Bombyx Mori Research Articles

Downregulation of BmSTAT transcription factor promoted nucleopolyhedrovirus replication in Bombyx mori.

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays a crucial role in the viral immune processes of organisms, with STAT being a key transcription factor downstream in this pathway. The STAT gene of Bombyx mori has two splicing forms, named BmSTAT-S and BmSTAT-L. This study compared the effects of the two splicing forms on Bombyx mori nucleopolyhedrovirus (BmNPV) infection through cell-level interference and further explored whether BmSTAT participates in the immune response to BmNPV infection via transgenic intervention at the individual level. Our research results indicated that BmNPV upregulates the expression of BmSTAT-S and BmSTAT-L in Bombyx mori BmE cells and larvae. Furthermore, BmE cells with interference of BmSTAT-S or BmSTAT-L displayed significantly higher expression levels of the viral gene GP41 and increased viral fluorescence compared to the control group after 48 h of infection with BmNPV. Then, we constructed transgenic silkworms with genetic interference, and the results showed that both the transgenic silkworms with systemic interference and midgut-specific interference of the two splice forms of BmSTAT exhibited significantly reduced survival rates and increased viral replication numbers after infection with BmNPV. The above results indicated that the BmSTAT gene is involved in the immune response of Bombyx mori to BmNPV and these findings lay the foundation for further research on the mechanism of JAK/STAT signaling pathway involvement in BmNPV infection.

Anti-Diabetic Activity of Cordyceps-Fermented Edible Insects by the Promotion of Glucose Absorption.

Insects are considered important food resources for future diet due to diverse nutrients and pharmacological effects. Fermentation is an important strategy of food processing with various beneficial effects such as increasing nutrients, promoting bioavailability, and reducing anti-nutrients. Cordyceps is a mushroom that grows on insects and produces various active ingredients. Therefore, we investigated the effect of Cordyceps-fermentation of insects on the nutritional composition and functional benefits of the insects. Six edible insects: Bombyx mori, Protaetia brevitarsis, Caelifera, Gryllus bimaculatus, Tenebrio molitor, and Allomyrina dichotoma were fermented with Cordyceps militaris to produce mycelia and fruiting bodies. Analysis of nutritional components showed that protein content was increased whereas carbohydrate content was decreased by the fermentation with Cordyceps. In addition, the fermented insects showed anti-diabetic efficacy by the promotion of glucose absorption as evaluated using differentiated L6-GLUT4myc cells. Quantitation using HPLC analysis suggested that cordycepin was produced in both mycelium and fruiting bodies in Cordyceps-fermented edible insects with different amounts depending on insect type and cultivation conditions. Therefore, the fermentation of insects with Cordyceps is expected to increase nutritional values and bioactive constituents and exert anti-diabetic effects.

Review on Silk Worm Pupal Meal: A Protein Source in Aquatic Animal Nutrition as a Replacement of Fish Meal

Amongst the insect meals, silkworm pupal meal is the most efficient fish meal replacement. It can be utilized in aquatic animal feeds as pupal meal, deoiled silkworm pupal meal and fermented pupal meal, to increase growth performance. It also has a significant impact on animal body composition and digestive enzyme activity. India is the world's second-largest producer of silk, and a vast amount of silkworm pupae waste is discharged into the environment, which can be used as protein in feed. It has a strong nutritional profile that is near or equal to fish meal, consists of 50-80% of crude protein on dry matter, rich in important amino acid profile, (methionine 3.5, lysine 7.0, aspartic acid 10.4, threonine 5.2, serine 5.0, glutamic acid 13.9, proline 5.2, glycine 4.8, alanine 5.8, cystine 1.0, valine 5.5, isoleucine 5.1, leucine 7.5, tyrosine 5.9, phenylalanine 5.1, histidine 2.6, and arginine 5.6) and lipid content ranges from 20-40% in freshly spent pupa and less than 10% in defatted pupa. The oil derived from pupa is high in alpha-linolenic acid, oleic acid, palmitic acid and the gross energy values range from 5.09 to 6.28 MJ/kg. It also contains interesting minerals and vitamins, which piqued the interest of researchers in its use in aqua feed. Aside from the nutritional profile, nutrient digestibility, and the effect of diet on organoleptic quality revealed that SWP meal is equivalent to fish meal in all respects. So, the current review focuses on the effects of silkworm pupal meal on aquatic animals when supplied through diets.

