Molecular Markers (RAPD) Associated with Growth, Yield, and Origin of the Silkworm, Bombyx mori L. in India

To identify the molecular markers associated with growth and yield parameters in silkworm, Bombyx mori, RAPD profiles generated with seven UBC primers for fourteen silkworm stocks, originated from China, Japan, India, and Russia, were statistically analyzed. Stepwise multiple regression analysis establishes significant association of 45 markers with larval span, growth indices and four cocoon yield parameters relevant for silk production and t-test attest significance of the association of 89.5_{1500 bp} and 54.13_{300 bp}, respectively, with longer larval duration and high cocoon weight. The validity of this selection of markers was further supported with discriminant function analysis (DFA) done on the basis of Mahalanobis D ² statistics. The two indices of yield/growth were also tested with DFA, which helped in identifying a few markers and thereby opened scope of using such marker (e.g., 91.11_{900 bp}) for incorporating molecular markers in the breeding program for crop improvement in silkworm.     

Identification of ISSR markers associated with productivity traits in silkworm, Bombyx moni L.

Bombyx mori L., commonly recognised around the world as the mulberry silkworm, is characterized by a wide variability in yield and developmental traits, which have been proven through conventional genetic analysis to be of polygenic nature. A large number of morpho-biochemical traits and RFLP and RAPD markers are mapped on different linkage groups, but to this point very little attention has been given to unravelling the genetics of yield traits. To address this issue, polymorphic profiles of 147 markers generated with 12 ISSR primers on the genomic DNA of 20 silkworm stocks of diverse yield status were subjected to multiple regression and discriminant function analyses (DFA). This led to the identification of eight markers generated by six primers, which demonstrated high beta-coefficient indices of -0.451 to -0.940. Furthermore, a significant difference between the yield traits for stocks with and without the specific marker could also be established. The inheritance pattern of one marker, L13800bp, identified at the first step of selection of markers through stepwise regression analyses for five yield parameters is discussed in the context of applying multiple regression analysis for establishing association, if not linkage, between a group of DNA markers and a particular yield trait of polygenic nature and using such markers in molecular marker-assisted breeding programs.     

Molecular genetic approach for identifying markers associated with yield traits in the silkworm, Bombyx mori using RFLP-STS primers

Bombyx mori, the mulberry silkworm, exhibits wide variability in yield and developmental attributes. The genetics of yield expression, shown to be of polygenic nature, is poorly studied in silkworm. To identify markers associated with 10 selected yield traits, multiple regression analysis (MRA) and discriminant function analysis (DFA) were applied on 64 markers generated with eight RFLP-derived sequence-tagged-site (STS) primers on the genomic DNA of 20 silkworm stocks of different origin and diverse yield potential. The analyses led to the identification of ten markers showing significant association with the different yield traits. The markers could classify the stocks according to yield potential, irrespective of their origin and status of diapause. Trait means were significantly different for stocks with and with out the associated marker. The inheritance of a marker G2(1300bp), selected at the first step of MRA for five yield traits was shown to segregate in 1:1 ratio in the F2 progeny from a cross between two divergent stocks. The relevance of the STS primers is discussed in the context of applying multiple regression model for identifying markers associated with yield expression and suitability for molecular breeding work in B. mori for yield improvement.     

Identification of RAPD and SCAR markers associated with yield traits in the Indian tropical tasar silkworm Antheraea mylitta drury

The tropical tasar silkworm, Antheraea mylitta, is a semi-domesticated vanya silk-producing insect of high economic importance. To date, no molecular marker associated with cocoon and shell weights has been identified in this species. In this report, we identified a randomly amplified polymorphic DNA (RAPD) marker and examined its inheritance, and also developed a stable diagnostic sequence-characterized amplified region (SCAR) marker. Silkworms were divided into groups with high (HCSW) and low (LCSW) cocoon and shell weights, and the F₂ progeny of a cross between these two groups were obtained. DNA from these silkworms was screened by PCR using 34 random primers and the resulting RAPD fragments were used for cluster analysis and discriminant function analysis (DFA). The clustering pattern in a UPGMA-based dendogram and DFA clearly distinguished the HCSW and LCSW groups. Multiple regression analysis identified five markers associated with cocoon and shell weights. The marker OPW16_{905 bp} showed the most significant association with cocoon and shell weights, and its inheritance was confirmed in F₂ progeny. Cloning and sequencing of this 905 bp fragment showed 88% identity between its 134 nucleotides and the Bmc-1/Yamato-like retroposon of A. mylitta. This marker was further converted into a diagnostic SCAR marker (SCOPW 16_{826 bp}). The SCAR marker developed here may be useful in identifying the right parental stock of tasar silk-worms for high cocoon and shell weights in breeding programs designed to enhance the productivity of tasar silk.     

