Bivoltine cocoon color sex-limited breeds of Bombyx mori (Lepidoptera: Bombycidae) show enhanced economic performance and fecundity following spermidine supplementation.

Sericulture has immense economic significance. Separating male and female silkworm pupae for egg production in grainage is a laborious task; hence, sex-limited breeds for cocoon color are advantageous for this process. The major constraint in sex-limited breeds is their low fecundity. Sex-limited female moths lay a lower number of eggs than nonsex-limited breeds. Polyamine, spermidine was shown to improve fecundity in several organisms, including the silkworm, Bombyx mori L. In the present study, cocoon color sex-limited breeds, HTO2SL, APS27SL, and SLFC27, were selected and fed with spermidine to improve fecundity and nutritional efficiency. The fifth-instar silkworm larvae of the selected breeds were subjected to standard rearing conditions with and without spermidine supplementation up to spinning. The spermidine-supplemented sex-limited breeds exhibited a significant increase in fecundity, nutritional indices, and economic parameters compared with the control sex-limited breeds. Among the 3 sex-limited breeds tested, the performance of APS27SL improved significantly.

Thermal tolerance role of novel polyamine, caldopentamine, identified in fifth instar Bombyx mori.

Silkworms have limited ability to regulate their body temperature; therefore, environmental changes, such as global warming, can adversely affect their viability. Polyamines have shown protection to various organisms against heat stress. This study evaluated the qualitative and quantitative changes in heat-stressed Bombyx mori larvae polyamines. Fifth instar Bombyx mori larvae were divided into two groups; control group, reared at room temperature, i.e., 28 ± 2 °C, and the heat shock group, exposed to 40 °C. Dansylation of the whole worm polyamines and subsequent thin-layer chromatography revealed the presence of components with the same Rf value as dansyl-putrescine, spermidine, and spermine. The dansyl-putrescine, spermidine, and spermine polyamines were identified by mass spectrometric analyses. After heat shock, the thin-layer chromatography of the whole-larvae tissue extracts showed qualitative and quantitative changes in dansylated polyamines. A new polyamine, caldopentamine, was identified, which showed elevated levels in heat-stressed larvae. This polyamine could play a role in helping the larvae tolerate various stress, including thermal stress. No significant changes in silk fiber's economic and mechanical properties were observed in our study. This study indicated that PA, caldopentamine, supplementation could improve heat-stress tolerance in Bombyx mori.

Spermidine alleviates heat shock and promotes the growth of Bombyx mori.

Silkworms have limited ability to regulate their body temperature upon exposure to varying atmospheric temperatures. Environmental changes, such as global warming, adversely affect silkworm viability. Spermidine, a polyamine, protects various organisms against heat stress. This study aimed to evaluate the effect of spermidine on the thermotolerance of Bombyx mori larvae. 5th instar Bombyx mori larvae were divided into the control and spermidine groups and reared at 28 ± 1 °C and 80%-85% relative humidity. To induce heat stress, the larvae were exposed to various temperatures 32 °C, 36 °C, and 40 °C for 1 h on day 5 and subsequently allowed to recover at 28 ± 1 °C. Growth characteristics were evaluated by examining larval viability and quantifying proteins and carbohydrates. The thermotolerance of the spermidine group was higher than that of the control group at 40 °C heat stress conditions. The spermidine feeding increased the protein content and reduced the carbohydrate content significantly under heat stress condition. For the first time, this study demonstrated that spermidine alleviated thermal stress by enhancing the nutritional indices and antioxidant potential of the Bombyx mori larvae. A significant increase in economic properties was observed in spermidine fed groups. Thus, foliar feeding of spermidine to B. mori larvae markedly improved silkworm thermotolerance.

Dynamics of nuclear matrix attachment regions during 5th instar posterior silk gland development in Bombyx mori.

BACKGROUND: Chromatin architecture is critical for gene expression during development. Matrix attachment regions (MARs) control and regulate chromatin dynamics. The position of MARs in the genome determines the expression of genes in the organism. In this study, we set out to elucidate how MARs temporally regulate the expression of the fibroin heavy chain (FIBH) gene during development. We addressed this by identifying MARs and studying their distribution and differentiation, in the posterior silk glands of Bombyx mori during 5th instar development. RESULTS: Of the MARs identified on three different days, 7.15% MARs were common to all 3 days, whereas, 1.41, 19.27 and 52.47% MARs were unique to day 1, day 5, and day 7, respectively highlighting the dynamic nature of the matrix associated DNA. The average chromatin loop length based on the chromosome wise distribution of MARs and the distances between these MAR regions decreased from day 1 (253.91 kb) to day 5 (73.54 kb) to day 7 (39.19 kb). Further significant changes in the MARs in the vicinity of the FIBH gene were found during different days of 5th instar development which implied their role in the regulation and expression of the FIBH gene. CONCLUSIONS: The presence of MARs in the flanking regions of genes found to exhibit differential expression during 5th instar development indicates their possible role in the regulation of their expression. This reiterates the importance of MARs in the genomic functioning as regulators of the molecular mechanisms in the nucleus. This is the first study that takes into account the tissue specific genome-wide MAR association and the potential role of these MARs in developmentally regulated gene expression. The current study lays a foundation to understand the genome wide regulation of chromatin during development.