Acetylation of DnaJ facilitates the proliferation of BmNPV by affecting the transport of nucleocapsids

DnaJ (Orf40), a late-expressed factor of Bombyx mori nucleopolyhedrovirus (BmNPV), is essential for the budding of virions and influences the transfer of the nucleocapsid from the nucleus to the cytoplasm. Previous studies showed that the knockdown of dnaj could prevent the nucleocapsid from exiting the nucleus, but the underlying regulatory mechanism remains unknown. In our previous acetylomic analysis of BmN cells infected with wild-type BmNPV, we found that a lysine residue (K17) was acetylated 36h post-infection, and the acetylation level of this site was upregulated about 3.5-fold. Here, we found that deacetylation of DnaJ K17 significantly inhibited viral proliferation without affecting viral DNA replication. Furthermore, deacetylation of DnaJ K17 affected the interaction with two nucleocapsid-associated proteins, Ac66 and VP80, which in turn affected the production of nucleocapsids, as well as their transport within the nucleus along F-actin fibers, leading to a decrease in the export of nucleocapsids from the nucleus. The reduced amount of nucleocapsids in the cytoplasm ultimately led to a decrease in the production of budded virions and consequently inhibited viral proliferation. In conclusion, acetylation of DnaJ affects nucleocapsid production and transport, thereby influencing viral proliferation.

Multi-targets cleavage of BmNPV genome through genome-wide repeat sequence using CRISPR/Cas9 antiviral system.

The escalating severity of Bombyx mori nuclear polyhedrosis virus (BmNPV) infections poses significant challenges to the silkworm industry, especially when massive production shifts occur from the eastern regions to western regions with lower labor costs. Education and experience levels are different and disease control is badly needed. To solve the problems, we have developed an innovative CRISPR/Cas9 system specifically targeting BmNPV to enhance viral resistance. For the system, we selected BmNPV genes linked to virus replication and proliferation as targets, designing 2 sites for each gene. Mutating the target sequence renders the system incapable of efficiently cleaving the virus genome, hence decreasing cleavage efficiency. We conducted a search for "NGG" or "CCN" target sequences in the BmNPV genome, excluding non-recurring and potential targets in the B.mori genome. We successfully identified 2 distinct target sequences in the BmNPV genome-one being repeated 12 times and the other three times. These sequences lead to fragmentation of virus genome into multiple large segments that are difficult to repair. Transgenic silkworms demonstrate robust resistance to viruses, significantly boosting their survival rates compared with wild-type silkworms under various virus infection concentrations. Our system efficiently targets dozens of viral genomes with just 2 sequences, minimizing transposable elements while ensuring cutting effectiveness. This marks a pioneering advancement by using repetitive elements within the virus genome for targeted CRISPR cleavage, aiming for antiviral effects through genome fragmentation rather than disrupting essential viral genes. Our research introduces innovative concepts to CRISPR antiviral investigations and shows promise for the practical application of gene editing in industrial silkworm strains.

Orientation Distribution of Crystalline β-Sheet Domains in Bombyx mori Silk Fiber Studied with Vibrational Sum Frequency Generation Spectroscopy.

Silk fibers have good biocompatibility and mechanical properties, which make them attractive in biomaterial applications as well as textile industries. It is believed that the superior mechanical property is associated with the crystalline β-sheet structure in the fiber; but a deeper understanding of the structure-property relationship is still needed for full exploitation of its physical properties. Especially, accurate information on hydrogen-bonding interactions within β-sheet domains at the nanoscale and their spatial distributions at the mesoscale are critically needed. In this study, we demonstrate the selective detection of crystalline β-sheet domains in Bombyx mori silk fiber using sum frequency generation (SFG) spectroscopy and its use to determine the angular distribution of the β-sheet crystallites with respect to the fiber axis. Numerical simulations of the SFG signal of the amide-I band were carried out using tensors based on the B2 symmetry of the D2 point group and compared with experimental data. This comparison found that the crystalline β-sheet domains are aligned along the fiber axis with a standard deviation of ∼27° and parallel to the fiber surface with a standard deviation of ∼5°. It was also found that the amide bands in the SFG spectra cannot be fully explained with the assumption that the crystalline β-sheet vibrations can be described with the D2 point group. Being able to monitor the amide group vibrations sensitive to both interchain hydrogen bonding and crystallite orientations, SFG analysis has a potential to unveil the structure-mechanical property relationship that may not be readily assessable with other characterization techniques.

