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Silk gland size in silkworms (Bombyx mori) affects silk output. However, the molecular mechanisms by which genes regulate silk gland size remain unclear. In this study, silk glands from three pure silkworm strains (A798, A306 and XH) with different silk gland weight phenotypes were compared using transcriptomics and proteomics to identify differentially expressed genes (DEGs) and proteins (DEPs). When comparing A798 to A306 and A798 to XH, 830 and 469 DEGs were up‐regulated, respectively. These genes were related to the gene ontology terms, metabolic process, transport activity and biosynthesis process. In addition, 372 and 302 up‐regulated differentially expressed proteins were detected in A798 to A306 and A798 to XH, respectively, related to the gene ontology terms, ribosome and protein export, ribosome and polypeptide biosynthesis processes. Moreover, combined transcriptomics, proteomics and weighted correlation network analyses showed that five genes (BGIBMGA002524, BGIBMGA002629, BGIBMGA005659, BGIBMGA005711 and BGIBMGA010889) were significantly associated with the silk gland weight. Reverse Transcription‐quantitative real‐time Polymerase Chain Reaction (RT‐qPCR) and Enzyme linked immunosorbent assay (ELISA) were used to verify the mRNA and protein expression of five genes in the silk glands and tissues of 18 silkworm strains. The results showed that four genes have higher expression levels in heavier silk glands. These genes are associated with glycogen metabolism, fatty acid synthesis and branched chain amino acid metabolism, thus potentially promoting growth and silk protein synthesis. These findings provide valuable insights into the molecular mechanisms underlying the relationship between silk gland weight and silk yield in silkworms. [ABSTRACT FROM AUTHOR]

Boron nitride nanosheets (BN NSs) have emerged as promising materials in a wide range of biomedical applications. Despite the extensive studies on these bio-nano interfacial systems, one critical concern is their toxicity, which is affected by a variety of factors, including size. This study aimed at assessing the relationship between BN NSs size and toxicity. Two silkworm strains (qiufeng × baiyu and Nistari 7019) were used as model organisms to investigate the effect of different sizes of BN NSs (BN NSs-1, thickness of 41.5 nm and diameter of 270.7 nm; BN NSs-2, thickness of 48.2 nm and diameter of 562.2 nm) on silkworm mortality, growth, cocoon weight, and tissue microstructure. The findings show that exposure to BN NSs in this work has no lethal adverse effects on silkworm growth or tissue microstructure. BN NSs have a higher effect on the growth rate of qiufeng × baiyu compared to Nistari 7019, demonstrating that the same treatment does not favorably affect the Nistari 7019 strain, as there is no significant increase in cocoon weight. Overall, the study suggests that the sizes of BN NSs employed in this study are relatively safe and have less negative impact on silkworms. This offers significant insights into the effect of BN NSs size, a crucial factor to consider for their safe use in biomedical applications. [ABSTRACT FROM AUTHOR]

Silkworm (Bombyx mori) is not only an economic insect but also a model organism for life science research. Bombyx mori nucleopolyhedrovirus (BmNPV) disease is a major infectious disease in the world's sericulture industry. The cocoon loss caused by this disease accounts for more than 60% of the total loss caused by all silkworm diseases. To date, there has been no effective solution for preventing and treating this disease. The most effective measure is to breed disease-resistant varieties. The quickest way to breed disease-resistant varieties is to apply genetic modification. However, this requires that we obtain disease resistance genes and know the mechanism of disease resistance. Since the discovery of disease-resistant resources in 1989, scholars in the sericulture industry around the world have been inspired to search for resistance genes. In the past two decades, with the help of multi-omics technologies, screening of resistance genes, gene localization, protein modification, virus–host interactions, etc., researchers have found some candidate genes that have been proposed to function at the cellular or individual level. Several disease-resistant varieties have been obtained and used in production through hybrid breeding, RNA interference, and genetic modification. This article summarizes and reviews the discovery of and research advances related to silkworm resistance to BmNPV. It is anticipated that the review will inspire scientific researchers to continue searching for disease resistance genes, clarify the molecular mechanism of silkworm disease resistance, and promote disease-resistant silkworm breeding. [ABSTRACT FROM AUTHOR]

We previously observed an over‐dominant silkworm heterosis of increased yield in a cross of Bombyx mori nuclear polyhydrosis virus‐resistant strain NB with a susceptible strain 306. In the present study, we found that heterosis also exists in crosses of NB with other susceptible strains, indicating it is a more general phenomenon. We performed comparative transcriptome and proteome and identified 1624 differentially expressed genes (DEGs) and 298 differentially expressed proteins (DEPs) in silk glands between parents and F1 hybrids, of which 24 DEGs/DEPs showed consistent expression at mRNA and protein levels revealed by Venn joint analysis. Their expressions are completely non‐additive, mainly transgressive and under low‐parent, suggesting recombination of parental genomes may be the major genetic mechanism for the heterosis. GO and KEGG analyses revealed that they may function in generally similar but distinctive aspects of metabolisms and processes with signal transduction and translation being most affected. Notably, they may not only up‐regulate biosynthesis and transport of silk proteins but also down‐regulate other unrelated processes, synergistically and globally remodelling the silk gland to increase yield and cause the heterosis. Our findings contribute insights into the understanding of silkworm heterosis and silk gland development and provide targets for transgenic manipulation to further increase the silk yield. [ABSTRACT FROM AUTHOR]

White cocoon is developed and used as a natural fiber, and different silkworm strains have different cocoon colors. Natural-colored cocoons are preferred by people, however, the cocoon color mainly settles on sericin and it basically falls off after reeling. Currently, there are no varieties applied to production due to the formation mechanism of cocoon color is not clear. The formation of cocoon color involves multiple gene regulations. Previous studies have shown that the main genes regulating cocoon traits are the yellow blood (Y) gene, yellow blood inhibitor (I) gene, and yellow cocoon (C) gene. Among them, the products of the Y gene and C gene have been studied, but the I gene is still unclear. In this study, the midgut tissues of the yellow (NB) and the white (306) cocoon silkworm were analyzed by whole transcriptome sequencing. The results showed that there are 1639 DE-circRNAs, 70 DE-miRNAs, and 3225 DE-mRNAs, including 1785 up-regulated genes and 1440 down-regulated genes. GO and KEGG annotation results indicated that DE-mRNAs are mainly involved in intracellular transport, signal transduction, lipid transport, and metabolic processes. Two key genes, KWMTBOMO10339 and KWMTBOMO16553, were screened out according to the annotation results, which were involved in amino acid transport and ion exchange function, respectively. The interaction analysis between ncRNA and target genes showed that there were five miRNAs regulating these two genes. The qPCR analysis showed that the I gene was down-regulated, and the miRNA expression profiles were most up-regulated. Therefore, during the yellow and white cocoon formation, KWMTBOMO10339 and KWMTBOMO16553 may be regulated by miRNA, resulting in the non-expression of KWMTBOMO10339 and KWMTBOMO16553 in yellow cocoon silkworm, and the pigment molecules can enter hemolymph from the midgut to form yellow blood, then transport to the middle silk gland to finally form yellow cocoons. [ABSTRACT FROM AUTHOR]

