Degumming Research Articles

Enhanced silk production and pupal weight in Bombyx mori through CRISPR/Cas9-mediated circadian Clock gene disruption.

The domesticated silkworm, Bombyx mori, is crucial for global silk production, which is a significant economic activity supporting millions of livelihoods worldwide. Beyond traditional silk production, the growing demand for insect larvae in cosmetics, biomedical products, and animal feed underscores the need to enhance B. mori productivity. This study investigates the role of the circadian clock gene Clock in B. mori using CRISPR/Cas9-mediated mutagenesis to establish the ClkΔ29 knock-out mutant strain. Dysregulation of the circadian clock in ClkΔ29 was demonstrated by altered temporal transcriptional profiles of core circadian clock genes in adult heads and disrupted circadian-controlled behaviors, including adult eclosion and egg hatching rhythms under constant darkness. By analysing larval development timing, as well as the weights of late instar larvae, pupae, and cocoon components in ClkΔ29 mutants and in ClkΔ1922 silkworms (carrying an independently generated Clk- null allele), we showed that CLK contributes to physiological processes regulating B. mori development and growth. Importantly, ClkΔ29 mutants reared on a standard sericulture diet exhibited significant increases in key economic traits, with silk production increasing by up to 7%, and pupal weight increasing by up to 25% compared to wild-type controls. This study highlights the potential of circadian clock gene manipulation to significantly enhance sericultural productivity. Future research should focus on elucidating the molecular mechanisms driving these phenotypes and determining whether they result from circadian clock functions or pleiotropic effects of B. mori Clk. These findings provide a foundation for advancing sustainable sericulture and developing new commercial applications for silkworm-derived products.

Nutritive Effects of Calcium Carbonate (caco3) on Selected Commercial Traits of Mulberry Silkworm (Bombyx mori L.)

Silkworm (Bombyx mori) is the primary producer of silk. It has also been noted that minerals affect the quality of the silkworm. Calcium, a mineral with vital metabolic functions, might be involved in betterment of silkworms resulting in high quality silk production. Hence, this study was designed to evaluate the nutritive effects of Calcium carbonate (CaCO3) on silkworms and subsequent silk production. Immediately after the completion of the third instar stage of silkworm (crossbreed race - BSR-95/14(M)SL), they were divided into the control group and treated groups. Both the treatment and control groups were replicated thrice with 60 larvae each. Then, the effect of Calcium carbonate was studied at 4th and 5th instar larvae of silkworm. The treated and the control group of 4th and 5th instar larvae of silkworm was fed four times a day with Mulberry leaves either dipped into different concentrations of Calcium carbonate (0.1%, 0.2%, 0.3%, 0.4% in distilled water) or dipped into distilled water only respectively. The results showed that the supplementation of Calcium Carbonate (CaCO3) with Mulberry leaves resulted in significantly higher larval, egg, pupal, and cocoon characteristics compared to the control group. Among the different concentrations, 0.2% Calcium Carbonate supplementation showed the most valuable effect on silkworm. It can be concluded that enrichment of mulberry leaves with calcium carbonate is suitable for improvement of economic characteristics of silkworms, hence it might be suggested for usage in boosting farmers' yields.

Comparison of Cocoon and Filament Quality of Bombyx mori L. Chinese Egg Seeds and Perum Perhutani Egg Seeds