Nuclear matrix associated RNAs in posterior silk glands show developmental dynamics in Bombyx mori in 5th instar larvae.

OBJECTIVES: The nuclear matrix maintains and regulates chromatin structure. RNA is an integral component of the nuclear matrix and is essential to its structural maintenance. Bombyx mori is a major economic contributor in the sericulture industry and produces fibroin-the most important silk protein in its posterior silk glands during 5th instar larval stage. The present study investigates the composition of nuclear matrix RNA prepared from the posterior silk glands of Bombyx mori during fifth instar larval stage where maximum silk production occurs. The datasets from which the analysis is carried out are part of data note titled "Nuclear matrix associated RNA datasets of posterior silk glands of Bombyx mori during 5th instar larval development". RESULTS: The results showed significant enrichment of nuclear matrix RNA from day 1, to day 5 and day 7. Nuclear RNA showed increased abundance from day 1 to day 5 and day 7. Nuclear matrix RNA exhibited repetitive RNA sequences, of which UGUCC and GCUGGU were the most abundant. Genes involved in metabolic pathways showed significant enrichment correlating with silk production. These results emphasize the role of dynamic, repetitive DNA transcripts in chromatin architecture and further reveal the close association between the nuclear matrix and gene expression.

Nuclear matrix associated RNA datasets of posterior silk glands of Bombyx mori during 5th instar larval development.

OBJECTIVES: Bombyx mori is the key contributor to industrial silk production. The maximum production of silk occurs during 5th instar. The posterior silk glands in the larvae are responsible for the production of the main component of silk fibre-fibroin. The expression of genes and their regulation are dependent on the chromatin architecture. The nuclear matrix supports its structure and function by anchoring specific regions to regulate gene expression. The major constituent of the nuclear matrix, crucial to its structural and temporal maintenance, is its RNA. Therefore, the study of nuclear matrix RNA of the posterior silk glands on different days of 5th instar larval development is essential to understand its association to differential expression of genes. DATA DESCRIPTION: The tissue-specific developmental association of nuclear matrix RNA (NuMat RNA) at the genome level has not been done so far for any organism. Bombyx mori, CSR2 X CSR4 is the most popular dihybrid strain in India. The nuclear matrix RNA was isolated from day 1, day 5 and day 7 of 5th instar posterior silk glands of Bombyx mori. The NuMat RNA was sequenced using Illumina platform. The reads obtained were processed and the datasets were deposited in NCBI.

16S rRNA sequence data of Bombyx mori gut bacteriome after spermidine supplementation.

OBJECTIVES: The silkworm Bombyx mori (B. mori) is an important domesticated lepidopteran model for basic and applied research. They produce silk fibres that have great economic value. The gut microbiome plays an important role in the growth of organisms. Spermidine (Spd) is shown to be important for the growth of all living cells. The effect of spermidine feeding on the gut microbiome of 5th instar B. mori larvae was checked. The B. mori gut samples from control and spermidine fed larvae were subjected to next-generation sequencing analysis to unravel changes in the bacterial community upon spermidine supplementation. DATA DESCRIPTION: The changes in gut bacteriota after spermidine feeding is not studied before. B. mori larvae were divided into two groups of 50 worms each and were fed with normal mulberry leaves and mulberry leaves fortified with 50 µM spermidine. The gut tissues were isolated aseptically and total genomic DNA was extracted, 16S rRNA region amplified and sequenced using Illumina platform. The spermidine fed gut samples were shown to have abundance and diversity of the phyla Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria.

Knockdown of Broad-Complex Gene Expression of Bombyx mori by Oligopyrrole Carboxamides Enhances Silk Production.