Melanin has been reported to have potential applications in industries such as cosmetics and food due to its anti-UV and antioxidative qualities. However, the corresponding data on its safety evaluation or biological consequences are fairly limited; such data are critical given its widespread use. The effect of different concentrations (1, 2, 3, and 4%) of melanin on growth status (larvae length and weight, cocoon weight, and morphology), the microstructure of the various tissues (fat body, silk gland, and midgut), and silk properties was studied by using the silkworm (bombyx mori) as the model organism. The weight and length of silkworm larvae fed with melanin were lower than the control, indicating that melanin appears to have a negative effect on the growth status of silkworms; however, the histophysiology analysis indicates that the cell morphologies are not changed, the XRD and FTIR spectra indicate that the secondary and crystalline structures of silks are also well preserved, and the thermogravimetric analysis and tensile test indicate that the thermal stability and mechanical properties are well maintained and even improved to some extent. Generally, it indicates that melanin has a certain inhibitory effect on the growth of silkworm larva but causes no harm to the cell microstructures or silk properties; this demonstrates that the safety of melanin as a food addictive should be considered seriously. The increase of thermal stability and mechanical properties shows that melanin may be a good chemical modifier in textile industries. [ABSTRACT FROM AUTHOR]

The reference genomes of Bombyx mori (B. mori), Silkworm Knowledge-based database (SilkDB) and SilkBase, have served as the gold standard for nearly two decades. Their use has fundamentally shaped model organisms and accelerated relevant studies on lepidoptera. However, the current reference genomes of B. mori do not accurately represent the full set of genes for any single strain. As new genome-wide sequencing technologies have emerged and the cost of high-throughput sequencing technology has fallen, it is now possible for standard laboratories to perform full-genome assembly for specific strains. Here we present a high-quality de novo chromosome-level genome assembly of a single B. mori with nuclear polyhedrosis virus (BmNPV) resistance through the integration of PacBio long-read sequencing, Illumina short-read sequencing, and Hi-C sequencing. In addition, regular bioinformatics analyses, such as gene family, phylogenetic, and divergence analyses, were performed. The sample was from our unique B. mori species (NB), which has strong inborn resistance to BmNPV. Our genome assembly showed good collinearity with SilkDB and SilkBase and particular regions. To the best of our knowledge, this is the first genome assembly with BmNPV resistance, which should be a more accurate insect model for resistance studies. [ABSTRACT FROM AUTHOR]

orf65 (Bm65) of Bombyx mori nucleopolyhedrovirus (BmNPV) codes for a putative 104-amino-acid protein containing three cysteine residues with a putative molecular mass of 12.2 kDa. Previous studies have showed that Bm65 accumulates mainly in nucleus and involved in the repair of UV-damaged DNA. However, the mechanism of nuclear import of Bm65 remains unclear. In this study, a SDS-stable Bm65 tetramer was found in BmNPV-infected BmN cells, and alanine substitutions for the three cysteine residues did not affect the formation of Bm65 tetramer. Additionally, a basic amino acid cluster of the Bm65 protein was identified as an efficient nuclear localization signal (NLS). Firstly, transient expression of GFP-fused truncated Bm65 variants revealed that the 76KRKCSK motif functions as the NLS. This was also confirmed by alanine substitution in the 76KRKCSK motif, which caused attenuated nuclear localization of Bm65. Next, the 76KRKCSK motif-mutated bacmid was generated and the 76KRKCSK motif was also found to be important for nuclear localization of Bm65 in BmNPV-infected conditions. Lastly, analyses of flag-tagged Bm65 expressing bacmids revealed that the mutations in 76KRKCSK motif did not affect the synthesis of Bm65 tetramer, but severely impaired production levels of infectious virions. In conclusion, Bm65 exists in mainly a tetrameric form in virus-infected cells, which may be involved with production levels of infectious virions. [ABSTRACT FROM AUTHOR]

Abstract The widespread use of silver nanoparticles (AgNPs) has raised public concern due to their potential toxic effects on humans and the environment. Although some studies have evaluated the toxicity of nanomaterials in vertebrates, studies on their hazardous effects on insects are limited. Here we focused on different concentrations of AgNPs to silkworms, a promising model organism, to evaluate their toxic effects by omics analysis. After the silkworms were fed with 100 mg L–1 AgNPs, transcriptomics analysis showed differential expression of 43 genes: 39 upregulated and 4 downregulated. These differentially expressed genes (DEGs) were involved in the digestion process, various metabolic pathways, transmembrane transport and energy synthesis. Proteomic results for silkworms fed with 400 mg L–1 AgNPs revealed 14 significantly differentially expressed proteins: 11 downregulated and 3 upregulated. Reverse transcription–polymerase chain reaction (RT-PCR) results showed that the expression levels of eight proteins were similar to the transcription levels of their corresponding genes. As the AgNPs concentration was increased, the expression of digestive enzymes was downregulated, which damaged the silkworm tissue and suppressed the activity of the enzyme superoxide dismutase and the protein HSP 1, causing oxidative stress and the production of reactive oxygen species, which had toxic effects on the silkworm digestive system. Histopathological results showed that treatment with 400 mg L–1 AgNPs destroyed the basal lamina and the columnar cells, caused adverse effects on tissues and had the potential to induce harmful effects on the digestive system. The data presented herein provide valuable information on the hazards and risks of nanoparticle contamination. Main finding: AgNPs would downregulate some digestive enzymes, damage the tissue of midgut in silkworm, meantime induce the accumulation of reactive oxygen species which may cause oxidative stress. Graphical abstract fx1 Highlights • Silver nanoparticles at 400 mg L−1 affect the midgut structure of silkworms. • Silver nanoparticles alter the expression of genes linked to extracellular matrix receptor interactions. • Silver nanoparticles at 400 mg L−1 suppress the activity of the enzyme superoxide dismutase and the HSP 1 protein. [ABSTRACT FROM AUTHOR]

Abstract: The silkworm Bombyx mori L., representing an important economic insect and one of the best models for studying insect immunity, possesses an efficient and sophisticated innate immune system against invasive microorganisms. The innate immune system basically includes humoural immunity and cellular immunity. The humoural immunity, which functions via molecules including humoural factors, lysozymes, phenoloxidase, hemolin, lectins and, in particular, antimicrobial peptides, plays a central role in eliminating the invading pathogens. The cellular immunity is primarily carried out and mediated by plasmatocytes and granular cells of haemocytes in the haemolymph, usually followed by melanization. Additionally, apoptosis, a primary viral defence for insects lacking adaptive immunity, comprises an important part of the silkworm immune system. Currently, there is still the lack of a comprehensive and systematic understanding of the molecular mechanisms of silkworm immunity. We review the latest research progress on silkworm immune mechanisms, including phenoloxidase‐dependent melanization and apoptosis, which is conducive to improving our understanding of the silkworm immune mechanism, clarifying the relationship of various immune mechanisms, and also providing a theoretical basis and reference for the future research of insect immunity. [ABSTRACT FROM AUTHOR]

Bm-SP142 is a 35 kDa protease in the silkworm, but its exact functions remain unknown. In this study, sequence alignment revealed that the His-Asp-Ser catalytic triad is embedded in the sequence motif, establishing Bm-SP142 as a serine protease. Soluble recombinant GST-BmSP142 was expressed and purified, and serine protease activity was confirmed in vitro. RT-qPCR results indicated that Bm-SP142 was mainly expressed in the middle part of the silkworm midgut, and Bm-SP142 transcripts were significantly up-regulated at 24 hours post infection (hpi) in BmBDV-resistant strains (798) inoculated with BmBDV and BmNPV-resistant strains (NB) inoculated with BmNPV, but not in BmBDV-susceptible strains (306). Surprisingly, transcripts were significantly down-regulated at 12 hpi in BmNPV-susceptible strains (HuaBa 35) inoculated with BmNPV, compared with healthy silkworms. Recombinant BmNPV treated with purified Bm-SP142 effectively impaired its ability to infect BmN cells, and Bm-SP142 decreases the efficiency of BmNPV and BmBDV propagation in silkworms. Furthermore, overexpression of Bm-SP142 in BmN cells inhibited viral propagation. [ABSTRACT FROM AUTHOR]