The sustainability of natural silk in South Sulawesi, especially in Soppeng Regency, has experienced a decline in production related to the quality of silkworms. The Indonesian government tried to import silkworm eggs from the Chinese to restore the silk business. It is necessary to know how the quality of silkworm eggs from the Chinese compares with local silkworm eggs produced by the State Forestry Public Company (Perum Perhutani). Therefore, this research aims to compare the quality of cocoons and filaments between Chinese egg seeds and Perum Perhutani egg seeds so that we can find out which egg seeds produce the best quality and production. The two egg seeds were tested in Pising Village, Donri-Donri District, Soppeng Regency. The parameters observed were fresh cocoon weight, cocoon shell weight, percentage, filament length, reelability and filament weight. The results of the research showed that there was a significant difference between egg seeds from Chinese and Perum Perhutani egg seeds in the reelability variable. Reelability of egg seeds from Chinese shows that the average cocoon weight is 74.06% higher compared to 69.31% for Perum Perhutani egg seeds. Apart from that, those produced from egg seeds from Chinese were also greater with differences in fresh cocoon weight of 1.56 g, cocoon shell weight of 0,29 g, cocoon shell percentage of 19.65%, filament weight of 0.25 g, while filament length was higher in seeds. Perum Perhutani eggs with a difference of 1118.39 m. The quality of cocoons and silkworm filaments from Chinese seeds and Perum Perhutani is classified into class C based on SNI: 2011. These results indicate that egg seeds from the Chinese have a higher potential to produce quality silk. This research emphasizes the importance of choosing the right egg seed to increase the quality and quantity of silk production. With the results obtained, it is hoped that the silk industry can choose more sources of egg seeds, especially to increase the competitiveness of silk products in the global market.

Molecular and Proteomic Analyses of Effects of Cadmium Exposure on the Silk Glands of Trichonephila clavata.

Cadmium (Cd) is a pervasive heavy metal pollutant released into the environment through industrial activities such as mining, smelting, and agricultural runoff. This study aimed to investigate the molecular and metabolic impacts of Cd exposure on the silk glands of Trichonephila clavata, a species renowned for producing silk with exceptional mechanical properties. Cd accumulation in spider bodies and silk glands was significantly higher in the low- and high-Cd groups compared to controls, with a dose- and time-dependent increase. Oxidative stress markers, including superoxide dismutase, glutathione peroxidase, peroxidase, and malondialdehyde, were significantly elevated, indicating a robust stress response. Proteomic analysis identified 2498 proteins, with 227 differentially expressed between Cd-treated and control groups. Key metabolic pathways, including glutathione metabolism, cysteine and methionine metabolism, and amino acid biosynthesis, were significantly disrupted. Downregulation of enzymes such as glutathione synthase and S-adenosylmethionine synthetase highlighted oxidative imbalance and impaired sulfur metabolism, indicating disruptions in redox homeostasis and energy metabolism critical for silk production. These findings demonstrate that Cd exposure alters oxidative stress responses, disrupts key metabolic pathways, and impairs silk gland functionality at multiple molecular levels. This study advances the understanding of the impact of heavy metal stress on spider physiology and provides a foundation for further research on the ecological implications of Cd contamination.

Muga Silk Production and Engagement of Family in Different Category of Silk Production: A Study in Lakhimpur District of Assam, India during 2013-23

Muga Silk Production and Engagement of Family in Different Category of Silk Production: A Study in Lakhimpur District of Assam, India during 2013-23

Evidence of Bombyx mori (Lepidoptera: Bombycidae) odorant receptors related to oviposition behavior

The silkworm, Bombyx mori,is an insect that is economically important for silk production, cosmetics, medical applications, food, and scientific research. The oviposition behavior of the female moth affects the number of eggs and the volume of silk production. This research aimed to investigate the relationship between the various treatment conditions of mulberry odor, the expression levels of Bombyx mori odorant receptor genes (BmOrs)in the antenna of female Bombyx mori moths, and the moths’ oviposition behavior. Four treatment conditions (fresh mulberry leaves, 2% mix, mulberry leaf juice, and 2% powder) showed a higher oviposition rate than that of the control. Our results revealed that the expression levels of BmOr44, BmOr54, BmOr56,and BmOr63 might play a major role in oviposition. The predicted three-dimensional structures of BmOr44, BmOr54, BmOr56, and BmOr63 proteins were found similar and some active compounds of mulberry leaf could virtually bind to these proteins. The expression patterns of BmOr19 and BmOr30,the specific female adult moth odorant receptor genes, were similar in almost every treatment.

Expression of spider silk protein in tobacco improves drought tolerance with minimal effects on its mechanotype.

