Proteotranscriptomic Integration analyses reveals new mechanistic insights regarding Bombyx mori fluorosis.

The commercial value of silkworms has been widely explored and the effects of fluoride exposure on silkworms' breeding and silk production cannot be ignored. Bombyx mori is a commonly used model to explore the mechanisms of fluorosis. In the present study, we analyzed the differences in physiological and biochemical indicators after exposing larva to NaF, then evaluated differential genes and proteins. Compared to control, larvae exposed to 600 mg L-1 NaF presented decreased bodyweight, damaged midgut tissue, and were accompanied by oxidative stress. The RNA-seq showed 1493 differentially expressed genes (574 upregulated and 919 downregulated). Meanwhile, the TMT detected 189 differentially expressed proteins (133 upregulated and 56 downregulated). The integrative analysis led to 4 upregulated and 9 downregulated genes and proteins. Finally, we hypothesized that fluoride exposure might affect the intestinal digestion of silkworms, inhibit the gene expression of detoxification enzymes and stimulate cellular immune responses. Our current findings provided new insights into insect fluorosis.

Bombyx mori Vps13d is a key gene affecting silk yield.

Bombyx mori is an important economic insect, its economic value mainly reflected in the silk yield. The major functional genes affecting the silk yield of B. mori have not been determined yet. Bombyx mori vacuolar protein sorting-associated protein 13d (BmVps13d) has been identified, but its function is not reported. In this study, BmVps13d protein shared 30.84% and 34.35% identity with that of in Drosophila melanogaster and Homo. sapiens, respectively. The expressions of BmVps13d were significantly higher in the midgut and silk gland of JS (high silk yield) than in that of L10 (low silk yield). An insertion of 9 bp nucleotides and two deficiencies of adenine ribonucleotides in the putative promoter region of BmVps13d gene in L10 resulted in the decline of promoter activity was confirmed using dual luciferase assay. Finally, the functions of BmVps13d in B. mori were studied using the CRISPR/Cas9 system, and the mutation of BmVps13d resulted in a 24.7% decline in weight of larvae, as well as a 27.1% (female) decline and a 11.8% (male) decline in the silk yield. This study provides a foundation for studying the molecular mechanism of silk yield and breeding the silkworm with high silk yield.

Geminin is essential for DNA re-replication in the silk gland cells of silkworms.

Endoreplication, known as endocycles or endoreduplication, is a cell cycle variant in which the genomic DNA is re-replicated without mitosis leading to polyploidy. Endoreplication is essential for the development and functioning of the different organs in animals and plants. Deletion of Geminin, a DNA replication licensing inhibitor, causes DNA re-replication or damage. However, the role of Geminin in endoreplication is still unclear. Here, we studied the role of Geminin in the endoreplication of the silk gland cells of silkworms by constructing two transgenic silkworm strains, including BmGeminin1-overexpression and BmGeminin1-RNA interference. Interference of BmGeminin1 led to body weight gain, increased silk gland volume, increased DNA content, and enhanced DNA re-replication activity relative to wild-type Dazao. Meanwhile, overexpression of BmGeminin1 showed an opposite phenotype compared to the BmGem1-RNAi strain. Furthermore, RNA-sequencing of the transgenic strains was carried out to explore how BmGeminin1 regulates DNA re-replication. Our data demonstrated a vital role of Geminin in the regulation of endoreplication in the silk gland of silkworms.

Mechanism of the growth and development of the posterior silk gland and silk secretion revealed by mutation of the fibroin light chain in silkworm.

Silkworm, as a model organism, has very high economic value due to its silk secretion ability. Although a large number of studies have attempted to elucidate the mechanism of silk secretion, it remains unclear. In this study, the fibroin light chain (Fib-L) gene of silkworm was subjected to CRISPR/Cas9 editing, which yielded premature termination of translation at 135 aa. Compared with those of the wild type, the posterior silk glands (PSGs) of the homozygous mutants on the third day of the fifth instar showed obvious premature degeneration. Comparative transcriptome and proteomic analyses of the PSGs of wild-type individuals, heterozygous mutants and homozygous mutants were performed on the fourth day of the fifth instar. A GO enrichment analysis showed that the differentially expressed genes (DEGs) between homozygous mutants and wild-type individuals were enriched in cytoskeleton-related terms, and a KEGG enrichment analysis showed that the upregulated DEGs between homozygous mutants and wild-type individuals were enriched in the phagosome and apoptosis pathways. These results indicated that apoptosis was activated prematurely in the PSGs of homozygous mutants. Furthermore, autophagy and heat shock response were activated in the PSGs of homozygous mutants, as demonstrated by an analysis of the DEGs related to autophagy and heat shock. A comparative proteomic analysis further confirmed that autophagy, apoptosis and the heat shock response were activated in the PSGs of homozygous mutants, which led to premature degradation of the PSGs. These results provide insights for obtaining a more in-depth understanding of the mechanism of silk secretion in silkworms.

Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, a vaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusion of the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity, including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Vero cells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production of trimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV.

Sage controls silk gland development by regulating Dfd in Bombyx mori.

Silk gland is an organ that produces and secretes silk proteins. The development of the silk gland is essential for high silk production yield and silk quality. Although Sage reportedly plays a pivotal role in embryonic silk gland development, the mechanism underlying its action remains unclear. Our study aimed to determine the genes downstream of Sage through which it regulates the development of the silk gland. After chromatin immunoprecipitation and sequencing, Dfd was identified as a downstream target gene of Sage and it was confirmed that Sage could inhibit Dfd expression by competing with SGF1. When Dfd was knocked down through RNA interference (RNAi), the number of cells in the middle silk gland decreased, and the posterior silk gland was straightened. Simultaneously, the expression of Ser1 and silk fibroin genes was no longer strictly regional. These changes eventually led to an alteration in the composition of the Dfd RNAi cocoon. In conclusion, our research contributes to a deeper understanding of the development of silk glands.

Gene mapping reveals the association between tyrosine protein kinase Abl1 and the silk yield of Bombyx mori.

The Z chromosome of the silkworm contains a major gene that influences silk yield. This major locus on chromosome Z accounts for 35.10% of the phenotypic variance. The location and identification of the gene have been a focus of silkworm genetics research. Unfortunately, identification of this gene has been difficult. We used extreme phenotype subpopulations and selected from a backcross population, BC1 M, which was obtained using the high-yield strain 872B and the low-yield strain IS-Dazao as parents, for mapping the gene on the chromosome Z. The candidate region was narrowed down to 134 kb at the tip of the chromosome. BmAbl1 in this region correlated with silk gland development by spatiotemporal expression analysis. This gene was differentially expressed in the posterior silk glands of the high- and low-yield strains. In BmAbl1, an insertion-deletion (indel) within the 10th exonic region and an SNP within the 6th intronic region were detected and shown to be associated with cocoon shell weight in 84 Bombyx mori strains with different yields. Nucleotide diversity analysis of BmAbl1 and its 50 kb flanking regions indicated that BmAbl1 has experienced strong artificial selection during silkworm domestication. This study is the first to identify the genes controlling silk yield in the major QTL of the Z chromosome using forward genetics.

Large scale production of synthetic spider silk proteins in Escherichia coli.

Spider silk, which has remarkable mechanical properties, is a natural protein fiber produced by spiders. Spiders cannot be farmed because of their cannibalistic and territorial nature. Hence, large amounts of spider silk cannot be produced from spiders. Genetic engineering is an alternative approach to produce large quantities of spider silk. Our group has produced synthetic spider silk proteins in E. coli to study structure/function and to produce biomaterials comparable to the silks produced by orb-weaving spiders. Here we give a detailed description of our cloning, expression, and purification methods of synthetic spider silk proteins ranging from ~30 to ~200 kDa. We have cloned the relevant genes of the spider Nephila clavipes and introduced them into bacteria to produce synthetic spider silk proteins using small and large-scale bioreactors. We have optimized the fermentation process, and we have developed protein purification methods as well. The purified proteins are spun into fibers and are used to make alternative materials like films and adhesives with various possible commercial applications.

Cribellate thread production as model for spider's spinneret kinematics.

Spider silk attracts researchers from the most diverse fields, such as material science or medicine. However, still little is known about silk aside from its molecular structure and material strength. Spiders produce many different silks and even join several silk types to one functional unit. In cribellate spiders, a complex multi-fibre system with up to six different silks affects the adherence to the prey. The assembly of these cribellate capture threads influences the mechanical properties as each fibre type absorbs forces specifically. For the interplay of fibres, spinnerets have to move spatially and come into contact with each other at specific points in time. However, spinneret kinematics are not well described though highly sophisticated movements are performed which are in no way inferior to the movements of other flexible appendages. We describe here the kinematics for the spinnerets involved in the cribellate spinning process of the grey house spider, Badumna longinqua, as an example of spinneret kinematics in general. With this information, we set a basis for understanding spinneret kinematics in other spinning processes of spiders and additionally provide inspiration for biomimetic multiple fibre spinning.

