Identification and association of ISSR markers for thermal stress in polyvoltine silkworm <Emphasis Type="Italic">Bombyx mori</Emphasis>

Evaluation of genetic resources is an essential prerequisite for their effective utilization. In India, the tropical climate prevails in most of the sericultural belts, where temperature goes beyond the ambient during summer, adversely affecting the silkworm rearing. Although polyvoltine silkworms are poor in silk content, they are mostly tolerant to tropical conditions and diseases. With an aim to identify potential silkworm races/breeds specific to thermotolerance for their effective utilization in breeding programme, 15 selected polyvoltine silkworm races were studied for their thermotolerance behaviour. Their genomic DNA samples were analyzed for ISSR-PCR using 15 selected primers. The UPGMA analysis based on Nei and Li algorithm has clustered the 15 silkworm races into five groups and one isolate. ALSCAL multidimensional scaling has not only supported the information generated by the dendrogram, but it has made the genetic distance among races more clear and substantiating their status in terms of thermal stress where pupation rate was taken as indicator. Further, discriminant function analysis (DFA) was done with three groups of silkworms classified for thermal stress viz. susceptible, moderately tolerant and tolerant. The canonical correlation value was estimated to be 0.987 (Wilk’s λ= 0.004; χ² = 36.044, p < 0.05). DFA clearly discriminated the above three groups. Beta statistics with t value and its significance for the markers identified through stepwise multiple regression analysis (MRA) revealed a total of five bands (807₁₃₀₀, 808₃₀₀₀, 808₄₀₀₀, 834₄₀₀₀, and 834₃₀₀₀) showing correlation with pupation rate after thermal treatment. Out of them, marker 808 3000 showed maximum and highly significant correlation (r = 0.757, p < 0.001, t = 4.182) with pupation rate among the silkworm races. The identified putative markers are being used to develop DNA marker to be used in evolving thermo-tolerant silkworm breeds using marker-assisted selection programme. The text was submitted by the authors in English.     

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.     

The profiles of red fluorescent proteins with antinucleopolyhedrovirus activity in races of the silkworm Bombyx mori

Partially purified red fluorescent proteins (RFPs) secured from the gut juice of 5th-instar multivoltine and bivoltine silkworm races were observed as several bands in electrophoretograms and chromatographic eluates. Interestingly, different races of silkworms had varying numbers of fluorescent protein bands: 11 in Pure Mysore (resistant), 11 in Nistari (resistant), 4 in CSR₂ (moderately susceptible) and 1 in NB₄D₂ (highly susceptible). Bioassay experiments indicated that the fluorescent bands had antinucleopolyhedrovirus (antiNPV) activity. The molar extinction coefficients and fluorescence quantum yields of all RFPs were estimated. The purified tetrapyrroles were characterized by UV–visible absorption and fluorescence spectral analyses. All tetrapyrrole moieties associated with RFPs were found to be different and characteristic of the fluorescent bands. The resulting qualitative and quantitative differences among the individual RFPs from various races of silkworm were related to the susceptibilities of the silkworms to the viral disease. Moreover, light was found to be essential for the synthesis of RFPs, and, therefore, the role of light in the synthesis of RFPs was evaluated. Thus, this work may elucidate the process of RFP synthesis in silkworm, which may be used as a biomarker to measure the degree of susceptibility of silkworm races to NPV. Therefore, the characteristic band pattern may be used as an indicator to define the relative resistance of a race towards the specific virus.     

Comparative study on the mechanical property of silk thread from cocoons of Bombyx mori L

Specimens of bave (undegummed silk thread) were collected from cocoons of various origins of parent silkworm races, such as Japanese, Chinese, European, Korean and tropical origins, and from as many races as possible. An apparatus was set up to measure the dynamic elastic modulus of these specimens. In all the categories of the races tested, the elastic modulus was linearly related to the size of bave, regardless of the portion of cocoon layer from which the specimens were taken. This correlation was concluded to be universal to the silk thread of Bombyx mori L. species; however, values of the regression coefficient and of the elastic modulus were susceptible to the origin of silkworm races, depending on whether they were native or improved.     

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.     