Spider silk, especially dragline silk from golden silk spiders (Trichonephila clavipes), is an excellent natural material with remarkable mechanical properties. Many studies have focused on the use of plants as biofactories for the production of recombinant spider silk. However, the effects of this material on the mechanical properties or physiology of transgenic plants remain poorly understood. Since glycine-rich proteins play key roles in plants, we evaluated the effects of a glycine-rich spider silk protein on plant mechanical properties (mechanotype) and physiology. We generated tobacco (Nicotiana tabacum) plants producing a nucleus- or plastid-encoded partial component of dragline silk, MaSp1 (major ampullate spidroin-1; MaSp1-tobacco), containing six repetitive glycine-rich and polyalanine tandem domains. MaSp1 accumulation had minimal effect on leaf mechanical properties, but improved drought tolerance. Transcriptome analysis of drought-stressed MaSp1-tobacco revealed the upregulation of genes involved in stress response, antioxidant activity, cellular metabolism and homeostasis, and phenylpropanoid biosynthesis. The effects of drought treatment differed between the nucleus- and the plastid-encoded MaSp1-tobacco, with the latter showing a stronger transcriptomic response and a higher total antioxidant status (TAS). Well-watered MaSp1-tobacco displayed elevated levels of the stress phytohormone ABA, leading to stomatal closure, reduced water loss, activation of stress response, and increased TAS. We show that the moderately enhanced ABA content in these plants plays a pivotal role in drought tolerance, alongside, ABA priming, which causes overall adjustments in multiple drought tolerance mechanisms. Thus, our findings highlight the potential of utilizing glycine-rich spider silk proteins to enhance plant resilience to drought.

Enhancing Silkworm (Bombyx mori L.) Larval Survivability Through Spermidine Supplementation

The silkworm, Bombyx mori L., is a species of great economic significance since it is essential to the manufacture of silk. Improving silkworms' growth, survival rates, and silk yield throughout their larval stages requires increasing their food intake. The purpose of this study is to evaluate the effects of adding spermidine to mulberry leaves on Bombyx mori L. larvae survival. The experimental groups in this experiment were given mulberry leaves treated with different doses of spermidine, such as Spd 25, 50, 75, and 100 µM. The larvae were separated into control and experimental groups. The findings demonstrated that larvae fed leaves laced with spermidine had higher survival rates than the control group. However, compared to the control group, the treated group's mortality rate was decreased. These findings indicate that spermidine could be a useful nutritional enhancement in sericulture, promoting better larval health and increased silk production.

Influence of Phyllanthus emblica Extract Fortified Mulberry Leaves on the Commercial Parameters of the Silkworm Bombyx mori. L

In recent years, a variety of plant extracts in varying concentrations have been utilized in commercial silk farming to observed their effects on commercial parameters of Bombyx mori L. In the present investigation, the effects of ethanolic extract of Phyllanthus emblica Linn. was studied with different concentrations on the larvae of silkworm, Bombyx mori L. The results show increasing trend in some important commercial parameters. The effective increase in single cocoon weight, filament length and filament weight was observed with the use of 1: 2, 1: 4 and 1: 8 concentrations of Phyllanthus emblica Linn.

Optimizing Silkworm Rearing: The Impact of Environmental Factors and Advanced Technologies on Silk Quality and Production

Optimizing Silkworm Rearing: The Impact of Environmental Factors and Advanced Technologies on Silk Quality and Production

Antiviral activity of red fluorescent proteins in silkworm, Bombyx mori L.

Silkworm is an important economic insect in the textile industry for the production of silk. During its larval period, it suffers from various types of diseases viz., fungal, viral, bacterial and protozoan which hamper its growth and development and negatively impact the raw silk production. To fight these pathogens, different types of proteins have been found in the silkworm larva. Among these proteins, red fluorescent proteins (RFPs) possess antiviral activity, found in the digestive juice of the midgut of silkworm larvae. These RFPs are found to be more effective against Bombyx mori L. nucleopolyhedrosis virus (BmNPV), which is the causal agent of the most dreadful disease known as Grasserie. The RFP is synthesised in the presence of light after silkworms are fed on fresh mulberry leaves having absorbance peaks (280 and 605 nm) wave length.