Phosphate-Driven Interfacial Self-Assembly of Silk Fibroin for Continuous Noncovalent Growth of Nanothin Defect-Free Coatings.

Silk fibroin is a fiber-forming protein derived from the thread of Bombyx mori silkworm cocoons. This biocompatible protein, under the kosmotropic influence of potassium phosphate, can undergo supramolecular self-assembly driven by a random coil to β-sheet secondary structure transition. By leveraging concurrent nonspecific adsorption and self-assembly of silk fibroin, we demonstrate an interfacial phenomenon that yields adherent, defect-free nanothin protein coatings that grow continuously in time, without observable saturation in mass deposition. This noncovalent growth of silk fibroin coatings is a departure from traditionally studied protein adsorption phenomena, which generally yield adsorbed layers that saturate in mass with time and often do not completely cover the surface. Here, we explore the fundamental mechanisms of coating growth by examining the effects of coating solution parameters that promote or inhibit silk fibroin self-assembly. Results show a strong dependence of coating kinetics and structure on solution pH, salt species, and salt concentration. Moreover, coating growth was observed to occur in two stages: an early stage driven by protein-surface interactions and a late stage driven by protein-protein interactions. To describe this phenomenon, we developed a kinetic adsorption model with Langmuir-like behavior at early times and a constant steady-state growth rate at later times. Structural analysis by FTIR and photoinduced force microscopy show that small β-sheet-rich structures serve as anchoring sites for absorbing protein nanoaggregates, which is critical for coating formation. Additionally, β-sheets are preferentially located at the interface between protein nanoaggregates in the coating, suggesting their role in forming stable, robust coatings.

Strong Silkworm Silk Fibers through CNT-Feeding and Forced Reeling.

High-performance silk fibers, with their eco-friendly degradability and renewability, have long captivated researchers as an alternative to synthetic fibers. Spider dragline silk, renowned for its exceptional strength (>1 GPa), has an extremely low yield, hindering its widespread use. While domesticated silkworms (Bombyx mori) can produce silk fibers industrially, their moderate strength (≈0.5 GPa) pales in comparison to the formidable spider dragline silk. In this study, naturally produced strong silkworm silk fibers are reported with a tensile strength of ≈1.2 GPa achieved through combining feeding carbon nanotubes (CNTs) to silkworms and in situ forced reeling for alignment. Molecular dynamics simulations confirm the interaction between the CNTs and silk fibroin, while the forced reeling process aligns these reinforcing fillers and the silk fibroin β-sheet nanocrystals along the fiber axis. Structural analysis reveals a significant enhancement in the content and alignment of β-sheet nanocrystals within the silk fibers, accounting for their superior mechanical properties, including tensile strength of ≈1.2 GPa and Young's modulus of 24.4 GPa, surpassing various types of silkworm silk and spider silk. This advancement addresses the historical trade-off between the strength and scalability of silk, potentially paving the way for eco-friendly, biodegradable, and renewable alternatives to synthetic fibers in a variety of applications.

Nanoparticles supplementation through foliar feed contributed to the growth and cocoon yield of silkworm (Bombyx mori)