Performance of polyvoltine silkworm Bombyx mori L. on different mulberry plant varieties

Five cultivated mulberry plant varieties, BSRM‐34, BSRM‐56, BSRM‐57, BSRM‐58 and BSRM‐59, were used to evaluate the effects of plant variety on larval and cocoon characteristics of mulberry silkworm Bombyx mori L. Silkworm larvae undergo their entire larval periods on leaves of mulberry varieties. Different larval characteristics of larval weight, larval length and larval breadth were measured in different growth stages. After rearing, the fifth instar larvae were transferred into a mountage (Chandraki) structure for cocoon rearing for spinning and cocoon production. The cocoons were then harvested and different cocoon characteristics like cocoon weight, shell weight and shell percentage; and other characters like pupal weight and effective rate of rearing were measured. The results showed statistically significant differences among different varieties on larval and cocoon characteristics. The highest performance was observed by feeding the variety BSRM‐34 in respect of the average weight of 10 mature larvae (40.5 g), single mature larval length (7.6 cm) and breadth (1.0 cm), single cocoon weight (1.4 g), shell weight (0.2 g) and pupal weight (1.2 g) while the average performance was recorded by feeding varieties BSRM‐57 and BSRM‐59. The poorest performance was showed by feeding the variety BSRM‐56 (in case of larval characters) and BSRM‐58 (in case of cocoon characteristics). These two varieties also showed the poorest performance for silk yield. Through comparing all the above parameters it was concluded that BSRM‐34 is the best mulberry variety and it is recommended that Bangladeshi farmers adopt that variety for better yield of silk.     

Effects of applaud on the growth of silkworm (Lepidoptera: Bombycidae)

An experiment was carried out to evaluate the effect of the insecticide Applaud (buprofezin 25% WP) on the silkworm Bombyx mori (L.). This insecticide belongs to the class of insect growth regulators (IGR). The larvae were fed on leaves treated with 3 different concentrations (0.5, 1, 2 g/liter) of Applaud on the 1st d of each instar. Analysis of data with the Tukey-Kramer test at 1% significant level revealed that mortality and larval duration did not differ among the treatments. On the contrary, the larval weight, which was estimated just before mounting (procedure during which the mature larva climbing on a branch or other material to spin the cocoon), differed among the treatments. Also, cocoon weight, shell weight, and cocoon sericin and fibroin content were different among the treatments, except the shell cocoon ratio. Maximum weight was observed in the controls and minimum in the last instar treatments. Our data suggest that supplementation of Applaud through food to larvae does not affect their mortality rate. On the contrary, it affects larval growth and cocoon parameters.     

Ecdysteroid as a mediator in the regulation of silk protein synthesis and its influence on silkworm (Bom., Lepidoptera) genome

Abstract:  Ecdysteroid is one of the two most important insect hormones, which controls growth and developmental activities of insect. In silkworm, Bombyx mori L., basically ecdysteroid induces molting and metamorphosis in larvae and exogenous administration hastens the silk synthetic activity and cocoon spinning process but it strictly depends on the time of administration. In the present study, we administered phytogenous ecdysteroid to the 48-h-old 5th instar silkworm, B. mori at a single dose of 2  μ g per larva to study its effects on the larval growth, cocoon and silk variables. The possible role of ecdysteroid in altering the developmental sequence of silkworm and thereby its effect on cocoon and silk production at molecular level have been tried to get elucidated. The genomic DNA was isolated from the posterior silk gland on day 5 and 7 of the 5th instar from the ecdysteroid treated and the control larvae and was randomly amplified with arbitrary primers. The result presented notable variation in the amplified product suggesting the participation of ecdysteroid in regulating the silk gene. The feeding period of treated larvae was unaffected while the cocoon characters exhibited considerable improvement. The filament traits also were improved notably in the treated larvae. The participation of ecdysteroid in the silk biosynthetic process with its physiological and molecular implications was discussed.     

An integrated database for the enhanced identification of silkworm gene resources

The National Academy of Agricultural Science (NAAS) has developed a web-based database to provide characterization information in silkworm. The silkworm database has four major function menus: variety searching, characterization viewing, general information and photo gallery. It provides 321 silkworm varieties characterization information for six different regions namely, Korean, Japanese, Chinese, European, Tropical and non-classified group. Additionally, the database provides 1,132 photo images regarding life cycle of various silkworm varieties. A specific characterization information table provides accession number, variety, strain and larval marking, blood color, cocoon color, cocoon shape, egg colors, remarks and image table provides photos which consist of shape and color in the different stages of larval, egg and cocoon stages. AVAILABILITY: The database is available for free at http://www.naas.go.kr/silkworm/english/     

Role of specific activators of intestinal amino acid transport in Bombyx mori larval growth and nutrition.