不同家蚕品系的耐热性特征

研究了家蚕Bombyx mori 3个品系(Pure Mysore,NB4D2和CSR2）5龄幼虫和蛹在不同温度（35,38和40℃）下的耐热性,采用Probit分析测定了它们在各温度下的LT50值和置信限。结果表明：多化性品系Pure Mysore在高温下的存活率高于两个二化性品系NB4D2和CSR2,而两个二化性品系中NB4D2表现出更好的耐热性。家蚕幼虫接触38℃高温6 h和40℃高温3 h后,其血淋巴中出现90,70和29 kDa的热激蛋白条带。在恢复过程中,NB4D2和CSR2的血淋巴中未见29 kDa蛋白条带,而Pure Mysore幼虫的血淋巴中29 kDa蛋白仍然表达。当幼虫置于高温下时,血淋巴中90和70 kDa蛋白表达,但是检测不到29 kDa蛋白。研究认为热激蛋白表达与热带家蚕不同品系的耐热性以及与同一品系不同发育阶段的耐热性具有相关性。     

基于线粒体COⅠ和NADH-6基因分子检测的中日家蚕和野桑蚕亲缘关系的研究

为了进一步明确家蚕和野桑蚕的亲缘关系,收集了中国和日本的一些家蚕和野桑蚕品种资源,提取了家蚕和野桑蚕的总mRNA,通过RT-PCR克隆了家蚕和野桑蚕线粒体DNA (mtDNA)的细胞色素氧化酶亚基1基因(cytochrome oxidase subunit 1 gene, COⅠ)和NADH-6基因,并制备探针进行了DIG-RFLP检测。结果表明,中国和日本的家蚕与中国野桑蚕的COⅠ和NADH-6基因的DIG-RFLP的分子多态性相同,但与日本野桑蚕存在差异。此结果从线粒体水平证实了中国和日本的家蚕都起源于中国野桑蚕,而不是起源于日本的野桑蚕。     

Comparative proteomic analysis reveals that caspase-1 and serine protease may be involved in silkworm resistance to Bombyx mori nuclear polyhedrosis virus

The silkworm Bombyx mori is of great economic value. The B. mori nuclear polyhedrosis virus (BmNPV) is one of the most common and severe pathogens for silkworm. Although certain immune mechanisms exist in silkworms, most silkworms are still susceptible to BmNPV infection. Interestingly, BmNPV infection resistance in some silkworm strains is varied and naturally existing. We have previously established a silkworm strain NB by genetic cross, which is highly resistant to BmNPV invasion. To investigate the molecular mechanism of silkworm resistance to BmNPV infection, we employed proteomic approach and genetic cross to globally identify proteins differentially expressed in parental silkworms NB and 306, a BmNPV-susceptible strain, and their F(1) hybrids. In all, 53 different proteins were found in direct cross group (NB♀, 306♂, F(1) hybrid) and 21 in reciprocal cross group (306♀, NB♂, F(1) hybrid). Gene ontology and KEGG pathway analyses showed that most of these different proteins are located in cytoplasm and are involved in many important metabolisms. Caspase-1 and serine protease expressed only in BmNPV-resistant silkworms, but not in BmNPV-susceptible silkworms, which was further confirmed by Western blot. Taken together, our data suggests that both caspase-1 and serine protease play a critical role in silkworm resistance against BmNPV infection.     

感染家蚕浓核病毒BmDNV1对家蚕抗性和敏感品种生化参数的影响

在家蚕疾病中,病毒性疾病是危害茧产品的主要且最普遍的疾病。病毒性蚕软腐病是由BmIFV, BmDNV1 和 BmDNV2引起的。对印度种质库（Indian Germ plasm stock）中的BmDN V1有抗性和敏感的家蚕品种进行了鉴定。利用标准方法对在BmDNV1感染过程中抗性和敏感品系的主要有机成分的变化（包括总蛋白、碳水化合物和脂类）进行了检测。结果表明： 随着幼虫年龄的增长,对照组和处理组中有机成分（即蛋白质和碳水化合物）的含量也随之增长,但是处理组的增长水平明显地低于各自对照组的增长水平。接种BmDNV1后,与对照组比较,敏感品种体内的血淋巴和中肠组织的总蛋白的量显著地下降。在抗性品种,接种4天后的总蛋白的量有了显著地下降,之后,下降水平小于各自的对照组。在抗性和敏感品种中,血淋巴和中肠组织中的总碳水化合物的量略有下降。在变化不显著的抗性和敏感品种中,血淋巴和中肠组织中的脂质的量有显著地提高。在敏感家蚕品种,生化变化清晰地显示： BmDNV1感染消耗作为主要能量来源的总蛋白和碳水化合物。这些成分的损耗导致了被感染家蚕的生长受阻。     