β-thymosin plays important roles in the development of the lymphatic system and the central nervous system in vertebrates. However, its role and function in invertebrates remain much less explored. Here, we firstly isolated a gene encoding β-thymosin in silkworm (Bombyx mori L.). Interestingly, this gene encodes two polypeptides, named as BmTHY1 and BmTHY2, via two different modes of RNA splicing. The recombinant proteins fused with an N-term GST tag were over-expressed in Escherichia coli (E. coli) and further purified to near homogenity to prepare mouse antibodies. The Western blot analysis showed that these proteins were expressed in various tissues and organs, as well as in different developmental stages. Amazingly, the expression of BmTHY2 was hugely increased during the pupae stage, indicating a specialized role in this period. The expression of these proteins was gradually decreased in BmN cells infected by BmNPV, suggesting they may play different roles in the virus infection. In addition, both BmTHY1 and BmTHY2 can interact with 14-3-3 of silkworm and Ubiquitin of BmNPV as shown by GST pull down and Co-IP assays, consistent with their roles in the regulation of the development of nervous system. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus ( BmBDV) VD1-ORF4 consists of 3,318 nucleotides, which codes for a predicted protein with molecular weight of about 127 kDa. However, the authentic proteins encoded by VD1-ORF4 in silkworm midguts infected with BmBDV and their interacting proteins are still unclear. In this study, Western blot analysis revealed that a 127-kDa protein was confirmed to be translated from the VD1-ORF4 transcript using polyclonal antibodies and monoclonal antibodies against VD1-ORF4 deduced amino acid. Moreover, four smaller proteins with molecular weight of about 70, 60, 53, and 42 kDa were also examined in the infected midguts. Transient expression assay indicated that the expression amount of VD1-ORF4 fused with egfp was at least 30-fold lower than that of egfp gene, and immunofluorescence staining result indicated that these proteins encoded by VD1-ORF4 were located in both the cytoplasm and nucleus. Co-immunoprecipitation result showed that Aminopeptidase and Heat shock protein 90 can be captured by these proteins encoded by VD1-ORF4. In conclusion, multiple proteins were produced from the transcripts of VD1-ORF4 gene by an uncertain expression strategy, which may play important roles in viral replication and assembly. [ABSTRACT FROM AUTHOR]

Orf101 ( Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) is a highly conserved gene in lepidopteran nucleopolyhedroviruses, but its function remains unknown. In this study, Bm101 was characterized. Transcripts of Bm101 were detected from 24 through 96 h post infection (h p.i.) by RT-PCR. The corresponding protein was also detected from 24 to 96 h p.i. in BmNPV-infected BmN cells by Western blot analysis using a polyclonal antibody against Bm101. Western blot assay of occlusion-derived virus and budded virus (BV) preparations revealed that Bm101 encodes a 28-kDa structural protein that is associated with BV and is located in the envelope fraction of budded virions. In addition, confocal analysis showed that the protein was localized in the cytosol and cytoplasmic membrane in virus-infected cells. In conclusion, the available data suggest that Bm101 is a functional ORF of BmNPV and encodes a protein expressed in the late stage of the infection cycle that is associated with the BV envelope. [ABSTRACT FROM AUTHOR]

Degradation performance of silk fibroin is an important property for its medical applications. Herein we constructed a shortened silk fibroin heavy chain protein fused with a matrix metalloproteinase cleavage site (SSFH-MMP) along with a glutathione S-transferase tag ahead. The digestion assay shows it can be cut by matrix metalloproteinase-2 (MMP-2) at its MMP cleavage site. Furthermore, we introduced the SSFH-MMP into silk fibroin by genetic modification of silkworms in order to increase the degradation rate of the silk fibroin. After acquisition of a race of transgenic silkworms with the coding sequence of the MMP cleavage site in their genomic DNA, we tested some properties of their silk fibroin designated TSF-MMP. The results show that the TSF-MMP has MMP cleavage sites and yields a quicker degradation rate during dilution in MMP-2 enzyme buffer or implantation into tumor tissues compared with that of normal silk fibroin. Moreover, the TSF-MMP is in vitro non-toxic to human bone marrow mesenchymal stem cells (hBM-MSCs) indicating that the TSF-MMP may become a biomaterial with a quicker degradation rate for its medical applications. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus ( BmBDV), which had been assigned to Densovirinae in Parvoviridae previously, replicates mainly in silkworm midgut olumnar cells and causes the fatal flacheric disease. In contrast to parvovirus, this virus possesses two single-stranded DNA genome segments and encodes a putative protein-primed DNA polymerase. The accumulating evidence suggests that it has unique characteristics different to parvovirus and adopts its own mechanisms for replication. So far, little is known about the replication mechanisms of BmBDV. In this review, we focus on the pathology associated with this virus and the viral biology such as viral genome structure, viral genes, and viral replication and expression strategies. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus ( BmBDV), which had been assigned to Densovirinae in Parvoviridae previously, replicates mainly in silkworm midgut olumnar cells and causes the fatal flacheric disease. In contrast to parvovirus, this virus possesses two single-stranded DNA genome segments and encodes a putative protein-primed DNA polymerase. The accumulating evidence suggests that it has unique characteristics different to parvovirus and adopts its own mechanisms for replication. So far, little is known about the replication mechanisms of BmBDV. In this review, we focus on the pathology associated with this virus and the viral biology such as viral genome structure, viral genes, and viral replication and expression strategies. [ABSTRACT FROM AUTHOR]

Bombyx mori nucleopolyhedrovirus ( BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms. BmNPV DNA polymerase ( Bm-DNAPOL), encoded by the ORF53 gene, plays a central role in viral DNA replication. In this work, a His-tagged Bm-DNAPOL fusion protein, constructed using a novel MultiBac expression system, was overexpressed in Sf-9 insect cells, purified to near homogeneity on Ni-NTA agarose beads and further purified by ion-exchange chromatography. About 0.4 mg of enzyme was obtained from about 1 × 10 9 infected Sf-9 cells in suspension culture. Characterization of the highly purified enzyme indicated that Bm-DNAPOL is a monomer with an apparent molecular mass of approximately 110,000 Da. It possessed a specific activity of 15,126.3 U/mg under optimal in vitro reaction conditions and behaved in the manner of a proliferating cell nuclear antigen (PCNA)-independent DNA polymerase on both poly(dA)/oligo(dT) primer/template and singly premiered M13 DNA. BmNPV viral replication may be independent of replication factor C and a PCNA complex, while single-stranded DNA binding protein might play an important role in BmNPV DNA replication. These findings will be significant in studies on BmNPV-based disease in silkworms and for using silkworms as a bioreactor for the production of biomolecules of commercial importance. [ABSTRACT FROM AUTHOR]

Open reading frame 17 ( Bm17) of Bombyx mori nucleopolyhedrovirus is a highly conserved gene in lepidopteran nucleopolyhedroviruses, suggesting that it performs an important role in the virus life cycle whose function is unknown. In this report, we describe the characterization of Bm17. Reversed transcriptive-PCR (RT-PCR) and Western blot analysis demonstrated that Bm17 was expressed as a late gen. Immunofluorescence analysis by confocal microscopy showed that BM17 protein was localized on cytoplasm and nucleus of infected cells. These results show that BM17 was a late protein localized in cytoplasm and nucleus. [ABSTRACT FROM AUTHOR]

It is difficult to obtain intact embryos, especially intact early embryos, from insect eggs because of their small sizes. Based on the means traditionally used to get silkworm embryos and the previous approaches used for getting Drosophila embryos, we established a novel method of silkworm embryo preparation. The new method is straightforward and easy to operate. Silkworm embryos could be prepared without severe damage in large quantities by this new protocol. In addition, the novel method of silkworm embryo preparation is quite suitable for immunohistochemistry. [ABSTRACT FROM AUTHOR]