Bombyx mori (B. mori) is important due to its major role in the silk production. Though DNA binding ligands often influence gene expression, no attempt has been made to exploit their use in sericulture. The telomeric heterochromatin of B. mori is enriched with 5'-TTAGG-3' sequences. These sequences were also found to be present in several genes in the euchromatic regions. We examined three synthetic oligopyrrole carboxamides that target 5'-TTAGG-3' sequences in controlling the gene expression in B. mori. The ligands did not show any defect or feeding difference in the larval stage, crucial for silk production. The ligands caused silencing of various isoforms of the broad-complex transcription factor and cuticle proteins which resulted in late pupal developmental defects. Furthermore, treatment with such drugs resulted in statistically enhanced cocoon weight, shell weight, and silk yield. This study shows for the first time use of oligopyrrole carboxamide drugs in controlling gene expression in B. mori and their long term use in enhancing silk production.

Effect of polyamines on mechanical and structural properties of Bombyx mori silk.

Silkworm, Bombyx mori (B. mori) belongs to the Lepidoptera family. The silk produced from this insect, mulberry silk, gained lot of importance as a fabric. Silk is being exploited as a biomaterial due to its surprising strength and biocompatibility. Polyamines (PA) are important cell growth regulators. In the present work the effect of treatment of polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm) on the quantity and quality of silk produced was assessed. Results showed that exogenous feeding of Spd at a concentration of 50 µM increased fiber length significantly. Analysis by Fourier transform infrared (FTIR) on the properties of silk obtained from Spd treated silkworms revealed an increase in percentage of absorption with no difference in peak positions of amide I and amide III groups. Scanning electron microscopy (SEM) revealed an increase in diameter of silk. Further, analysis at molecular level showed an increase in fibroin expression in Spd treated silk glands. However, the Spd treatment showed no significant difference with respect to fibroin to sericin ratio per unit weight of cocoon, silk tenacity, and percent elongation. Thus, the present results show that polyamine treatment would influence silk quality at structural, mechanical, and molecular level in the Bombyx mori, which can be exploited in silk biomaterial production.

Developmental proteome dynamics of silk glands in the 5th instar larval stage of Bombyx mori L (CSR2×CSR4).

UNLABELLED: Bivoltine breed of Bombyx mori (B. mori), CSR2×CSR4 is an Indian high yielding silkworm strain. Silk gland proteome of this strain was not studied till now. Methods of improving silk production by chemical approaches have reached saturation and transgenic methods are needed in further to boost silk production. An understanding of proteomic changes during silk gland development helps in designing experiments to enhance silk production by transgenic approaches. The present study reports comprehensive developmental proteomic analysis of CSR2×CSR4, 5th instar whole silk glands. Eighty six unique protein IDs were obtained from the analysis of one hundred and twenty protein spots. Among the identified proteins, majority of the proteins were involved in metabolism (41%) followed by proteins involved in protein homeostasis (30%). Sixty percent of the identified proteins showed dynamic nature by expression analysis from day 1, day 3, day 5 and day 7 gels. In comparison to the published data till now on silk gland proteomics this study reports identification of 20 new proteins from the silk glands for the first time. SIGNIFICANCE: The paper reports for the first time proteomic analysis of high yielding silkworm strain of India. The study analyzes whole silk glands to understand the tissue in total during 5th instar development. Lowering fibroin content made us to identify a large number of new proteins which were not reported till now in the silk gland proteome. Proteins which are involved in silk synthesis and release were found to be developmentally regulated. The study identified alanine, serine and glycine tRNA ligases for the first time and also showed their up-regulation on day 7 of 5th instar larval stage. The amino acid repeat of fibroin protein is enriched with the three amino acids, glycine, serine and alanine. The identified proteins could be studied further to understand their functional role in-depth.

Spermidine enhances the silk production by mulberry silkworm.

Polyamines are ubiquitous low molecular weight polycationic aliphatic amines involved in diverse cellular processes. Spermidine (Spd), a polyamine, has been proved to be crucial for cell survival in various organisms. Our study reports the effect of Spd on the growth of Bombyx mori. Silkworms showed improved silk gland weight and economic parameters in the fifth instar larval stage when treated with different concentrations of Spd, in the range of 25-75 µM. The worms treated with Spd produced 31% more silk when compared with the control worms. Altogether, this study establishes that Spd-treated leaves can be fed into the larvae for better silk production.

The effect of bovine milk on the growth of Bombyx mori.