An experiment was conducted during 2021–22 and 2022–23 at University of Agricultural Sciences, Dharwad, Karnataka to study the effect of metal nanoparticles on silkworm (Bombyx mori) growth and cocoon yield. The experiment was laid out in completely randomized design (CRD) comprised of 11 treatments with 3 replications. The various concentrations (50, 100 and 200 ppm) of nanoparticles, viz. nano silver, nano zinc and nano copper were extrafoliated to mulberry leaves and were administered to silkworms daily once at morning (first feed) from second day of fifth instar. The study revealed that, silver nanoparticles at 200 ppm concentration emerged as the best supplement with improved larval, cocoon and silk parameters by recording shorter larval duration (616.42 h), fifth instar duration (166.12 h), higher mature larval weight (40.626 g/10 larvae), effective rate of rearing (97.58%), silk productivity (6.225 cg/day), silk gland weight (1.885 g/larva), pupal weight (13.757 g/10 pupae), cocoon yield (794.85 g/dfl), cocoon weight (18.102 g/10 cocoons), shell weight (4.307 g/10 shells), cocoon shell ratio (23.79%), cocoon filament length (948.52 m), finer denier (2.75 d), highest fibroin (80.23%) and lowest sericin protein (19.78%).

Gut bacteria of lepidopteran herbivores facilitate digestion of plant toxins

Lepidopterans commonly feed on plant material, being the most significant insect herbivores in nature. Despite plant resistance to herbivory, such as producing toxic secondary metabolites, herbivores have developed mechanisms encoded in their genomes to tolerate or detoxify plant defensive compounds. Recent studies also highlight the role of gut microbiota in mediating detoxification in herbivores; however, convincing evidence supporting the significant contribution of gut symbionts is rare in Lepidoptera. Here, we show that the growth of various lepidopteran species was inhibited by a mulberry-derived secondary metabolite, 1-deoxynojirimycin (DNJ); as expected, the specialist silkworm Bombyx mori grew well, but interestingly, gut microbiota of early-instar silkworms was affected by the DNJ level, and several bacterial species responded positively to enriched DNJ. Among these, a bacterial strain isolated from the silkworm gut (Pseudomonas fulva ZJU1) can degrade and utilize DNJ as the sole energy source, and after inoculation into nonspecialists (e.g., beet armyworm Spodoptera exigua), P. fulva ZJU1 increased host resistance to DNJ and significantly promoted growth. We used genomic and transcriptomic analyses to identify genes potentially involved in DNJ degradation, and CRISPR-Cas9-mediated mutagenesis verified the function of ilvB, a key binding protein, in metabolizing DNJ. Furthermore, the ilvB deletion mutant, exhibiting normal bacterial growth, could no longer enhance nonspecialist performance, supporting a role in DNJ degradation in vivo. Therefore, our study demonstrated causality between the gut microbiome and detoxification of plant chemical defense in Lepidoptera, facilitating a mechanistic understanding of host-microbe relationships across this complex, abundant insect group.

Identification and characterization of a novel small viral peptide (VSP59) encoded by Bombyx mori cypovirus (BmCPV) that negatively regulates viral replication.

A novel small open reading frame (sORF) from the viral genome was identified and characterized. The sORF could encode a small viral peptide (VSP59) that targeted mitochondria and induced prohibitin 2-related apoptosis, further attenuating Bombyx mori cypovirus replication.

Preparation and study of properties of sorbents based on the fibrous waste of “Bombyx mori” natural silk

The work presents information on obtaining hydrolyzed fibroin powder with high sorption properties under the influence of ultra-high frequency rays from the fibrous waste of the silk industry in an acidic environment, as well as information on its composition, structure, and sorption properties. During the research, it was found that the formation of hydrolyzed fibroin powder is caused by the hydrolysis of alpha- and random chains of fibroin. In differential scanning calorimetry analysis, it was shown that hydrolyzed fibroin is thermally structurally stable up to 210 °C and capable of sorption of gold and silver ions in large quantities. Using the samples obtained by treating the hydrolyzed fibroin powders in different medium solutions, cottonseed oil was whitened, and the peroxide content of the oil was reduced from 12 mmol/kg to 2.2 mmol/kg. By lowering it to this point, it is possible to increase the oil's shelf life.

Sharp decline in male fertility in F2 hybrids of the female-heterogametic silk moth Bombyx.