With the continuous improvement of the living standards of human society, the number of diabetics worldwide is growing rapidly. To date, the main effective therapy for diabetic is intravenous injection of insulin, which is accompanied by a lot of shortage such as high cost and side effects. To obtain long-term bioactive anti-diabetic drug for oral administration, we used human proinsulin ( hpi) as a foreign gene to construct a recombinant plasmid pJS700-HPI with an enterokinase site Asp-Asp-Asp-Asp-Lys suitable for digestion. Plasmid pJS700-HPI was transformed into Bacillus subtilis by double cross-over and an amylase inactivated mutant was produced. After induction of spores formation, western blot was used to monitor HPI surface expression on spores. Oral administration to the silkworms with spores implied that the HPI protein displayed on recombinant spores may be digested and absorbed into the silkworm's hemolymph due to the resistant characters of spores and the addition of enterokinase site. [ABSTRACT FROM AUTHOR]

Silkworms are usually susceptible to the infection of Bombyx mori (B. mori) nucleopolyhedrovirus (BmNPV), which can cause significant economic loss. However, some silkworm strains are identified to be highly resistant to BmNPV. To explore the silkworm genes involved in this resistance in the present study, we performed comparative real-time PCR, ATPase assay, over-expression and sub-cellular localization experiments. We found that when inoculated with BmNPV both the expression and activity of V-ATPase were significantly up-regulated in the midgut column cells (not the goblet cells) of BmNPV-resistant strains (NB and BC8), the main sites for the first step of BmNPV invasion, but not in those of a BmNPV-susceptible strain 306. Furthermore, this up-regulation mainly took place during the first 24 hours post inoculation (hpi), the essential period required for establishment of virus infection, and then was down-regulated to normal levels. Amazingly, transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm cells could significantly inhibit BmNPV proliferation. To our knowledge this is the first report demonstrating clearly that V-ATPase is indeed involved in the defense response against BmNPV. Our data further suggests that prompt and potent regulation of V-ATPase may be essential for execution of this response, which may enable fast acidification of endosomes and/or lysosomes to render them competent for degradation of invading viruses. [ABSTRACT FROM AUTHOR]

Bombyx mori Bidensovirus ( BmBDV), a bipartite virus possesses two single-stranded linear DNAs (VD1 and VD2) and shows high pathogenic ability to Bombyx mori. Previous research found that the genes of nonstructural protein ns1 and ns2 were in the same transcript. To investigate the mechanism of transcriptional regulation of ns1 and ns2 genes, the 5′-flanking sequence (289 nt) of ns1 gene, encompasses the regions of the common terminal sequence (CTS) and the predicted P5 promoter from the 5′-terminus of the viral genome to the transcription initiation site of the ns1 gene was cloned and fused to the upstream of the luciferase reporter gene. The luciferase reporter assay showed that the 53 nt CTS of VD1 and VD2 can downregulate the activity of P5 by 13.3 %. The comparison in different cell lines showed that P5 possessed high promoter activity in BmN and Hi5 cell lines. Interestingly, P5 also had high activity in Hela cells, a kind of cancer cell of human. Subsequent truncated promoter analysis showed that the 31 nt (−236 to −206 nt) sequence is very important to P5 for the activity down to 36.5 % after deletion of it. While the activity also remained 26.5 % after the deletion of the TATA box, suggesting that the promoter is TATA independent. Moreover, in order to further understand the activity intensity of P5, a comparison with other three promoters, B. mori actin3 ( Bm-actin3), B. mori nuclear polyhedrosis virus ( BmNPV) immediate early 1 gene promoter ( BmNPV-ie-1), and a synthetic promoter (3xP3) was carried out, the result indicated that the activity of P5 was weaker than that of anyone of them. [ABSTRACT FROM AUTHOR]

In order to improve the classic techniques of silkworm breeding and achieve the integration of good economic traits and disease resistance traits, a cross-breeding and molecular marker assisted breeding technology combined was developed in this experiment. The practical strain Jingsong and the strain NB, highly resistant to the BmNPV found from the library of silkworm races and strains in China, were selected as the breeding material. Through cross-breeding and backcrossing, the disease resistant gene was introducted into the economic strain Jingsong. At the same time, the strain Qiufeng, resistant to the BmDNV, was used as the donor material of resistance gene. In this experiment, a new strain resistant to the two virus, BmNPV and BmDNV-Z, was obtained through the method of virus administrion and molecular marker assisted breeding technology. [ABSTRACT FROM AUTHOR]

Abstract: All lepidopteran baculovirus genomes sequenced encode a homolog of the Bombyx mori nucleopolyhedrovirus orf61 gene (Bm61). To determine the role of Bm61 in the baculoviral life cycle, we constructed a Bm61 knockout virus and characterized it in cells. We observed that the Bm61 deletion bacmid led to a defect in production of infectious budded virus (BV). Quantitative PCR analysis of BV in the media culturing the transfected cell indicated that BV was not produced due to Bm61 deletion. Electron microscope analysis showed that in the knockout of Bm61, nucleocapsids were not transported from the nucleus to the cytoplasm. From these results we concluded that BM61 is required in the BV pathway for the egress of nucleocapsids from the nucleus to the cytoplasm. [Copyright &y& Elsevier]

Bombyx mori parvo-like virus ( BmPLV) has two complementary single-stranded DNA genome (VD1 and VD2) and owns a self-encoding DNA polymerase motif, but its replication mechanism is unclear . In our previous research, a protein encoded by VD1-ORF1 was indentified in the midgut of BmPLV China Zhenjiang isolate-( BmPLV-Z) infected silkworm larvae via two-dimensional gel electrophoresis (2-DE). This protein was named as non-structural protein 2 (NS2), which showed no similarity to that of parvoviruses. To date, little is known about it. In this study, sequence alignment results showed that NS2 shared homology with some chromosomal replication initiator protein dnaA and DNA-binding response regulators. The ns2 was cloned and expressed in E. coli, and then a polyclonal antibody of the NS2 protein was prepared successfully. The data from real-time quantitative PCR displayed that the transcription of VD1-ORF1 from BmPLV-Z-infected midguts started from 28-h post inoculation (h p.i.) in low amounts, but in high amounts at late stages of infection. Immunofluorescence showed that NS2 ultimately concentrated on the nuclear membrane in BmN cells at late stages, indicating that NS2 might be associated with integral membrane protein. [ABSTRACT FROM AUTHOR]

Alcohol dehydrogenases (ADHs) are oxidoreductases catalyzing the reversible oxidation of alcohols to corresponding aldehydes or ketones accompanied by nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as coenzyme. ADHs attract major scientific and industrial interest for the evolutionary perspectives, afforded by their wide occurrence in nature, and for their use in industrial synthesis. However, the low activity of ADHs under extremes of pH and temperature often limits their application. To obtain ADH with high activity, in this study, we used Bombyx mori alcohol dehydrogenases (BmADH) as foreign gene and constructed a recombinant integrative plasmid pJS700-BmADH. This pJS700-BmADH was transformed into Bacillus subtilis by double cross-over and produced an amylase inactivated mutant. The fusion protein containing BmADH was expressed on the spore surface and recognized by BmADHspecific antibody. We also assayed the alcohol dehydrogenase activity of the fusion protein together with the native BmADH at different pH and temperature levels, which indicated the recombinant enzyme exhibits activity over wider ranges of temperature and pH than its native form, perhaps due to the resistance properties of B. subtilis spores against adverse conditions. [ABSTRACT FROM AUTHOR]