Cyclic Expansion/Compression of the Air-Liquid Interface as a Simple Method to Produce Silk Fibers.

Self-assembly of recombinant spider silk protein at air-liquid interfaces is used as a starting point to produce homogeneous fiber bundles. The film that is formed on a silk protein solution in a vertically placed syringe is subjected to repeated controlled extension and compression by an oscillating vertical motion. Thereby, a precise breakup of the film can be achieved, followed by transport and roll-up against the syringe wall prior to extraction. Advantages of the method are that it 1) is simple to use; 2) requires a small volume of protein solution (1 mL) at relatively low concentration (1 mg mL-1 ); 3) can be performed under sterile conditions; 4) does not require any use of coagulants; and 5) is compatible with the addition of viable cells during the process, which thereby are integrated uniformly throughout the fiber.

Effect of pyriproxyfen exposure on cocooning and gene expression in the silk gland of Bombyx mori （Linnaeus, 1758）.

Bombyx mori（Linnaeus, 1758） is an important economical insect, and the sericulture is a flourishing industry in many developing countries. Pyriproxyfen, a juvenile hormone pesticide, is often applied to cultivations widely in the world, and its exposure often resulted in silk yield reduction and non-cocooning. However, the effect of pyriproxyfen exposure on cocooning and gene expression level in the silk gland of B. mori has not been studied yet, and this study focused on the above issues. The result indicated that pyriproxyfen exposure can lead to silk gland injury, reduction of silk yield and cocooning rate. Furthermore, the expression levels of silk protein synthesis related genes were down regulated significantly. The same change trends were shown between PI3K/Akt and CncC/Keap1 pathway, which is the expressions of key genes can be elevated by pyriproxyfen exposure. In addition, the activity of detoxification enzymes (P450, GST and CarE) and the expression levels of detoxification genes were elevated after pyriproxyfen exposure, suggesting that detoxification enzymes may play an important role in detoxification of pyriproxyfen in silk gland. These results provided possible clues to the silk gland injury and gene transcriptional level changes in silkworm after pyriproxyfen exposure.

Enhanced silk yield in transgenic silkworm (Bombyx mori) via ectopic expression of BmGT1-L in the posterior silk gland.

The silkworm is an economically important insect producing plentiful silk fibre in the silk gland. In this study, we reported a cross-talk between the fat body, silk gland and midgut through a glycine-serine biosynthetic pathway in the silkworm. Amino acid sequence and functional domains of glycine transporter gene BmGT1-L were mapped. Our results indicated that BmGT1-L was specifically expressed in the midgut microvilli and persistently expressed during the feeding stages. RNA interference of BmGT1-L activated glycine biosynthesis, and BmGT1-L overexpression facilitated serine biosynthesis in the BmN4-SID1 cell. In addition, silkworms after FibH gene knock-out or silk gland extirpation showed markedly decreased BmGT1-L transcripts in the midgut and disturbed glycine-serine biosynthesis as silk yield decreased. Finally, BmGT1-L ectopic expression in the posterior silk gland promoted glycine biosynthesis, and enhanced silk yield via increasing fibroin synthesis. These results suggested that cross-talk between tissues can be used for enhancing silk yield in the silkworm.

Photoelectric Silk via Genetic Encoding and Bioassisted Plasmonics.

Genetically encoded photoelectric silk that can convert photons to electrons (light to electricity) over a wide visible range in a self-power mode is reported. As silk is a versatile host material with electrical conductivity, biocompatibility, and processability, a photoelectric protein is genetically fused with silk by silkworm transgenesis. Specifically, mKate2, which is conventionally known as a far-red fluorescent protein, is used as a photoelectric protein. Characterization of the electrochemical and optical properties of mKate2 silk allows designing a photoelectric measurement system. A series of in situ photocurrent experiments support the sensitive and stable performance of photoelectric conversion. In addition, as a plasmonic nanomaterial with a broad spectral resonance, titanium nitride (TiN) nanoparticles are biologically hybridized into the silk glands, taking full advantage of the silkworms' open circulatory system as well as the absorption band of mKate2 silk. This biological hybridization via direct feeding of TiN nanoparticles further enhances the overall photoelectric conversion ability of mKate2 silk. It is envisioned that the biologically derived photoelectric protein, its ecofriendly scalable production by transgenic silkworms, and the bioassisted plasmonic hybridization can potentially broaden the biomaterial choices for developing next-generation biosensing, retina prosthesis, and neurostimulation applications.