Sexual selection drives rapid evolution of morphological, physiological, and behavioral traits, especially in males, and it may also drive the rapid evolution of hybrid male sterility. Indeed, the faster male theory of speciation was once viewed as a major cause of Haldane's rule in male-heterogametic XY taxa, but is increasingly being replaced by the genetic conflict hypothesis partly because it cannot explain the faster evolution of hybrid female sterility in female-heterogametic ZW taxa. The theory nonetheless predicts that there should be more genes for hybrid male sterility than for hybrid female sterility even in such taxa, but this remains untested. Thus, finding evidence for the faster male theory of reproductive isolation beyond the F1 generation in ZW systems still represents a challenge to studying the impact of sexual selection. In this study, we examined F2 hybrids between the domesticated silkworm Bombyx mori and the wild silk moth Bombyx mandarina, which have ZW sex determination. We found that although only females showed reduced fertility in the F1 generation, the F2 hybrid males had a significant reduction in fertility compared with the parental and F1 males. Importantly, 27% of the F2 males and 15% of the F2 females were completely sterile, suggesting the presence of recessive incompatibilities causing male sterility in female-heterogametic taxa.

Bioconjugation of Serratiopeptidase with Titanium Oxide Nanoparticles: Improving Stability and Antibacterial Properties.

Antimicrobial resistance (AMR) poses a significant global health threat, necessitating the development of novel antibacterial strategies. Serratiopeptidase (SP), a metalloprotease produced by bacteria such as Serratia marcescens, has gained attention not only for its anti-inflammatory properties but also for its potential antibacterial activity. However, its protein nature makes it susceptible to pH changes and self-proteolysis, limiting its effectiveness. This study aimed to increase both the enzymatic stability and antibacterial activity of serratiopeptidase through immobilization on titanium oxide nanoparticles (TiO2-NPs), leveraging the biocompatibility and stability of these nanomaterials. Commercial TiO2-NPs were characterized using TGA/DTG, FT-IR, UV-Vis, and XRD analyses, and their biocompatibility was assessed through cytotoxicity studies. Serratiopeptidase was produced via fermentation using the C8 isolate of Serratia marcescens obtained from the intestine of Bombyx mori L., purified chromatographically, and immobilized on carboxylated nanoparticles via EDC/NHS coupling at various pH conditions. The optimal enzymatic activity was achieved by using pH 5.1 for nanoparticle activation and pH 5.5 for enzyme coupling. The resulting bioconjugate demonstrated stable proteolytic activity at 25 °C for 48 h. Immobilization was confirmed by FT-IR spectroscopy, and the Michaelis-Menten kinetics were determined. Notably, the bioconjugate exhibited two-fold greater antibacterial activity against E. coli than the free enzyme or TiO2-NPs at 1000 µg/mL. This study successfully developed a serratiopeptidase-TiO2 bioconjugate with enhanced enzymatic stability and antibacterial properties. The improved antibacterial activity of the immobilized enzyme presents a promising approach for developing new tools to combat antimicrobial resistance, with potential applications in healthcare, food safety, and environmental protection.

Screening of Economically Sustainable Strains of Bivoltine Silkworm, Bombyx mori L. by Assessing the Comparative Fibroin Gene Expression

Screening of Economically Sustainable Strains of Bivoltine Silkworm, Bombyx mori L. by Assessing the Comparative Fibroin Gene Expression

Biomimetic bilayer scaffold from Bombyx mori silk materials for small diameter vascular applications in tissue engineering.

Enhancing the biocompatibility and mechanical stability of small diameter vascular scaffolds remain significant challenges. To address this challenge, small-diameter tubular structures were electrospun from silk fibroin (SF) from silk textile industry discarded materials to generate bilayer scaffolds that mimic native blood vessels, but derived from a sustainable natural material resource. The inner layer was obtained by directly dissolving SF in formic acid, while the middle layer (SF-M) was achieved through aqueous concentration of the protein. Structural and biological properties of each layer as well as the bilayer were evaluated. The inner layer exhibited nano-scale fiber diameters and 57.9% crystallinity, and degradation rates comparable with the SF-M layer. The middle layer displayed micrometer-scale fibers diameters with an ultimate extension of about 274%. Both layers presented contact angles suitable for cell growth and cytocompatibility, while the bilayer material displayed an intermediate mechanical response and a reduced enzymatic degradation rate when compared to each individual layer. The bilayer material emulates many of the characteristics of native small-diameter vessels, thereby suggesting further studies towards in vivo opportunities.