Artificial parthenogenesis and control of voltinism to manage transgenic populations in Bombyx mori

In order to improve the management of transformed populations in a routine application of transgenesis technology in Bombyx mori, we modified its mode of reproduction and its voltinism. On one hand, after a stable integration of the gene of interest by transgenesis, it is preferable to maintain this gene in an identical genomic context through successive generations. This can be obtained by artificial parthenogenetic reproduction (ameiotic parthenogenesis) giving isogenic females identical to their transformed mother. On the other hand, it is essential to obtain continuous generations (polyvoltinism) after microinjection, in order to screen positive transgenic insects and study genetics and insertion of the transgene. Thereafter, it is more convenient to store these populations, as diapause eggs before their use in biotechnology application. We obtained such polyvoltine parthenoclones, first by selection for a parthenogenetic character in polyvoltine races, and second, by selection for a polyvoltine character in a parthenogenetic, but diapausing clone of B. mori. As diapause was directly under the control of diapause hormone (DH), we also tested direct injection of DH in female pupae of polyvoltine strains, as well as anti-DH antibody treatment to eliminate diapause in univoltine strains. We discussed the advantages and limitations of these methods and proved the feasibility in obtaining polyvoltine parthenoclones and determining the voltinism in B. mori. These methods would permit us to improve the management of populations used in transgenesis technology.     

Transcriptome analysis of the response of silkworm to drastic changes in ambient temperature

Bombyx mori is a poikilothermic insect and is economically important for silk production. Drastic changes in the ambient temperature have a negative impact on sericulture. However, the reason as to why high temperature is associated with the occurrence of diseases in silkworm and the response of silkworm to low temperature remain unclear and were the focus of the present study. Dazao silkworm exposed to 13 °C (DZ-13), 25 °C (DZ-25), and 37 °C (DZ-37) were used for RNA-seq analysis. There were 478 and 194 upregulated differentially expressed genes (DEGs) in DZ-13 and DZ-37 while 49 and 273 downregulated DEGs in DZ-13 and DZ-37, respectively. Eight DEGs were co-upregulated, in which seven genes were for heat shock proteins (Hsps), implying that Hsps play important roles in the tolerance of silkworm to high and low temperature. Gene ontology analysis revealed that the developmental process was downregulated in DZ-13. All the DEGs in the oxidative phosphorylation and insulin signaling pathways were upregulated in DZ-13. Several cuticular proteins and ATP synthesis-related genes were upregulated in DZ-13, suggesting that thickening of the cuticle and increase in the ATPase expression would help silkworms to protect themselves from low temperature-induced stress. Several immune-related genes, such as BmRel and BmSerpin-2, were downregulated in DZ-37, revealing that the resistance of silkworm is decreased under high temperature shock resulting in susceptibility to pathogens. Thus, the increase in the thermo-tolerance of silkworm should be related to the enhancement in the pathogen resistance.

     

Proteolytic enzymes as markers of productivity and heterosis of silkworm

A positive correlation between the activity level of cysteine proteinases in developing eggs of common silkworm moth (Bombyx mori L.), on the one hand, and a set of commercial characteristics, on the other, was found. This allows the determination of cysteine proteinase activities (pH optima of 3.0, 3.6, and 8.6) to be recommended as a biochemical test for an early prediction of potential productivity of silkworm breeds. A positive correlation between the activity level of acid cysteine proteinases in eggs of parental breeds and a set of commercial characteristics of their hybrids was detected, indicating a principal possibility of predicting the degree of heterosis.     