Hers and his: Silk glands used in egg sac construction by female spiders potentially repurposed by a 'modern' male spider.

Cylindrical silk gland (CY) spigots distinguish a large clade of modern spiders, the CY spigot clade, which includes all entelegyne spiders and their closest relatives. Following a widespread paradigm, CYs and their spigots are only known to occur in female spiders and they produce silk used in the construction of egg sacs. Here we report the occurrence of a CY spigot or CY nubbin on each posterior median spinneret (PMS) in males (5th stadium and later) of the spider Australomimetus maculosus. Late juvenile males had a CY spigot on each PMS, whereas adult males either had a CY spigot or, more often, a non-functional CY nubbin. This indicates that potential CY use by males is at least largely limited to late juvenile instars and is not involved with egg sac construction. Despite the presence of CY spigots in both sexes, sexual dimorphism with respect to CYs was still evident since males lacked the CY spigot on each posterior lateral spinneret present in late juvenile and adult females, and CY spigots of males never had the wide shaft and opening of adult females. This study adds to our knowledge of spinning apparatus variability in modern spiders and demonstrates an exception to the paradigm that, in the CY spigot clade, such spigots are restricted to female spiders.

Spider Silk for Tissue Engineering Applications.

Due to its properties, such as biodegradability, low density, excellent biocompatibility and unique mechanics, spider silk has been used as a natural biomaterial for a myriad of applications. First clinical applications of spider silk as suture material go back to the 18th century. Nowadays, since natural production using spiders is limited due to problems with farming spiders, recombinant production of spider silk proteins seems to be the best way to produce material in sufficient quantities. The availability of recombinantly produced spider silk proteins, as well as their good processability has opened the path towards modern biomedical applications. Here, we highlight the research on spider silk-based materials in the field of tissue engineering and summarize various two-dimensional (2D) and three-dimensional (3D) scaffolds made of spider silk. Finally, different applications of spider silk-based materials are reviewed in the field of tissue engineering in vitro and in vivo.

Let-7 microRNA is a critical regulator in controlling the growth and function of silk gland in the silkworm.

The silk gland is characterized by high protein synthesis. However, the molecular mechanisms controlling silk gland growth and silk protein synthesis remain undetermined. Here we demonstrated that CRISPR/Cas9-based knockdown of let-7 or the whole cluster promoted endoreduplication and enlargement of the silk gland, accompanied by changing silk yield, whereas transgenic overexpression of let-7 led to atrophy and degeneration of the silk gland. Mechanistically, let-7 controls cell growth in the silk gland through coordinating nutrient metabolism processes and energy signalling pathways. Transgenic overexpression of pyruvate carboxylase, a novel target of let-7, resulted in enlargement of the silk glands, which is consistent with the abnormal phenotype of the let-7 knockdown. Overall, our data reveal a previously unknown miRNA-mediated regulation of silk gland growth and physiology and shed light on involvement of let-7 as a critical stabilizer and booster in carbohydrate metabolism, which may have important implications for understanding of the molecular mechanism and physiological function of specialized organs in other species.

Novaluron impairs the silk gland and productive performance of silkworm Bombyx mori (Lepidoptera: Bombycidae) larvae.

This study investigates the effects of the insect growth regulator Novaluron on the silk gland (SG) and silk cocoon production in a nontarget insect, the silkworm Bombyx mori, which is a model research insect among Lepidoptera and of great economic importance for the commercial production of silk threads. Larvae were segregated into experimental groups: the control group (CG) and the treatment group (TG), which was exposed to a Novaluron concentration of 0.15 mL/L. Following exposure, we analyzed the cytotoxic effects on the epithelial cells of the anterior, middle and posterior regions of the SG of B. mori larvae in the 3rd, 4th, and 5th instars, as well as the quality of the cocoons from larvae in the 5th instar. Cytotoxic effects were observed in the TG, such as the dilation of cells, emission of cytoplasmic protrusions, extreme rarefaction of the cytoplasm and nuclei, dilation of the endoplasmic reticulum, intracellular and intercellular spaces, spacing between the epithelial cells and the basal lamina and detachment of some cells towards the lumen of the SG, and decreased protein in the lumen, with faults in its composition. In addition, we verified ultrastructural changes in the production of fibers and silk cocoons, including a reduction in the weight of the cocoons constructed by both males and females in the TG and the construction of defective cocoons. Novaluron exposure impairs the SG and may affect the physiological functions of this organ; additionally, it compromises the quality of silk cocoons, potentially causing serious damage to sericulture.