16S rRNA Sequencing Analysis Uncovers Dose-Dependent Cupric Chloride Effects on Silkworm Gut Microbiome Composition and Diversity.

Copper-based pesticides are extensively used in agriculture, yet their impacts on beneficial insects remain poorly understood. Here, we investigate how cupric chloride exposure affects the gut microbiome of Bombyx mori, a model organism crucial for silk production. Using 16S rRNA sequencing, we analyzed the gut bacterial communities of fifth-instar silkworm larvae exposed to different concentrations of cupric chloride (0, 4, and 8 g/kg) in an artificial diet. The high-dose exposure dramatically altered the microbial diversity and community structure, where the Bacteroidota abundance decreased from 50.43% to 23.50%, while Firmicutes increased from 0.93% to 18.92%. A network analysis revealed complex interactions between the bacterial genera, with Proteobacteria and Firmicutes emerging as key players in the community response to copper stress. The functional prediction indicated significant shifts in metabolic pathways and genetic information processing in the high-dose group. Notably, the low-dose treatment induced minimal changes in both the taxonomic composition and predicted functions, suggesting a threshold effect in the microbiome response to copper exposure. Our findings provide novel insights into how agricultural chemicals influence insect gut microbiota and highlight potential implications for silkworm health and silk production. This work contributes to understanding the ecological impacts of copper-based pesticides and may inform evidence-based policies for their use in sericulture regions.

Comparative Physiological, Proteomic, and Metabolomic Insights into a Promising Low-Pruning Mulberry Cultivar for Silkworm Rearing.

Mulberry (Morus spp.) is an economically significant plant in the production of silk through feeding leaves to silkworm larvae. Traditional silkworm rearing is heavily labor-intensive, particularly in leaf collection, which leads to low efficiency and impedes the development of sericulture. Here, to assess the feasibility and effectiveness of a novel low-pruning mulberry cultivar, ZJ1, in the silkworm rearing industry, a comprehensive investigation integrating physiological, proteomic, and metabolomic analyses was conducted in comparison with the traditionally high-pruning cultivar, N14. The low-pruning mulberry variety ZJ1 exhibited a notable increase in annual leaf yield of 43.94%, along with a significant enrichment of serine and isoleucine contents, in contrast to those of the high-pruning variety N14. Through iTRAQ proteomics and LC-MS/MS metabolomics analyses, a total of 561 reduced and 803 increased differentially expressed proteins (DEPs), as well as 332 differential expressed metabolites (DEMs) in positive ions and 192 DEMs in negative ions, were identified in the ZJ1 group relative to the N14 group, respectively. The observed features in amino acid profiles and the enrichment of the sucrose-related metabolic pathway provided interesting insights for future endeavors in mulberry variety improvement and the optimization of silkworm diet formulations. Collectively, the low-pruning cultivar ZJ1, characterized by its rapid growth, high leaf productivity, and suitability for mechanized operations, is expected to be an efficient substitute in improving the future sericultural industry, especially in urbanized and industrialized regions.

Genetic Code Expansion of the Silkworm Bombyx mori Using a Pyrrolysyl-tRNA Synthetase/tRNAPyl Pair.

The domesticated silkworm Bombyx mori, an essential industrial animal for silk production, has attracted attention as a host for protein production due to its remarkable protein synthesis capability. Here, we applied genetic code expansion (GCE) using a versatile pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pair from Methanosarcina mazei to B. mori; GCE enables synthetic amino acid incorporation into proteins to give them non-natural functions. Transgenic B. mori lines expressing M. mazei PylRS and its cognate tRNAPyl were generated and cross-mated to obtain their F1 hybrid. Orally administering a click-compatible synthetic amino acid, trans-cyclooctene-lysine (TCO-Lys), to the F1 hybrid has led to the production of silk fiber incorporated with TCO-Lys. TCO-Lys incorporation in silk fiber was verified by selective labeling of the TCO group by click chemistry. The developed system is available for large-scale protein production with a wide variety of synthetic amino acids.