Identification of function modules in the co-expression protein–protein interaction network of Bombyx mori in response to Beauveria bassiana infection

Beauveria bassiana (B. bassiana) is a common fungal disease in sericulture. Previous research has primarily focused on investigating genes involved in innate immunity. However, the response of Bombyx mori (B. mori) to B. bassiana requires the coordination of other biological processes in addition to the immune system. We measured protein expression profile of B. mori after inoculating B. bassiana using iTRAQ technology in previous. Here we constructed a co-expression protein–protein interaction network of B. mori in response to B. bassiana infection. Subnetworks and modules were analyzed, and the functions of these modules were annotated. The results revealed the identification of numerous proteins associated with cellular immunity, including those involved in phagosomes, lysosomes, mTOR signaling, sugar metabolism, and the ubiquitin–proteasome pathway. Meanwhile, we observed that the pathways involved in protein synthesis were activated, including pyruvate and purine metabolism, RNA transport, ribosome, protein processing in endoplasmic reticulum, and protein export pathways, during B. bassiana infection. Based on this analysis, we selected six candidate genes (shock protein, ribosome, translocon, actin muscle-type A2, peptidoglycan recognition protein, and collagenase) that were found to be related to the response to B. bassiana. Further verification experiments demonstrated significant changes in their expression levels after inoculation with B. bassiana. These research findings provide new insights into the molecular mechanism of insect immune response to fungal infection.

RNA-seq analysis of LPS-induced immune priming in silkworms (Bombyx mori) and the role of cytochrome P450 detoxification system in the process

While immune priming has been identified in many invertebrates, the intricate mechanisms that drive this process in insects continue to be a subject of mystery. In this study, we exposed silkworm larvae to varying doses of lipopolysaccharide (LPS) to induce immune priming and assessed their survival upon challenge with Bacillus thuringiensis (Bt). Transcriptome analysis was performed to identify differentially expressed genes (DEGs) associated with immune priming. The role of CYP450 genes in this process was further explored using RNA interference (RNAi) to knockdown CYP9E2 and CYP6K1, followed by measurements of detoxification enzyme activities and reactive oxygen species (ROS) levels. We found that LPS exposure significantly increased silkworm survival rates upon Bt challenge, indicating the induction of immune priming. Transcriptome analysis revealed 549 DEGs, including a large number involved in detoxification, immunity, and metabolism, suggesting a complex regulatory network that encompasses immune responses and metabolic pathways. Functional enrichment and gene set enrichment analysis (GSEA) highlighted the activation of immune signaling pathways and the involvement of detoxification processes. Knockdown of CYP9E2 and CYP6K1 resulted in increased ROS levels, decreased detoxification enzyme activities, and reduced survival rates post-Bt challenge, implicating the critical role of these genes in immune priming and detoxification. Our findings demonstrate that LPS-induced immune priming in silkworms involves the upregulation of CYP450 genes, which play a critical role in detoxification and immune response modulation. The study provides insights into the molecular mechanisms of immune priming in insects and highlights the potential of CYP9E2 and CYP6K1 as targets for enhancing disease resistance and pest management in insects.

Effect of different growth regulators on biology and economic traits of silkworm (Bombyx mori L.)

Effect of different growth regulators on biology and economic traits of silkworm (Bombyx mori L.)

Distribution and genetic diversity of Bombyx mori nucleopolyhedrovirus in mass-reared silkworms in Thailand

Silk is an economically and culturally important product of Thailand that is threatened by the outbreaks of the Bombyx mori nucleopolyhedrovirus (BmNPV) in silkworm rearings. BmNPV infects the larval stages of the silkworm Bombyx mori, causing fatal viral infection and preventing the larvae from reaching the pupal stage. Numerous BmNPV isolates have been described from silk producing countries from all over the world. In this study, the geographic distribution of BmNPV in Thailand was analyzed by collecting 18 samples from different regions. The BmNPV samples were analyzed by PCR and whole genome sequencing was performed for eight specimens. Their genome size ranged from 125,888 bp to 126,783 bp, comprising 138 open reading frames. Although the sequenced BmNPV genomes could be phylogenetically differentiated, a correlation between geographic and genetic distance was not observed indicating a close relationship between the BmNPV from Thailand.