Bombyx mori L. (Lepidoptera: Bombycidae) is a well-studied Lepidopteran model system because of its morphology, life cycle, and economic importance. Many scientists have placed importance on enhancing the economic traits of B. mori because it's larvae, silkworms, are vital in the production of silk. In this study, the effect of bovine milk on B. mori growth was tested. Bovine milk contains several components that aid in healthy growth. The treatment was given to fifth instar B. mori larvae because the fifth instar period is when B. mori eats voraciously and shows maximum growth among all its larval stages. The larvae were treated with fresh mulberry, Morus L. (Rosales: Moraceae), leaves and mulberry leaves dipped in milk from the first day of the fifth instar. Treatments were given on alternate days, and the silkworms were weighed every day to determine whether milk had any role in enhancing the weight of the larvae. Cocoon weights were measured, as the weight indicates the approximate amount of silk that can be reeled. The results showed that larvae gained 82.5% more weight by the end of fifth instar larval when fed with mulberry leaves dipped in milk than when fed with fresh mulberry leaves without milk. The larvae fed with milk-treated leaves gained 310% weight from day 1 to day 7 of the fifth instar, while the larvae fed with fresh leaves gained 153% weight in the same timespan. In addition, cocoon weight increased by 8% when milk was added compared to when it was not. These results suggest that B. mori larvae can be fed mulberry leaves treated with bovine milk for better growth rate and increased silk production.

AAGAG repeat RNA is an essential component of nuclear matrix in Drosophila.

Eukaryotic nucleus is functionally as well as spatially compartmentalized and maintains dynamic organization of sub-nuclear bodies. This organization is supported by a non-chromatin nuclear structure called the nuclear matrix. Although the precise molecular composition and ultra-structure of the nuclear matrix is not known, proteins and RNA molecules are its major components and several nuclear matrix proteins have been identified. However, the nature of its RNA component is unknown. Here we show that in Drosophila melanogaster, transcripts from AAGAG repeats of several hundred nucleotide in length are critical constituents of the nuclear matrix. While both the strands of this repeat are transcribed and are nuclear matrix associated, the polypurine strand is predominantly detected in situ. We also show that AAGAG RNA is essential for viability. Our results reveal the molecular identity of a critical RNA component of the nuclear architecture and point to one of the utilities of the repetitive part of the genome that has accumulated in higher eukaryotes.

Transposable element 'roo' attaches to nuclear matrix of the Drosophila melanogaster.

The genome of eukaryotes is organized into structural units of chromatin loops. This higher order organization is supported by a nuclear skeleton called the nuclear matrix. The genomic DNA associated with the nuclear matrix is called the matrix associated region (MAR). Only a few genome-wide screens have been attempted, although many studies have characterized locusspecific MAR DNA sequences. In this study, a MAR DNA library was prepared from the Drosophila melanogaster Meigen (Diptera: Drosophilidae) genome. One of the sequences identified as a MAR was from a long terminal repeat region of 'roo' retrotransposon (roo MAR). Sequence analysis of roo MAR showed its distribution across the D. melanogaster genome. roo MAR also showed high sequence similarity with a previously identified MAR in Drosophila, namely the 'gypsy' retrotransposon. Analysis of the genes flanking roo MAR insertions in the Drosophila genome showed that genes were co-ordinately expressed. The results from the present study in D. melanogaster suggest this sequence plays an important role in genome organization and function. The findings point to an evolutionary role of retrotransposons in shaping the genomic architecture of eukaryotes.

Polymorphism and epitope sharing between the alleles of merozoite surface protein-1 of Plasmodium falciparum among Indian isolates.

BACKGROUND: The C-terminal region of merozoite surface protein-1 (MSP-1) is one of the leading candidates for vaccination against the erythrocytic stages of malaria. However, a major concern in the development of MSP-1 based malaria vaccine is the polymorphism observed in different geographical Plasmodium falciparum isolates. To explore whether the sequence heterogeneity of PfMSP-1 leads to variation in naturally acquired anti-MSP-119 antibodies, the present study was undertaken to study PfMSP-119 sequence polymorphism in malaria-endemic villages in eastern India and also carried out a competition enzyme-linked immunosorbent assay using three PfMSP-119 variant forms. METHODS: The sequence variations in the C-terminal region of PfMSP-119 were determined in a malaria endemic region. Three PfMSP-119 variants were produced in Escherichia coli (PfMSP119QKNG-L, PfMSP119EKNG-L and PfMSP119ETSR-F) and an immunodepletion assay was carried out using the corresponding patients' sera. RESULTS: Results revealed predominance of PfMAD20 allele among Indian field isolates. Seven PfMSP-119 variant forms were isolated in a singe geographical location. Three of PfMSP-119 variant forms when expressed in E. coli showed presence of cross-reaction as well as variant specific antibodies in malaria infected patient sera. CONCLUSION: The present study demonstrates the existence of allele specific antibodies in P. falciparum-infected patient sera, however their role in protection requires further investigation. These results thereby, suggest the importance of a multi-allelic PfMSP-119 based vaccine for an effective malaria control.