Mechanisation applied to entomological production cannot ignore insect reactivity: a case study on Bombyx mori in the context of the 'Serinnovation' project.

In December 2017 the Venetian Region (local Authority), financed the creation of the Operational Group (OG) 'Serinnovation', within the framework of the Rural Development Plan supported by the European Community. The OG aims at coordinating and spreading innovation in sericulture through mechanisation of processes and centralisation of some rearing steps, the use of waste as by-products and traceability to promote local productions. The project acts on perceived quality by increasing the added value, through production cost efficiency, and on the recovery of the waste material for further applications (circular economy). The final target was to develop a niche-process to obtain traceable 'Made-in-Italy' silk for the luxury market and non-textile applications. A first strategy to increase the efficiency of the process was to build an automatic leaf cutting machine to prepare the feed for the first three instars of the silkworm (Bombyx mori Linnaeus). This new machine - based on a patent - was validated through several tests and compared with the cutting system previously used. The study was completed by a bioassay of production and survival rate associated with the introduction of this innovation. The results showed that labour saving is in the order of 10% compared to a semi-manual process, the leaf quality is not affected, survival of larvae and silk production are not significantly different from the control. This methodology is proposed as a study case for other similar mechanisation processes in entomological production, as the impact of innovations on insect physiology should be carefully considered.

Implementation of Blue Economy to Increase Lobster Production through Natural Feeding Efficiency

Community Service Activities entitled Freshwater Lobster and Silkworm Cultivation, were carried out from September to November 2024. The need for freshwater lobster feed that is difficult to obtain makes natural feed such as silkworm cultivation, an alternative to be used as feed for freshwater lobster cultivation. In addition, Freshwater Lobster cultivation is still very rarely carried out in the Gorontalo area, thus providing opportunities and benefits for inmates to be able to become independent after leaving prison. The need for freshwater lobster feed that is difficult to obtain makes natural feed for larvae/fry such as silkworms, an alternative to be used as feed for freshwater lobster cultivation. This PKM activity is a new hope for inmates to get a sustainable livelihood and income.

β-fructofuranosidase regulation in silkworm silk gland development: Implications for silk gland morphogenesis and silk production

This study investigates the impact of β-fructofuranosidase (Bmsuc1) on the development of the silk gland in silkworms (Bombyx mori). Previous research shows that Bmsuc1 is highly expressed in the silk glands and may be involved in silk gland development and protein synthesis. However, the precise mechanism by which Bmsuc1 regulates silk gland development remains unclear. This study specifically used RNA interference to inhibit Bmsuc1 expression in silkworm larvae. The results revealed that silencing Bmsuc1 led to significant shortening of the anterior silk gland cells, left and right side size asymmetrical development of the middle silk gland, and alterations in cellular and inner membrane layer thickness. Furthermore, the glucose and fructose levels in the silk gland were significantly reduced, reducing cocoon weight. The interference of Bmsuc1 also triggers carbohydrate metabolism-related genes, beta-hexosaminidase subunit (HEXA-β) and glucose-6-phosphatase (G6Pase), which were upregulated. In addition, cell cycle-related genes, Cyclin E and cyclin-dependent kinase 2 (CDK2), were downregulated. These findings provide a new theoretical foundation for understanding the molecular mechanisms of silk gland development and offer insights into improving silk yield.

Diversity of Eri Silkworm Samia ricini donovan, its host plants in north east India and their utilization for sustainable livelihood