Effect of Honey (Apis dorsata [Hymenoptera: Apidae]) on Larval Growth and Silk Cocoon Yield of Bombyx mori (Lepidoptera: Bombycidae).

The present study was carried out to determine the influence of 2% aqueous honey (Apis dorsata Fabricius, 1793 [Hymenoptera: Apidae]) on larval growth and silk cocoon yield of fifth-instar larvae of the silkworm, Bombyx mori (Linnaeus, 1758) (Lepidoptera: Bombycidae). The larvae of silkworms (Chinese HUAKAND2) were divided into a control and an experimental groups (n = 20 in each group). Control group was fed with plain mulberry leaves throughout the fifth instar, whereas the experimental group was offered mulberry leaves dipped in 2% aqueous solution of honey every other day for 4 d (days 1, 3, 5, and 7). On the other days (days 2, 4, 6, and 8), plain mulberry leaves were offered to larvae. Results showed that the average weight gain in larvae of the experimental group was 348.23 and 204.54% in case of the control group. Uneaten mulberry leaves were weighed; the control group left 34.05% of their leaves and the treated group 28.54%. The cocoon formation in the honey-treated larvae was more uniform in shape than the control group. Furthermore, honey-treated larvae began to form cocoons 7.8 ± 0.23 h earlier than the control group. We also recorded an increase of 15.34% in average weight of cocoons of the experimental group when compared with the control. Average shell percentage of fresh silk cocoons of the control and experimental groups was 20.5 and 23.5%, respectively. It is concluded from the study that 2% aqueous honey has positive impact on the larval growth and cocoon yield of B. mori.

Bmsage is involved in the determination of cell number in the silk gland of Bombyx mori.

The number of cells in tissues is under strict genetic control, and research on the determination of cell number is of great importance to understand the growth and development of organs. Bmsage, a bHLH transcription factor, is involved in the development of the silk gland during the embryonic stage in Bombyx mori. However, the mechanism by which it influences silk gland development is unclear. In the present study, we determined via immunofluorescence staining during the embryonic stage of Bombyx mori that Bmsage is expressed in silk gland cells from the beginning of development of the silk gland until its complete formation. By comparing different silkworm strains, we found that Bmsage expression is positively correlated with the number of silk gland cells. Bmsage knockdown by RNAi resulted in shorter silk glands and lower cell numbers, especially in the posterior silk gland. The silk gland lumen also shriveled, and the silk protein content was significantly lower than that in the control. Further investigation revealed that all cyclins decreased after knock down of Bmsage, and cyclin B and cyclin 3 were significantly down-regulated. Bmsage may be involved in the regulation of the cyclin pathway to control silk gland development. Taken together, it can be concluded from our results that Bmsage is involved in the determination of cell number in silk glands. Our results help clarify the process of cell number determination in silk gland and identify a potential target for silkworm breeding.

A proteotranscriptomic study of silk-producing glands from the orb-weaving spiders.

Orb-weaving spiders can produce different silk fibers, which constitute outstanding materials characterized by their high strength and elasticity. Researchers have tried to reproduce the fibers of these proteins synthetically and/or by using recombinant DNA technology, but only a few of the natural physicochemical and biophysical properties have been obtained to date. Female orb-web-spiders present seven silk-glands, which synthesize the spidroins and a series of other proteins, which interact with the spidroins, resulting in silk fibers with notable physicochemical properties. Despite the recognized importance of the silk-glands for understanding how the fibers are produced and processed, the investigation of these glands is at a nascent stage. In the current study we present the assembled transcriptome of silk-producing glands from the orb-weaving spider Nephila clavipes, as well as develop a large-scale proteomic approach for in-depth analyses of silk-producing glands. The present investigation revealed an extensive repertoire of hitherto undescribed proteins involved in silk secretion and processing, such as prevention of degradation during the silk spinning process, transportation, protection against proteolytic autolysis and against oxidative stress, molecular folding and stabilization, and post-translational modifications. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among three groups of silk-producing organisms - (i) Araneomorphae spiders, (ii) Mygalomorphae spiders, and (iii) silk-producing insects. A common orthologous gene, which was annotated as silk gland factor-3 is present among all species analysed. This protein belongs to a transcription factor family, that is important and related to the development of the silk apparatus synthesis in the silk glands of silk-producing arthropods.