Nutrient absorption and its modulation are critical for animal growth. In this paper, we demonstrate that leucine methyl ester (Leu-OMe) can greatly increase the activity of the transport system responsible for the absorption of most essential amino acids in the larval midgut of the silkworm Bombyx mori. We investigated leucine uptake activation by Leu-OMe in brush border membrane vesicles and in the apical membrane of epithelial cells in the midgut incubated in vitro. Moreover, the addition of this strong activator of amino acid absorption to diet significantly affected larval growth. Silkworms fed on artificial diet supplemented with Leu-OMe reached maximum body weight 12-18 h before control larvae, and produced cocoon shells up to 20% heavier than those of controls. The activation of amino acid absorption plays an essential role in larval development so that larval growth and cocoon production similar to controls reared on an artificial diet with 25% of dry mulberry leaf powder were observed in silkworms fed on an artificial diet with only 5% of mulberry powder. Arch.     

In-situ observation of tryptophan in larval cocoon silk of the hornet Vespa simillima xanthoptera cameron by ultraviolet resonance raman spectroscopy

We performed in-situ ultraviolet resonance Raman (UVRR) spectroscopy of the larval cocoon silk of the hornet, Vespa simillima xanthoptera Cameron, and compared the result with that of the silkworm, Bombix mori. The UVRR spectrum of the hornet cocoon differed markedly from that of the B. mori cocoon: peaks attributable to tyrosine (Tyr) were observed strongly, and tryptophan (Trp) peaks weakly, in the spectrum of the B. mori cocoon, whereas peaks attributable to Trp exclusively appeared in the spectrum of the hornet cocoon.     

Screening of molecular markers for NPV resistance in Bombyx mori L. (Lep., Bombycidae)

Abstract: Silkworm ( Bombyx mori L.) is one of the important economic insects. Silkworm rearing and silk industry plays an important role in China, India and other developing countries. In the long history of sericultural practice, introduction of silkworm strains with high resistance to diseases has greatly improved cocoon and silk quality and productivity. However, current silkworm breeding is mainly based on traditional method that involves high input of time and labour. In order to increase the selection efficiency and accuracy for future silkworm breeding, it is necessary to establish a molecular marker-assisted selection system. In our study, three silkworm near isogenic lines that had different resistance to nuclear polyhedrosis virus (NPV) were established by means of different hybridization methods. A total of 150 random amplified polymorphic DNA (RAPD) random primers were used to screen molecular markers. Among them, two molecular markers OPA-18₇₀₀ and OPY-11₄₀₀ were found linked to major genes resistant and susceptible to NPV, respectively. Validity of the molecular markers was proved in F2 populations.     

不同水分含量桑叶对二化性家蚕的营养效应

研究了取食不同水分含量桑叶对二化性家蚕(CSR3×CSR6)5龄幼虫的影响。实验设嫩叶(水分含量80%～85%)、半成熟叶(水分含量65%～70%)和成熟叶(水分含量55%～60%)3个不同处理。采用标准重量分析法分析和计算各种生长、营养和营养效率指数。结果表明,在取食较高水分含量桑叶的处理中,营养指数(包括取食量、消化量和近似消化率等)和营养效率指数(包括食物利用率和转化率等)均显著较高,但不同处理中消化食物向茧壳的转化率及产生每克茧壳所需食物消化量差异不明显。结论是取食水分含量高的桑树嫩叶有助于提高家蚕的取食和营养。     

Effect of Cordyceps, Paecillomyces sp., Cordyceps pruinosa and Paecillomyces japonica, on the Development of Domestic Silkworm, Bombyx mori

ABSTRACT This study was carried out to investigate pathogenicity of cordyceps on the silkworm larva, Bombyx mori . For experiment, Cordyceps, Pacillomyces sp. strain collected from Chiak mountain area in Gangwon Province and Pacillomyces japonica collected from dead silkworm larva at the rearing room in Sangju National University were used. Spores of cordyceps were propagated on sterile PDA (potato dextrose agar 39g, water 1,000mL) at 25°C for 21 days. Spores of cordyceps isolated from media were inoculated on integuments of 3rd instar newly exuviated silkworm for pathogenicity on the silkworm larvae. The cordyceps used in this study was highly infectious to the silkworms. Virulence of cordyceps was different depending on species. Pacillomyces japonica was the most efficacious with 70% mortality on silkworm larvae. Cordyceps had adverse effect on the overall rearing from larval period to mounting of matured silkworm larvae, cocoon making, pupation and moth emergence. The result suggests that cordyceps used in this study may be useful for the tracking of biocontrol.     