North Eastern Region of India occupies a distinct position in the country with flourishing green forests and conducive climatic condition harbouring varied flora and fauna including sericigenous insects and their host plants producing Muga, Eri, Mulberry and Oak Tasar silks. The region is recognized as one of the biodiversity hotspots among 34 biodiversity hotspots of the world. The Indian Sub-Himalayan belt extending from Jammu and Kashmir in the North West and North Eastern states in the extreme east is the natural abode of many wild sericigenous species. Among the culture of vanya silks, ericulture is emerging as one of the most vibrant socio-economic activities among all vanya silk culture in North Eastern India providing gainful occupation to about 4.00 lakh families with 70% involvement of women from weaker sections of the society. During 2022-23, total production of raw silk in the country was 36,582MT comprising of 27654 MT Mulberry (75.59%) and 8928 MT Vanya or wild silk (24.41% ) and production of eri raw silk was 7349 MT contributing about 82.31% of total vanya silk produced in the country. Production of eri raw silk in the country has increased manifold from 3116 MT during 2012-13 to 7349 MT during 2022-2023 in the last ten years with CAGR of 8.96% and ericulture is expanding in other states of the country other than north eastern states by virtue of its feasible economic implications. Presently, seven eco-races and six strains of eri silkworm are being maintained at R& D institutes of CSB for commercial exploitation while wild eri silk moth Samia canningi (Hutton) available in the foothills of the region needs to be conserved for future exploration. The paper highlights the diversity of eri silkworm and its host plants in North East India, needs for conservation of bio-resources along with prospects of utilizing their products and by-products for sustainable livelihood and upliftment of rural economy.. KEYWORDS :Ser-biodiversity, Samia ricini, Samia canningi, Conservation, Sustainable development.

Study on Wax Secretion and Wax‐Secreting System of Mulberry Pest Paurocephala sauteri Enderlein (Hemiptera: Psyllidae)

ABSTRACTPaurocephala sauteri Enderlein is one of the most important pests of mulberry trees in the tropical and subtropical regions of China. This species by penetrating the veins on the back of leaves and buds of mulberry trees, nymphs and female adults can secrete wax, these wax accumulated on the mulberry leaves significantly affects photosynthesis and silkworm cultivation. Therefore, this study aimed to understand the wax‐secreting system and characteristics of wax secretion, expecting a potential strategy to control the species population. The main findings were as follows: scanning electron microscopy revealed that the nymphs and female adults secrete wax in long tubes with spherical ends, and male adults secrete honeydew. Additionally, the wax‐secreting system of the nymphs and female adults differ, the nymph owned a bending moon‐shaped wax‐secreting system, the wax‐secreting system first instar nymph consisted of one circle of rectangular, depressed cell, whereas the wax‐secreting system of second to fifth instar nymph made up of one circle of rectangular, depressed cell and one circle of round, depressed cell. There were a certain number of white pores inside of this depressed cell. Wax filaments produced by these pores are converted into a dense, tight and hollow the wax tube. Gas chromatography/mass spectrometer showed that the main constituents of P. sauteri wax are hydrocarbons. The wax tube as a water conservation pool during the P. sauteri developmental process. This physiological feature may be a key point for a potential strategy to control P. sauteri, even other secreting insects.

High Absorption and Elasticity of a Novel Transgenic Silk with Egg Case Silk Protein from Nephila clavata.

Spider silk is part of a special class of natural protein fibers that have high strength and toughness: these materials have excellent comprehensive properties that are not found in other natural fibers (including silk) or most synthetic fibers. Spider egg case filaments have good hardness, can resist water, can protect spider eggs from external threats, have a significantly high initial modulus and high moisture absorption rate, and are expected to be used as a new generation of environmentally friendly natural polymer fibers and biomaterials. However, spiders are predatory and difficult to rear in large numbers, and it is also difficult to obtain spider egg case filaments in large quantities. Silkworms and spiders have a similar spinning system, and the use of transgenic technology in silkworms can obtain stable and high-yield exogenous gene proteins for a long time, representing an ideal bioreactor for the production of spider silk. In this study, the eukaryotic bioreactor and piggyBac transposon system were employed to recombinantly introduce the egg case silk protein of Nephila clavata (Nc-CYSP1) into the silkworm in the silkworm heavy-chain expression system. The results revealed that the silk glands produced a new type of transgenic silk with a significantly high initial modulus and high moisture absorption. In summary, this study provides an experimental reference for future research on the large-scale production and application of spider egg case filamentous protein, with great application prospects in the development of new environmentally friendly materials.