Genetic divergence and allelic-specificity in relation to expression of voltinism in silkworm using ISSR and RAPD fingerprinting

The silkworm B. mori is a multicellular organism revealing genetic resources which makes an ideal model for lepidoptera for the present investigation. With the objective of targeting distinctive markers for utilization in future breeding programmes, Bivoltine and Polyvoltine silkworm strains were used by inter-simple sequence repeats (ISSR) and random amplified polymorphic-DNA (RAPD) fingerprinting to detect their genetic versatility and volatility. Six ISSR primers generated 99 markers, of which 76.76% were found to be polymorphic with an average number of observed alleles (N _a) (1.86 ± 0.40), an effective number of alleles (N _e) (1.43 ± 0.30) as well as six RAPD primers that produced a total of 95 bands, developing 61.05% polymorphism with N _a (1.93 ± 0.51) and N _e (1.18 ± 0.30). The dendrogram produced by UPGMA analysis, based on Dice’s coefficient, clustered four races into two major groups which accurately segregated them according to their inheritance of voltinism. In this research, the ISSR markers were more accurate than the RAPD markers and ISSR also displayed better polymorphism. The outcome showed that the bivoltine strains exhibited higher allelic expressions with ISSR primers when compared to the polyvoltine strains. Despite exhibiting their unique race by certain DNA markers, most of the primers represented voltinism-specificity. Hence molecular marker amplification is a beneficial approach to reveal genetic divergence among closely related strains, and molecular characterization of phylogenetic relationships in addressing evolutionary evidences of individuals.     

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 D2 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.     

Development of a heat shock inducible and inheritable RNAi system in silkworm

A heat shock inducible and inheritable RNA interference (RNAi) system was developed in the silkworm (Bombyx mori). RNAi transgenic silkworms were generated by injecting silkworm eggs with a piggyBac transposon plasmid carrying RNAi sequence against target gene driven by the Drosophila heat shock protein 70 (HSP70) promoter and the helper plasmid expressing piggyBac transposase. The transgenic EGFP gene and the endogenous eclosion hormone (EH) gene were chosen respectively as the target genes. In the RNAi transgenic silkworms, heat shock at 42 degrees C significantly and specifically reduced the expression of EGFP or EH gene in silkworms according to the corresponding RNAi targeting sequence but not in silkworms with the irrelevant RNAi sequence demonstrating the efficiency and specificity of the RNAi effect. Heat shock in the pupal stage hampered pupal-adult eclosion and reduced egg fertility in EH RNAi transgenic silkworms but not in the wild type or EGFP RNAi transgenic silkworms. The establishment of this heat inducible and inheritable conditional RNA interference system in silkworms provided an approach for the first time to dissect the functions of target genes in silkworms at different stages.     

Generation of transgenic silkworms for production of erythropoietin in Bombyx mori

There have been many attempts to generate various essential proteins using transformed E. coli systems. However, prokaryote systems are not equipped with the protein maturation mechanisms necessary to generate eukaryotic proteins. In this sense, among the eukaryotes, silkworms have major merits in overcoming the difficulties. Such protein maturation mechanisms are available in silkworms. In this study, a transgenic silkworm producing rhEPO in the cocoon was generated and purified. Specifically, we constructed a transgenic silkworm using a vector system that could be controlled to the next generation. To accomplish this, we microinjected the system into eggs laid during the preblastoderm stage. The rhEPO was then purified from transgenic silkworm cocoons using a Con A affinity column. The biological activity of rhEPO isolated from the cocoon of transgenic silkworms was then assessed in a cell culture system using an EPO-dependent cell line, F-36E. Next, PCR analysis was used to demonstrate that stable gene expression can occur in the embryos of the silkworm, Bombyx. mori. Transgenic silkworms were then selected and observed to ensure that the transgenic silkworm was maintained and transmitted to their progeny. The rhEPO was subsequently purified from the transgenic silkworm cocoon and the electrophoretic pattern of the purified rhEPO revealed a protein band with a molecular weight of approximately 20 kDa. A total of 3 mg of rhEPO was eluted from 10 g of cocoons. The proliferation of F36E cells for 25 ng/ml rhEPO was 1.32, while the proliferation for 2.5 IU/ml hEPO was 1.32. The proliferation of these cells could be induced by commercial hEPO, as well as by rhEPO from transgenic silkworm cocoons. An in vivo test of mice treated with rhEPO revealed relatively high RBC values when compared to normal mice. These results indicated that purified glycosylated EPO from transgenic silkworms had biological activities. Overall, the transgenic silkworm technique will be very useful for the large scale production of proteins for diagnostic and therapeutic purposes.     