Biological relevance of host plant-derived terpenoid in the cocoons of the tropical tasar silkworm Antheraea mylitta

We have characterized and studied the biological functions of a terpenoid derivative in the Indian tropical tasar silkworm, Antheraea mylitta reared on the primary host plant Arjun, Terminalia arjuna. The compound from insect cocoon turned out to be a terpenoid derivative which resembled oleanane type triterpene (Arjunolic acid) present in the host plant. The plant and cocoon compounds were anti-oxidative as determined by bleaching of beta carotene in vitro. UV-exposure is the major form of peroxidative insult encountered by this wild tropical silkworm. The life cycle comprising five larval stages and the cocoon stage lasts for about 30–45 days. Hence the sequestration of antioxidant and UV-protectant molecule from the host plant commands great biological significance.     

Elementary research of the formation mechanism of sex-related fluorescent cocoon of silkworm, Bombyx mori

To understand mechanisms for the difference of uptaking and transporting the pigments between the male and female in the silkworm, Bombyx mori strain of sex-related fluorescent cocoon, the fluorescent pigments in the midgut lumen, midgut, blood, silk glands and cocoon were analyzed with thin-layer chromatography, and showed that fluorescent colors of cocoons consisted with that of blood and silk glands. The different fluorescent colors of cocoons between the male and female may be mainly caused by the difference of accumulation and transportation for fluorescent pigments in the midgut and in the silk glands. Furthermore the midgut proteins were separated with Native-PAGE, and the proteins respectively recovered from three fluorescent regions presenting on a Native-PAGE gel for the female silkworms were determined using shotgun proteomics and mass spectrometry sequencing, of which 60, 40 and 18 proteins respectively from the region 1, 2 and 3 were identified. It was found that the several kinds of low molecular mass 30 kDa lipoproteins and the actins could be detected in all three regions, troponin, 30 kDa lipoprotein and 27 kDa glycoprotein precursor could be detected in the region 2 and 3, suggesting these proteins may be fluorescent pigments binding candidates proteins. Analysis of gene ontology indicated that the identified proteins in the three regions linked to the cellular component, molecular function, and biological process categories. These results provide a new clew to understand the formation mechanism of sex-related fluorescent cocoon of silkworm.     

SSR based linkage and mapping analysis of C,a yellow cocoon gene in the silkworm,Bombyx mori

The yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y (Yellow haemolymph), I (Yellow inhibitor) and C (Outer‐layer yellow cocoon), which are located on linkage groups 2, 9 and 12, respectively. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F₁ (BC₁) progeny were used for linkage analysis and mapping of the C gene using silkworm strains C108 and KY, which spin white and yellow cocoons, respectively. DNA was extracted from individual pupae and analyzed for simple sequence repeat (SSR) markers. The C gene was found to be linked to seven SSR markers. All the yellow cocoon individuals from a female heterozygous backcross (BC₁ F) showed a heterozygous profile for SSR markers on linkage group 12, whereas individuals with light yellow cocoons showed the homozygous profile of the strain C108. Using a reciprocal heterozygous male backcross (BC₁ M), we constructed a linkage map of 36.4 cM with the C gene located at the distal end, and the closest SSR marker at a distance of 13.9 cM.     