Objective: ORF134 of Bombyx mori nucleopolyhedrovirus (BmNPV) is a homolog of Autographa californica multiple NPV ORF5, but its function is unknown so far. The aim of this study is to characterize BmNPV ORF134 (Bm134). Methods: Transcription, protein expression and subcellular localization of Bm134 in BmN cells and silkworm midguts were analyzed using real-time polymerase chain reaction (RT-PCR), Western blot and immunofluorescence, respectively. Results: Both the transcription and protein expression of Bm134 were not detected until 24 h post-infection (p.i.) by RT-PCR and Western blot, indicating that Bm134 is a late gene. Western blot revealed that Bm134 encodes an expected 12.4-kDa structural protein that is associated with occlusion-derived virus (ODV), not with budded virus. Immunofluorescence analysis showed that the Bm134 was first detected in the cytoplasm 24 h p.i. and then transported to the nucleus during later infection. Transcripts of Bm134 and the corresponding protein were only detected 48-72 h p.i. in BmNPV-infected larvae of 306, a highly BmNPV-susceptible silkworm strain, but not in the NB strain that is resistant to BmNPV infection. Conclusion: Taken together, our data suggest that Bm134 is a late gene of BmNPV and may function as an ODV structural protein. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Objective:Bombyx mori parvo-like virus is a type of virus containing two single-stranded linear DNA molecules (VD1, VD2). In the present work, the structural proteins of B. mori parvo-like virus (China Zhenjiang isolate) (BmDNV-Z) were identified. Methods: The virions were purified by cesium chloride density gradient centrifugation from the feces of B. mori larvae infected with BmDNV-Z and analyzed by SDS-PAGE, Western blotting and MALDI-TOF/mass spectometry. Results: There were seven polypeptides of the virions, named P1 to P7, which reacted with the anti-virion serum. In addition, two polypeptides, P5 and P6, were recognized by the antibody against a viral protein encoded by VD1-ORF3. Conclusion: The results showed that the viral polypeptides were mainly composed of 7 proteins named P1 to P7. Western blotting with anti-virion serum indicated that structural proteins P5, P6 and P7 had molecular weights of around 50, 55 and 130 kDa, respectively. They were the major immunogens of the virion. Among them, P5 and P6 were coded by VD1-ORF3 and P7 was encoded by VD2-ORF1. Taken together, three viral structural proteins encoded by VD1-ORF3 and VD2-ORF1 were identified in BmDNV-Z for the first time. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The nanos gene encodes a zinc-finger protein which is required for the migration and differentiation of primordial germ cells as well as for their fate maintenance. In this study, a 1913bp nanos gene was cloned and characterized in silkworm (Bombyx mori). RT-PCR and Western blot analysis showed that the nanos was expressed in developing embryos and various silkworm larval tissues. The expression patterns of Nanos and Vasa in silkworm larval gonads were analyzed using immunohistochemistry. It was found that, in silkworm larval ovaries, the Nanos and Vasa proteins were expressed in oocytes. While in testes, high expression of Nanos and Vasa was detected in spermatogonia and relatively weaker expression was found in spermatocytes at latter stages. [Copyright &y& Elsevier]

Bacterial contamination has caused a major public health problem worldwide. Bacillus cereus is a conditional environmental pathogenic bacteria that can cause food poisoning. Whether environmental pathogens can cause widespread transmission in the insect kingdom is unclear. In this study, a Bacillus cereus ZJ-4 was isolated from the hospital environment of Zhenjiang City, Jiangsu Province, China. It was fatal by injection into the silkworm hemolymph. To investigated the potential toxic factors of ZJ-4 and clarified the toxicity response mechanism of silkworm by the ZJ-4 infection. Then, the whole genome of ZJ-4 was sequenced, and the immune mechanism of silkworm fat body to ZJ-4 pathogen was studied by HE pathological section and proteomics. Bacterial genome sequencing indicated that ZJ-4 had 352 drug resistance genes and 6 virulence genes. After 36 h of subcutaneous puncture with ZJ-4 suspension, the pathological changes were obviously found in HE pathological sections of fat body tissue. Comparative proteomic results indicated that differentially expressed proteins are mainly involved in stress reactions, biological regulation, and innate immunity. The qRT-PCR analysis showed that the expressions of β-GRP , Spaetzle , MyD88 , Tube and Dorsal genes in Toll pathway were up-regulated, while Pell and Cactus genes were down-regulated; in the antimicrobial peptide pathway, Glv2 , Lzm , Mor, and Leb3 genes were up-regulated, while attacin1 and defensin genes were down-regulated; Sod gene was up-regulated, while Cat gene was down-regulated in the antioxidant pathway; Ldh , Sdh , and Mdh genes were down-regulated in glucose metabolism pathway. These results indicated that ZJ-4 can damage the innate immune pathway of silkworm, and also affect the normal immune function of fat body cells. [Display omitted] • A new B. cereus strain named Bacillus cereus ZJ-4 was isolated from hospital environmental. • The whole genome information of B. cereus ZJ-4 was sequenced. • The virulence genes of B. cereus ZJ-4 were difference from other Bacillus cereus. • B. cereus ZJ-4 could damage the innate immune response of silkworm fat body. [ABSTRACT FROM AUTHOR]

Background: The silkworm (Bombyx mori) is an important lepidopteran model insect worldwide which undergoes a complete metamorphosis developmental process. Although genome sequencing has been long performed, no transcriptome data covering the complete life cycle are available. Methods: Herein, a total of 10 samples were collected consecutively at four different developmental stages, including eggs of 24 h after oviposition (Ed) and eggs of 24 h after artificial egg-hatching (E); larvae from fist to fifth instar (L1–L5); early and late pupa (P4 and P8); and adult moth (M), were subjected to Illumina RNA-Seq and time-course analysis. Results: The summations of the gene expression of the silkworm ten developmental stages show: at Ed stage, eggs develop towards diapause status, the total gene expression level is relatively low; at E stage, after artificial egg-hatching, the expression level improves rapidly; during larval stages from L1–L5, the expression level rises gradually and reaches a peak at L5 stage; during pupae and moth stages, the total gene expression decline stage by stage. The results revealed a dynamical gene expression profile exhibiting significant differential expressions throughout the silkworm life cycle. Moreover, stage-specific key genes were identified at different developmental stages, suggesting their functions mainly characterized in maintaining insect development and immunity homeostasis or driving metamorphosis. GO annotation and KEGG enrichment analysis further revealed the most significantly enriched and fundamentally biological processes during silkworm growth. Conclusion: Collectively, our omics data depicted the first comprehensive landscape of dynamic transcriptome throughout complete developmental processes of B. mori. Our findings also provide valuable references and novel insights into understanding the molecular developmental remodeling events for other Lepidoptera species. [ABSTRACT FROM AUTHOR]

The silkworm, Bombyx mori, uses a complex olfactory system to determine whether the food is edible. As an odor degrading enzyme, UDP-glycosyltransferase (UGT) participates in the degradation of odor molecules in the olfactory system of the silkworm. By sequencing the whole genome of the silkworm NB and using comparative genomics methods, we found that UGT46A1 is unique in species that eat mulberry leaves. Bioinformatics shows that its function may be related to the feeding habits of the silkworm. In this study, it was found through quantitative real-time polymerase chain reaction (qRT-PCR) that UGT46A1 was highly expressed in the heads of silkworms, which was consistent with the conjecture that UGT46A1 was involved in silkworm olfactory recognition. RNA interference (RNAi) was used to knock down the expression of UGT46A1. By observing the silkworm's tendency toward mulberry leaves and food selectivity, it was found that the silkworms that successfully knocked down the UGT46A1 gene altered their feeding habits and that their ability to find food was weakened, but they could eat more leaves of plants other than mulberry leaves. This evidence indicates that UGT46A1 may affect the silkworm's feeding by influencing the olfactory system of the silkworm. [ABSTRACT FROM AUTHOR]