酿酒酵母乙酸耐受性相关的微卫星分子标记筛选

【目的】筛选与酵母乙酸耐受性状紧密相关的微卫星分子标记。【方法】以两株表型差异菌株YHA和YLA作为亲本构建F2代菌株共计160株,选取15个微卫星位点通过PCR方法在40株子代中扩增产物,利用SPSS 11.5软件分析耐酸性状与微卫星序列间的相关性。【结果】找到3个与乙酸耐受性性状相关的微卫星位点,其中位点14P2与酵母乙酸耐受性状有极显著的正相关性(P<0.01),15P2和15P3与酵母乙酸耐受性具有显著的负相关性(P<0.01和P<0.05);此外对于微卫星位点14P2,耐酸亲本YHA在该位点的基因片段(344 bp)在子代耐酸菌株中出现频率达到70.6%,而不耐酸亲本YLA的基因片段(331 bp)在子代不耐酸菌株中出现的频率达91.3%。【结论】微卫星14P2的等位基因在子代菌株中的遗传具有明显的偏好性,该微卫星位点与某种耐酸基因存在一定的连锁遗传,为酵母分子标记辅助育种提供了有价值的遗传标记。     

Fat body catalase activity as a biochemical index for the recognition of thermotolerant breeds of mulberry silkworm, Bombyx mori L.

The fifth instar larvae of the silkworm Bombyx mori L. were exposed to selected high temperatures (35 and 40 °C) in order to understand the changes in the level of catalase activity in the three tissues of fat body, midgut and haemolymph of the five selected bivoltine breeds and their 9 quantitative traits, namely larval weight, cocoon weight, shell weight, shell ratio, filament length, filament weight, denier, renditta and effective rearing rate (ERR), also the correlation between them under high temperature conditions were examined. Catalase activity resulting in fat body revealed a positive correlation between the control (28±1 °C) and 40±1 °C. The CSR₂ breed showed the most level of thermotolerance and catalase activity, compared with the CSR₄, JROP, NB₄D₂ and KA breeds. It was found that the level of catalase activity in fat body may be a reliable biochemical index to recognize thermotolerant breeds in order to develop resistant hybrids for tropical areas.     

Proteolytic enzymes as markers of productivity and heterosis of silkworm

A positive correlation between the activity level of cysteine proteinases in developing eggs of common silkworm moth (Bombyx mori L.), on the one hand, and a set of commercial characteristics, on the other, was found. This allows the determination of cysteine proteinase activities (pH optima of 3.0, 3.6, and 8.6) to be recommended as a biochemical test for an early prediction of potential productivity of silkworm breeds. A positive correlation between the activity level of acid cysteine proteinases in eggs of parental breeds and a set of commercial characteristics of their hybrids was detected, indicating the possibility of predicting the degree of heterosis.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 1, 2005, pp. 99–106.Original Russian Text Copyright © 2005 by Krylova, Yarygin, Filippovich.     

Efficient strategies for changing the diapause character of silkworm eggs and for the germline transformation of diapause silkworm strains

Abstract To overcome the disadvantages of current silkworm Bombyx mori transgenic technology, such as costly and time‐consuming to maintain non‐diapause transgenic silkworms, we report here on the development of treatments for the germline transformation of diapause silkworm strains. Our results showed that HCl treatment within 3 h of oviposition was able to prevent the diapause of eggs from Japanese lineage diapause silkworm strains and was also suitable for germline transformation of the same strains. By incubating developing mother eggs from Chinese lineage diapause silkworm strains at 15°C (15°C‐IME), we were able to prevent the diapause of their daughter eggs; a similar strategy (15°C‐IMES) for the germline transformation of the same strains was that the mother eggs were incubated at 15°C, and the daughter eggs were then microinjected according to the conventional microinjection methods used for non‐diapause eggs. By combining temperature and light controls, the improved 15°C‐IMES strategy prevented diapause in daughter eggs, and also enabled the germline transformation of both Japanese and Chinese lineage diapause silkworm strains. Although each of the strategies developed here has advantages and disadvantages, we suggest that the 15°C‐IMES strategy is a good reference for the establishment of germline transformation technologies of other egg diapause insects. These new strategies for the efficient germline transformation of diapause silkworm strains are likely to improve the practical use of silkworm transgenic lines in sericulture and also highlight silkworm functional genomics research and its modeling.