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.

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.

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.

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.

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.

Hierarchical spidroin micellar nanoparticles as the fundamental precursors of spider silks.

Many natural silks produced by spiders and insects are unique materials in their exceptional toughness and tensile strength, while being lightweight and biodegradable-properties that are currently unparalleled in synthetic materials. Myriad approaches have been attempted to prepare artificial silks from recombinant spider silk spidroins but have each failed to achieve the advantageous properties of the natural material. This is because of an incomplete understanding of the in vivo spidroin-to-fiber spinning process and, particularly, because of a lack of knowledge of the true morphological nature of spidroin nanostructures in the precursor dope solution and the mechanisms by which these nanostructures transform into micrometer-scale silk fibers. Herein we determine the physical form of the natural spidroin precursor nanostructures stored within spider glands that seed the formation of their silks and reveal the fundamental structural transformations that occur during the initial stages of extrusion en route to fiber formation. Using a combination of solution phase diffusion NMR and cryogenic transmission electron microscopy (cryo-TEM), we reveal direct evidence that the concentrated spidroin proteins are stored in the silk glands of black widow spiders as complex, hierarchical nanoassemblies (∼300 nm diameter) that are composed of micellar subdomains, substructures that themselves are engaged in the initial nanoscale transformations that occur in response to shear. We find that the established micelle theory of silk fiber precursor storage is incomplete and that the first steps toward liquid crystalline organization during silk spinning involve the fibrillization of nanoscale hierarchical micelle subdomains.

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.

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.

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.

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.

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.

Deep Insight into the Transcriptome of the Single Silk Gland of Bombyx mori.

The silk gland synthesizes and secretes a large amount of protein and stores liquid silk protein at an extremely high concentration. Interestingly, silk proteins and serine protease inhibitors are orderly arranged in the silk gland lumen and cocoon shells. Silk fiber formation and the spinning mechanism have not been fully elucidated. Therefore, we conducted a comparative transcriptome analysis of seven segments of the single silk gland to characterize internal changes in the silk gland during the 5th instar of mature larvae. In total, 3121 differentially expressed genes were identified in the seven segments. Genes highly expressed in the middle silk gland (MSG) were mainly involved in unsaturated fatty acid biosynthesis, fatty acid metabolism, apoptosis-fly, and lysosome pathways, whereas genes highly expressed in the posterior silk gland (PSG) were mainly involved in ribosome, proteasome, citrate cycle, and glycolysis/gluconeogenesis pathways. Thus, the MSG and PSG differ greatly in energy source use and function. Further, 773 gradually upregulated genes (from PSG to MSG) were involved in energy metabolism, silk protein synthesis, and secretion, suggesting that these genes play an important role in silk fiber formation. Our findings provide insights into the mechanism of silk protein synthesis and transport and silk fiber formation.

The origins of the Psechridae: Web-building lycosoid spiders.

Psechrids are an enigmatic family of S.E. Asian spiders. This small family builds sheet webs and even orb webs, yet unlike other orb weavers, its putative relatives are largely cursorial lycosoids - a superfamily of approximately seven spider families related to wolf spiders. The orb web was invented at least twice: first in a very ancient event, and then second, within this clade of wolf-like spiders that reinvented this ability. Exactly how the spiders modified their silks, anatomy, and behaviors to accomplish this transition requires that we identify their precise evolutionary origins - yet, thus far, molecular phylogenies show poor support and considerable disagreement. Using phylogenomic methods based on whole body transcriptomes for psechrids and their putative relatives, we have recovered a well-supported phylogeny that places the Psechridae sister to the Ctenidae - a family of mostly cursorial habits but that, as with all psechrids, retains some cribellate species. Although this position reinforces the prevailing view that orb weaving in psechrids is largely a consequence of convergence, it is still possible that some components of this behavior are retained or resurrected in common with more distant true orb weaving ancestors.

In Vivo Coating of Bacterial Magnetic Nanoparticles by Magnetosome Expression of Spider Silk-Inspired Peptides.

Magnetosomes are natural magnetic nanoparticles with exceptional properties that are synthesized in magnetotactic bacteria by a highly regulated biomineralization process. Their usability in many applications could be further improved by encapsulation in biocompatible polymers. In this study, we explored the production of spider silk-inspired peptides on magnetosomes of the alphaproteobacterium Magnetospirillum gryphiswaldense. Genetic fusion of different silk sequence-like variants to abundant magnetosome membrane proteins enhanced magnetite biomineralization and caused the formation of a proteinaceous capsule, which increased the colloidal stability of isolated particles. Furthermore, we show that spider silk peptides fused to a magnetosome membrane protein can be used as seeds for silk fibril growth on the magnetosome surface. In summary, we demonstrate that the combination of two different biogenic materials generates a genetically encoded hybrid composite with engineerable new properties and enhanced potential for various applications.

Improved strength of silk fibers in Bombyx mori trimolters induced by an anti-juvenile hormone compound.