The BbRib gene participates in the infection process of Beauveria bassiana (B. bassiana). It also helps pathogenic fungi to escape and defeat the insect host immune defense system by regulating the innate immune response. However, model insects are rarely used to study the mechanism of fungal ribosomal toxin protein. In this study, BbRib protein was produced by prokaryotic expression and injected into silkworm (Bombyx mori) larvae. The physiological and biochemical indexes of silkworm were monitored, and the pathological effects of BbRib protein on immune tissues of silkworm were examined by Hematoxylin and Eosin (HE) staining. BbRib protein can significantly affect the growth and development of the silkworm, causing poisoning, destroying the midgut and fat body and producing physiological changes. The ROS stress response in the adipose tissue and cells of the silkworm was activated to induce apoptosis. These results indicated that the BbRib gene not only participates in the infection process of B. bassiana, it also helps the pathogenic fungi escape the immune system by regulating the innate immune system of the silkworm, allowing it to break through the silkworm's immune defense. This study reveals the potential molecular mechanism of BbRib protein to insect toxicity, and provides a theoretical basis and material basis for the development and use of novel insecticidal toxins. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applications due to its scalability and predictable biosafety. To establish a more efficient production system, Bombyx mori pupae were used as a new platform and infected with recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV). The production of a chimeric recombinant adeno-associated virus (rAAV) serotype 2/human bocavirus type-1 (HBoV1) vector was used to evaluate the efficiency of this new baculovirus expression vector (BEV)–insect expression system. For this purpose, we constructed two recombinant BmNPVs, which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. The yields of rAAV2/HBoV1 derived from the rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP co-infected BmN cells exceeded 2 × 104 vector genomes (VG) per cell. The rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP can express stably for at least five passages. Significantly, rAAV2/HBoV1 could be efficiently generated from BmNPV-infected silkworm larvae and pupae at average yields of 2.52 × 1012 VG/larva and 4.6 × 1012 VG/pupa, respectively. However, the vectors produced from the larvae and pupae had a high percentage of empty particles, which suggests that further optimization is required for this platform in the future. Our work shows that silkworm pupae, as an efficient bioreactor, have great potential for application in the production of gene therapy vectors. [ABSTRACT FROM AUTHOR]

Uridine diphosphate (UDP)‐glycosyltransferases (UGTs) are important metabolizing enzymes functioning by adding a sugar moiety to a small lipophilic substrate molecule and play critical roles in drug/toxin metabolism for all realms of life. In this study, the silkworm Bombyx mori UGT33D1 gene was characterized in detail. UGT33D1 was found localized in the endoplasmic reticulum (ER) compartment just like other animal UGTs and was mainly expressed in the silkworm midgut. We first reported that UGT33D1 was important to BmNPV infection, as silencing UGT33D1 inhibited the BmNPV infection in silkworm BmN cells, while overexpressing the gene promoted viral infection. The molecular pathways regulated by UGT33D1 were analysed via transcriptome sequencing upon UGT33D1 knockdown, highlighting the important role of the gene in maintaining a balanced oxidoreductive state of the organism. In addition, proteins that physically interact with UGT33D1 were identified through immunoprecipitation and mass spectrometry analysis, which includes tubulin, elongation factor, certain ribosomal proteins, histone proteins and zinc finger proteins that had been previously reported for human UGT‐interacting proteins. This study provided preliminary but important functional information on UGT33D1 and is hoped to trigger deeper investigations into silkworm UGTs and their functional mechanisms. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensovirus (BmBDV) is a single-stranded DNA (ssDNA) virus from the genus Bidensovirus of the Bidnaviridae family, which, thus far, solely infects insects. It has a unique genome that contains bipartite DNA molecules (VD1 and VD2). In this study, we explored the detailed transcription mapping of the complete BmBDV genome (VD1 and VD2) by rapid amplification of cDNA ends (RACE), reverse transcription quantitative real-time PCR (RT-qPCR), and luciferase assays. For the first time, we report the transcription map of VD2. Our mapping of the transcriptional start sites reveals that the NS genes in VD1 have separate transcripts that are derived from overlapping promoters, P5 and P5.5. Thus, our study provides a strategy for alternative promoter usage in the expression of BmBDV genes. [ABSTRACT FROM AUTHOR]

Bombyx mori nucleopolyhedrovirus (BmNPV) is the most important virus that threatens sericulture industry. At present, there is no effective treatment for BmNPV infection in silkworms, and lncRNA plays an important role in biological immune response and host-virus interaction, but there are relatively few studies in silkworms. In this study, the four midgut tissue samples of the resistance strain NB (NB) and susceptible strain 306 (306) and the NB and 306 continuously infected with BmNPV for 96 h are used for whole transcriptome sequencing to analyze the differences in the genetic background of NB and 306 and the differences after inoculation of BmNPV, and the significantly different mRNA, miRNA and lnRNA between NB and 306 after BmNPV inoculation were screened. By comparing NB and 306, 2651 significantly different mRNAs, 57 significantly different miRNAs and 198 significantly different lncRNAs were screened. By comparing NB and 306 after BmNPV inoculation, 2684 significantly different mRNAs, 39 significantly different miRNAs and 125 significantly different lncRNAs were screened. According to the significantly different mRNA, miRNA and lncRNA screened from NB and 306 and NB and 306 after virus inoculation, the mRNA-miRNA-lncRNA regulatory network was constructed before and after virus inoculation, and the BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis was screened from them, and it was found that BmBCAT was not Bomo_chr7_8305 regulated in the genetic background, after viral infection, MSTRG.3236.2 competes for binding Bomo_chr7_8305 regulates BmBCAT. The whole transcriptome sequencing results were verified by qPCR and the time-series expression analysis was performed to prove the reliability of the regulatory network. The BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis may play a potential role in the interaction between silkworms and BmNPV. These results provide new insights into the interaction mechanism between silkworms and BmNPV. • At present, there have been studies on small RNA regulation by using sensitive silkworm varieties, but no studies have conducted whole transcriptome analysis by comparing resistant and sensitive silkworm strans inoculated with virus. • The mRNA-miRNA-lncRNA ceRNA regulatory network was constructed, from which the BmBCAT-Bomo_chr7_8305-MSTRG.3236.2 regulatory axis was screened, and the whole transcriptome sequencing results were verified by qPCR and the time-series expression analysis was performed to prove the reliability of the regulatory network. • From the perspective of lncRNA to explain the significantly different genes, our research results may provide new clues to the molecular mechanism that reveals the interaction between silkworm and virus. [ABSTRACT FROM AUTHOR]

