a gene,sw11114,that is expressed in both testis and ovary of Bombyx mori,and can be used to establish novel gonad-specific expression systems in transgenic silkworms.This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.

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.

浙江省家蚕微粒子病成品卵检疫的回顾和展望

家蚕微粒子病是养蚕业的毁灭性病害,长期以来通过检疫进行预防和控制。本文回顾了浙江家蚕微粒子病成品卵检疫的历史,解析了开展成品卵检疫的必要性,总结了取得的工作成效,找出了现行成品卵检疫存在的问题,最后展望成品卵检疫工作并提出技术研究方向以及建设管理体系的建议。

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.

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.