基于超高效液相色谱-质谱技术对野蚕茧和家蚕茧化学成分进行比较

鉴定并比较野蚕茧与家蚕茧的化学成分对于理解家蚕的驯化具有重要的意义。利用高温高压结合甲醇-水提取的方法获得蚕茧中的化学成分,利用UHPLC-MS技术对野蚕、家蚕大造品种和皓月品种3种蚕茧丝中的小分子成分进行鉴定和比较分析。通过阳离子模式和阴离子模式的UHPLC-MS获得了野蚕、大造和皓月蚕茧丝的代谢指纹图谱,对鉴定到的高丰度化合物进行注释,发现其中包括了氨基酸、黄酮、生物碱、萜类、有机酸和木脂素等成分。PLS-DA的得分图表明,野蚕、家蚕大造品种和皓月品种的3种蚕茧的代谢组存在显著差异。发现脯氨酸、亮氨酸/异亮氨酸和苯丙氨酸在大造茧中的含量显著高于在野蚕和皓月茧中的含量,黄酮类植物次生代谢物在大造茧中的含量显著提高,包括槲皮素、异槲皮素、槲皮素-3-O-槐糖苷、槲皮素-3-O-L-鼠李糖苷、槲皮素-3-O-芸香糖苷和山奈酚;而神经碱、白桥楼碱、毛果芸香次碱、美洲豚草内酯、线叶泽兰素和中缅木莲素等生物碱、萜类和木脂素类的植物次生代谢物在野蚕茧中的含量显著高于在家蚕茧中的含量。在紫外光的激发下观察黄酮的绿色荧光发现家蚕大造茧中的黄酮含量最高,家蚕皓月茧中的黄酮含量最低,而野蚕茧中的黄酮含量居中。生物碱和有机酸是良好的抗虫抗菌剂,它们在野蚕茧中的含量较高,能够提高野蚕茧的防护能力。黄酮类物质在家蚕大造茧中的含量较高,是导致家蚕大造茧呈黄绿色的主要原因。     

Application of Multi-vitamins as Supplementary Nutrients on Biological and Economical Characteristics of Silkworm Bombyx mori L

In order to investigate the effects of supplementary nutrients on silkworm, Bombyx mori, an experiment was conducted with multi-vitamins treatments. Leaves enriched with multi-vitamins (1, 2.5 and 5%) were fed once a day to 4th and 5th instar silkworm larva. The supplementation of the leaves was done by spraying the treatments on them. These treatments resulted in a significant increase in biological and economical parameters such as larval weight, female and male cocoon weight, pupal weight and egg productivity when compared with normal control. Multi-vitamins of 2.5% treatment increased the larval weight by 10.2%, which had the most weight enhancement in the fifth day of 5th instar larvae. However, 5% concentration of multi-vitamins decreased some biological characteristics. These treatments resulted in 4.7% increase of cocoon's weight. In most of the moths the number and weight of eggs had significant difference compared to control, but these supplementary materials caused relatively less egg hatching.     

Hierarchical clustering of 54 races and strains of the mulberry silkworm,Bombyx mori L: Significance of biochemical parameters

Summary A detailed analysis was undertaken to test the efficacy of hierarchical agglomerative clustering (UPGMA method) in grouping the races and strains of the mulberry silkworm, Bombyx moti L., and to ascertain the importance of biochemical parameters in the clustering process. The analysis was based on data from two rearing seasons with 54 selected races/strains of different geographic origin and varying yield potentials. The results indicate that seven clusters can be realised with yield parameters alone, whereas the inclusion of biochemical parameters in clustering resulted into two broad groups: one having all the breeds with high cocoon weight and shell weight, the other having all the low-yielding silkworm strains both from India and from other countries. Further sub-grouping under these two groups highlights genetical differences associated with the differentiation of various groups of races in temperate and tropical areas as well as their significance for silkworm breeding. Estimates of all ten variables were further subjected to quick clustering and the results showed that cluster 5, constituted by 38 lowyielding strains of India, China and Europe, had the highest values of the final cluster centre for amylase and the effective rate of rearing (ERR), while clusters 1 and 4 had the highest values for invertase and alkaline phosphatase. The evolutionary aspect of the genetic channelisation of silkworm races from various countries is discussed against the background of differences in the biochemical parameters and yield variables.     

广西桑蚕茧丝质量与性能指标的测定分析

本研究对广西桑蚕茧的茧丝质量与性能指标进行了测定分析.结果表明,广西桑蚕茧外形略小,茧层丝胶含量、茧层易溶丝胶含量、丝胶溶解性以及茧丝纤度等主要指标均与江浙夏秋用桑蚕茧有明显差异.桑蚕茧的茧层丝胶含量、易溶丝胶含量随庄口、季节不同而有显著差异;春季桂南地区的蚕茧茧层丝胶含量高于桂西北地区,秋季刚好相反.茧层易溶丝胶含量在11.6%～14.4%之间,约占丝胶总含量的41%～45%,茧层易溶丝胶含量和解舒率呈显著的正相关关系,即茧层易溶丝胶含量越多,蚕茧解舒越好.庄口和季节是影响桑蚕茧层易溶丝胶含量高低与蚕茧解舒优劣的重要因素.广西桑蚕茧层丝胶对煮茧温度更敏感,茧丝纤度粗细变化开差大.因此,我们认为目前困扰广西桑蚕茧加工技术突破的原因之一是缫丝工艺技术,采用传统缫丝工艺技术难以正常发挥广西桑蚕茧的茧丝优势与特性.