Diagram illustrating the potential mechanism of BmNPV infection in the 306 strain. The three-color modules represent distinct signaling pathways: NF-kB (depicted in purple), MAPK (illustrated in green), and JAK/STAT (highlighted in pink). Genes outlined in red signify upregulation, while those outlined in green denote downregulation. Genes outlined in black represent expression levels of uncertainty. Illustrations were generated using BioRender. [Display omitted] • Integration of third-generation and second-generation high-throughput sequencing technologies for precise identification and analysis of alternative splicing (AS) patterns. • Identification of five pivotal genes (Dsclp, LOC692903, LOC101743583, LOC101742498, LOC101743809) associated with the response to BmNPV infection. • Discovery of novel transcripts, expanding the current understanding beyond what is available in SilkBase. The silkworm, Bombyx mori, stands out as one of the few economically valuable insects within the realm of model organisms. However, Bombyx mori nucleopolyhedrovirus (BmNPV) poses a significant threat, decreasing the quality and quantity of silkworm cocoons. Over the past few decades, a multitude of researchers has delved into the mechanisms that underlie silkworm resistance to BmNPV, employing diverse methodologies and approaching the problem from various angles. Despite this extensive research, the role of alternative splicing (AS) in the silkworm's response to BmNPV infection has been largely unexplored. This study leveraged both third-generation (Oxford Nanopore Technologies) and second-generation (Illumina) high-throughput sequencing technologies to meticulously identify and analyze AS patterns in the context of BmNPV response, utilizing two distinct silkworm strains—the susceptible strain 306 and the resistant strain NB. Consequently, we identified five crucial genes (Dsclp , LOC692903, LOC101743583, LOC101742498, LOC101743809) that are linked to the response to BmNPV infection through AS and differential expression. Additionally, a thorough comparative analysis was conducted on their diverse transcriptomic expression profiles, including alternative polyadenylation, simple sequence repeats, and transcription factors. [ABSTRACT FROM AUTHOR]

[Display omitted] • A near-isogenic line preserving BmNPV resistance was constructed to reduce the interference of genetic background. • RNA-seq of the BmNPV-infected silkworm midgut revealed 1350 differential expression genes and 32 BmNPV defense responsive genes. • Genes involved in metabolism, ROS, signal transduction and immune response, and insect hormones were upregulated. • Paved the way in the breeding of BmNPV resistance dominant silkworms. Bombyx mori nucleopolyhedrovirus (BmNPV) caused serious economic losses in sericulture. Analyzing the molecular mechanism of silkworms (B. mori) resistance to BmNPV is of great significance for the prevention and control of silkworm virus diseases and the biological control of agricultural lepidopteran pests. In order to clarify the defense mechanisms of silkworms against BmNPV, we constructed a near isogenic line BC8 with high resistance to BmNPV through the highly BmNPV-resistant strain NB and the highly BmNPV-susceptible strain 306. In this study, RNA-Seq technique was used to analyze the transcriptome level differences in the midgut of BC8 and 306 following BmNPV infection. A total of 1350 DEGs were identified. Clustering analysis showed that these genes could be divided into 8 clusters with different expression patterns. Functional annotations based on GO and KEGG analysis indicated that they were involved in various metabolism pathways. Finally, 32 BmNPV defense responsive genes were screened. They were involved in metabolism, reactive oxygen species (ROS), signal transduction and immune response, and insect hormones. The further verification shows that HSP70 should participate in resistance responses of anti-BmNPV. These findings have paved the way in further functional characterization of candidate genes and subsequently can be used in breeding of BmNPV resistance dominant silkworms. [ABSTRACT FROM AUTHOR]

Baculoviruses have been developed as long-term and environmentally friendly biopesticides. However, solar ultraviolet radiation can reduce the activity of baculovirus. Radiation sensitive 23 (Rad23) can recognize DNA damage and is involved with nucleotide excision repair (NER). In the current study, BmRad23 was accumulated mainly within the nucleus. Host cell reactivation (HCR) assays have shown that BmRad23 significantly facilitated the expression of UV-damaged mCherry reporter gene. Reverse transcription quantitative PCR (RT-qPCR) result showed that the mRNA expression level of BmRad23 was increased in (Bombyx mori nuclear polyhedrovirus, BmNPV) BmNPV-infected BmN cells. However, the expression of BmRad23 was increased significantly when BmNPV budded viruses (BVs) or BmN cells were irradiated with UV light. Overexpression of BmRad23 promoted the mRNA levels of two UV-induced DNA damage repair genes which were from Bombyx mori and BmNPV, respectively. Meanwhile, the overexpression of BmRad23 in BmN cells was conducive to the proliferation of BmNPV and UV-damaged BmNPV. The recombinant BmNPV BVs expressing BmRad23 showed stronger resistance to UV radiation than the control virus. In conclusion, the results revealed that BmRad23 contributed to the proliferation of BmNPV and the repair of UV-damaged BmNPV, which would provide a reference for the development of efficient baculovirus pesticides against UV radiation. Unlabelled Image • BmRad23 was conducive to the repair of UV-damaged report gene. • The mRNA expression of BmRad23 was significantly increased if B. mori cells or BmNPV BVs were irradiated with UV light. • BmRad23 was conducive to the proliferation of BmNPV and the repair of UV-damaged BmNPV. [ABSTRACT FROM AUTHOR]

Interacting with cell surface attachment factors or receptors is the first step for virus infection. Glycans cover a thick layer on eukaryotic cells and are potential targets of various viruses. Bombyx mori nuclear polyhedrosis viruses (BmNPV) is a baculovirus that causes huge economic loss to the sericulture industry but the mechanism of infection is unclear. Looking for potential host receptors for the virus is an important task. In this study, we investigated the role of glycosaminoglycan (GAG) modifications, including heparan sulfate (HS) and chondroitin sulfate (CS), during BmNPV infection. Enzymatic removal of cell surface HS and CS effectively inhibited BmNPV infection and replication. Exogenous HS and CS can directly bind to BmNPV virion in solution and act as neutralizers for viral infection. Furthermore, the expression of enzymes involved in GAG biosynthesis was upregulated in the BmNPV susceptible silkworm after virus administration, but down-regulated in the resistant strain after virus treatment, suggesting that BmNPV was able to utilize host cell machinery to promote the biosynthesis of GAGs. This study demonstrated HS and CS as important attachment factors that facilitate the viral entry process, and targeting HS and CS can be an effective means of inhibiting BmNPV infection. • BmNPV infection was suppressed after cell surface heparin sulfate and chondroitin sulfate were reduced. • Heparan sulfate and chondroitin sulfate can directly bind to BmNPV in solution. • GAG biosynthesis was promoted in the susceptible silkworm but suppressed in the resistant strain upon BmNPV treatment. [ABSTRACT FROM AUTHOR]

• A rapid method for BmBDV detection in the field was developed. • The LAMP-LFD method requires only simple faeces-heating and is completed in 45 min. • The method can be adapted for rapid detection of other silkworm pathogens. Bombyx mori bidensovirus (BmBDV), is the only bipartite single-stranded DNA (ssDNA) insect virus reported to date. BmBDV causes fatal flacherie disease in silkworm, resulting in large economic losses to sericulture. We developed a loop-mediated isothermal amplification with lateral flow dipstick (LAMP-LFD) method that can successfully and rapidly detect BmBDV DNA with a low limit of detection (5 fg, 400 copies of the BmBDV genome). The method was evaluated and improved for direct field diagnosis using silkworm faeces. In the field, the actual limit of detection was ∼50 fg (4000 copies of the BmBDV genome). The results demonstrated that, compared with traditional methods for BmBDV detection, our new LAMP-LFD method was much more rapid, sensitive and cost-effective, with less dependence on equipment, making it easier to use. The method has potential to be translated into a new diagnostic product for field applications in the sericulture industry. [ABSTRACT FROM AUTHOR]

Dietary restriction (DR) leads to extended lifespan in many species ranging from yeasts to mammal, and it can also affect the immune system to some extent. Herein, we investigated whether DR can enhance the immunity of Bombyx mori suffering from acute pathogenic microorganism infection. The results showed that DR could accelerate the melanisation reaction, delay the early death in silkworms, meanwhile Staphylococcus aureus (SA) load was lower in the early stage of infection. Moreover, more immune-related genes were identified to be down-regulated in the DR group infected with SA compared with the ad libitum – fed (AL) group infected with SA through mRNA deep sequencing (RNAseq) and quantitation PCR. We speculate that rapid melanization may beneficial to the lower SA load and delay the time point of the early death, and the lower SA load may lead to many immune-related DEGs were down-regulated. These results may help us to understand the mechanisms by which DR affects the immune system in insects and other animals. [ABSTRACT FROM AUTHOR]

Silk proteins are synthesized and secreted by the silk gland. The differential gene expression in it leads to different silk yield among various silkworm strains. As crucial factors, microRNAs (miRNAs) regulate protein synthesis at post-transcriptional level in silk gland. MiRNAs expression level in the silk gland of three silkworm strains (Jingsong, Lan10 and Dazao) was analyzed and 33 differentially expressed miRNAs (DEMs) were discovered between JingSong (JS) and Lan10 (L10), 60 DEMs between JS and Dazao, 54 DEMs between L10 and Dazao respectively. The DEMs target genes were predicted combing with two different methods and their functions were annotated according to gene ontology. Our previous studies showed that a batch of genes related to silk yield were identified in JS and L10 strains by comparative transcriptome and quantitative trait loci (QTL) method. Thirteen DEMs whose target genes are related to protein biosynthesis processes were screened by combining with these researches. Twelve DEMs potentially regulate nineteen genes which exist in our QTL results. Six common DEMs potentially regulate the genes in both of previous results. Finally, five DEMs were selected to verify their expression levels between JS and L10 by qRT-PCR, which showed similar difference as the results of small RNA-sequencing. MiRNAs in the silk gland may directly affect silk protein biosynthesis in different silkworm strains. In current work, we identified a batch of DEMs which potentially regulate the genes related to silk yield. Further functionally study of these miRNAs will contribute to improve varieties and boost the silk yield. Our research provides a basis for studying these miRNAs and their functions in silk production. [ABSTRACT FROM AUTHOR]

The silkworm Bombyx mori ( B. mori ), a lepidopteran model organism, has become an important model for molecular biology researches with its genome completely sequenced. Silkworms confront different types of virus diseases, mainly including those caused by Bombyx mori nucleopolyhedrovirus (BmNPV), Bombyx mori densovirus type 1 (BmDNV-1), Bombyx mori bidesovirus (BmBDV) which was termed as Bombyx mori densovirus type 2 (BmDNV-2) or Bombyx mori parvo-like virus (BmPLV) before in sericulture. B. mori offers excellent models to study the molecular mechanisms of insect innate immune responses to viruses. A variety of molecules and pathways have been identified to be involved in the immune responses in the silkworm to viruses, such as the antimicrobial peptides, prophenoloxidase-activating system, apoptosis, ROS, small RNA and related molecules. Here in this review, we summarize the current research advances in molecules involved in silkworm anti-virus pathways. Moreover, taking BmNPV as an example, we proposed a schematic model of molecules and pathways involved in silkworm immune responses against virus infection. We hope this review can facilitate further study of antiviral mechanisms in silkworm, and provide a reference for virus diseases in other organisms. [ABSTRACT FROM AUTHOR]

It is unclear how, or to what extent, baculovirus DNA that has been damaged by ultraviolet (UV) light is repaired during infection and replication. In our previous study, expression of Bombyx mori nucleopolyhedrovirus (BmNPV) ORF Bm65, a homolog of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac79, correlated with decreased inactivation of virus by UV irradiation. In the current study, we accumulated more evidence pointing to a role for Bm65 in repair of UV-induced DNA damage. The localization of Bm65 was studied using enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bm65 expression plasmid. The results indicate that Bm65-EGFP accumulates in the nucleus. A host cell reactivation assay showed that Bm65 significantly increased the expression of UV-damaged mCherry reporter gene. An assay measuring cyclobutane pyrimidine dimers (CPDs) in UV-irradiated BmN cells found that CPD quantity was decreased in cells transfected with a Bm65 expression plasmid. We also showed that after UVC treatment, the viability of Bm65-transfected cells was higher than that of egfp-transfected cells. These results suggest that Bm65 may be involved in the repair of baculovirus DNA that has been damaged by UV light. [ABSTRACT FROM AUTHOR]

Bombyx mori bidensiovirus (BmBDV) is a species of Bidensovirus that has been was placed into a new genus within the new family Bidnaviridae by the International Committee on Taxonomy of Viruses. BmBDV causes fatal flacherie disease in silkworms, which causes large losses to the sericulture industry. BmBDV contains two sets of complementary linear single-stranded DNAs of approximately 6.5 kb (viral DNA 1, VD1) and 6.0 kb (viral DNA 2, VD2). VD1 and VD2 are encapsidated in separate icosahedral non-enveloped capsids, which are similar in size and shape. However, the strategies used to express BmBDV structural proteins remains unclear. In this work, a total of six structural proteins were separated by two-dimensional electrophoresis and shown to be encoded by the BmBDV VP gene via mass spectrometry. The transmission electron microscopy results showed that co-expression of the BmBDV VP and SP structural proteins in Spodoptera frugiperda sf9 cells resulted in the formation of 22–24 nm virus-like particles. Furthermore, a mutation of the major structural protein-encoding VP gene, in which the second in-frame ATG codon was mutated to GCG, abrogated the production of several structural proteins, indicating that this strategy of expressing BmBDV VP is dependent on a leaky scanning translation mechanism. [ABSTRACT FROM AUTHOR]

Tolfenpyrad is a broad spectrum of insecticide that can effectively kill different types of pests, including Lepidoptera. However, due to improper use, the adverse effects of tolfenpyrad on beneficial or economic insects have not been well studied. In this study, we systematically investigated the toxic effect of sublethal tolfenpyrad on silkworms. Sublethal tolfenpyrad exposure can affect the body weight, developments days, cocooning rate, eclosion rate and pupation rate. To further study the response mechanism of silkworms to tolfenpyrad stimulation, we compared the different expression genes by transcriptome sequencing and verified them by qRT-PCR. We found that significant changes in the genes expression was involved in xenobiotics biodegradation and metabolism, immune system and digestive system after tolfenpyrad treatment. To further investigate the possible mechanisms by which intestinal microbia in the response to tolfenpyrad, we analysed the microbia changes in the midgut of silkworms by 16S rRNA gene sequencing. The results showed that the relative abundances of Enterobacter and Staphylococcus were increased whereas the Tyzzerella and Methylobacterium-Methylorubrum were decreased after tolfenpyrad stimulation. Taken together, these results indicated that low concentration of tolfenpyrad affect the growth and development of silkworms. Silkworms respond to the toxicity of tolfenpyrad by inducing immune and detoxification-related gene expression or altering microbial composition in the midgut. [Display omitted] • Sublethal tolfenpyrad has serious toxic effect on silkworms. • Tolfenpyrad exposure can affect the growth and development of silkworms. • Silkworms detoxified by inducing immune, detoxification-related gene expression. • Gut microbial changes also involved in silkworms detoxication. [ABSTRACT FROM AUTHOR]

[Display omitted] Solar ultraviolet radiation (UV) can cause DNA damage in microorganisms. Flap endonuclease 1 (FEN1) is a structure-specific nuclease and plays important roles in DNA replication and repair. At present, the properties and functions of FEN1 have not been characterized in detail in invertebrates such as Bombyx mori. In this study, Bombyx mori FEN1 (BmFEN1) was expressed in E. coli, and was shown to have nuclease activity that nonspecifically cleaved DNA in vitro. However, inside the cell, BmFEN1 did not cleave DNA randomly. Truncated BmFEN1 missing the nuclear localization signal (346–380 aa) still had the nuclease activity, but was no longer precisely localized to the sites of UV-induced DNA damage. It was further found that BmFEN1 favored the faster repair of UV-damaged DNA. The present study will provide a reference for further understanding the functions of BmFEN1 and UV-induced DNA damage repair mechanisms in insects. [ABSTRACT FROM AUTHOR]