BACKGROUND: Bombyx mori silk fibers with thin diameters have advantages of lightness and crease-resistance. Many studies have used anti-juvenile hormones to induce trimolters in order to generate thin silk; however, there has been comparatively little analysis of the morphology, structure and mechanical properties of trimolter silk. METHODS: This study induced two kinds of trimolters by appling topically anti-juvenile hormones and obtained thin diameter silk. Scanning electron microscope (SEM), FTIR analysis, tensile mechanical testing, chitin staining were used to reveal that the morphology, conformation and mechanical property of the trimolter silk. RESULTS: Cocoon of trimolters were highly densely packed by thinner fibers and thus had small apertures. We found that the conformation of trimolter silk fibroin changed and formed more ß-sheet structures. In addition, analysis of mechanical parameters yielded a higher Young's modulus and strength in trimolter silk than in the control. By chitin staining of silk gland, we postulated that the mechanical properties of trimolters' silk was enhanced greatly during to the structural changes of silk gland. CONCLUSION: We induced trimolters by anti-juvenile hormones and the resulting cocoons were more closely packed and had smaller silk fiber diameters. We found that the conformation of trimolters silk fibroin had a higher content of ß-sheet structures and better mechanical properties. GENERAL SIGNIFICANCE: Our study revealed the structures and mechanical properties of trimolter silk, and provided a valuable reference to improve silk quality by influencing molting in silkworms.

Apoptosis of posterior silk gland of Bombyx mori during spinning period and the role of PI3K/Akt pathway.

Bombyx mori is an economic insect of the Lepidoptera. Its posterior silk gland (PSG) is an important organ for fibroin synthesis. In order to study the occurrence of apoptosis in PSG and the role of PI3K/Akt signaling pathway during spinning period, changes in morphology of silk gland, expressions of fibroin components Fib-H, Fib-L and P25 and Akt, TOR2, P70S6K and S6 in PI3K/Akt pathway, expressions of apoptosis related genes caspase-3, caspase-9 and activity of caspase-3 were explored. The results showed that the morphology of silk gland dramatically degenerated; transcription of Fib-H, Fib-L, and P25 gradually declined with time; and Fib-L protein level reduced by 0.6-fold at 72 h. Moreover, the transcription levels of Akt, TOR2, P70S6K, and S6 also decreased by 0.3-, 0.8-, 0.7-, and 0.1-fold, respectively, indicating that the downregulation of PI3K/Akt signaling pathway could lead to reduction in fibroin synthesis. In addition, the transcription levels of caspase-3 and caspase-9 increased by 1.3- and 3.6-fold, respectively, and the enzyme activity of caspase-3 grew at a maximum of 1.6-fold. The results showed the occurrence of apoptosis in PSG during spinning period. In conclusion, the present study indicated that both the decline in fibroin components and the increase in apoptosis-related genes were regulated by PI3K/Akt signaling pathway during spinning period, which shed new light on the functions of PI3K/Akt signaling pathway.

Titanium nanoparticles influence the Akt/Tor signal pathway in the silkworm, Bombyx mori, silk gland.

Various nanoparticles, such as silver nanoparticles (AgNPs) and titanium nanoparticles (TiO2 NPs) are increasingly used in industrial processes. Because they are released into the environment, research into their influence on the biosphere is necessary. Among its other effects, dietary TiO2 NPs promotes silk protein synthesis in silkworms, which prompted our hypothesis that TiO2 NPs influence protein kinase B (Akt)/Target of rapamycin (Tor) signaling pathway (Akt/Tor) signaling in their silk glands. The Akt/Tor signaling pathway is a principle connector integrating cellular reactions to growth factors, metabolites, nutrients, protein synthesis, and stress. We tested our hypothesis by determining the influence of dietary TiO2 NPs (for 72 h) and, separately, of two Akt/Tor pathway inhibitors (LY294002 and rapamycin) on expression of Akt/Tor signaling pathway genes and proteins in the silk glands. TiO2 NPs treatments led to increased accumulation of mRNAs for Akt, Tor1 and Tor2 by 1.6-, 12.1-, and 4.8-fold. Dietary inhibitors led to 2.6- to 4-fold increases in mRNAs encoding Akt and substantial decreases in mRNAs encoding Tor1 and Tor2. Western blot analysis showed that dietary TiO2 NPs increased the phosphorylation of Akt and its downstream proteins. LY294002 treatments led to inhibition of Akt phosphorylation and its downstream proteins and rapamycin treatments similarly inhibited the phosphorylation of Tor-linked downstream proteins. These findings support our hypothesis that TiO2 NPs influence Akt/Tor signaling in silk glands. The significance of this work is identification of specific sites of TiO2 NPs actions.

Transgenic Silkworm-Based Silk Gland Bioreactor for Large Scale Production of Bioactive Human Platelet-Derived Growth Factor (PDGF-BB) in Silk Cocoons.

Human platelet derived growth factor (PDGF) is a major therapeutic protein with great demand in the clinical setting; however, its rate of supply is far from meeting needs. Here, we provide an effective strategy to produce PDGF-BB in large quantities using a transgenic silkworm. The codon-optimized PDGF-B gene regulated by the highly efficient sericin-1 expression system was integrated into the genome of a silkworm. The high transcriptional expression of the PDGF-BB gene in the transgenic silkworm competitively inhibited the transcription expression of the endogenous sericin-1 gene which caused a significant 37.5% decline. The PDGF-BB synthesized in the middle silk gland (MSG) of transgenic silkworms could form a homodimer through intermolecular disulfide bonds, which is then secreted into sericin lumen and finally, distributed in the sericin layer of the cocoon. In this study, a protein quantity of approximately 0.33 mg/g was found in the cocoon. Following a purification process, approximately 150.7 µg of recombinant PDGF-BB with a purity of 82% was purified from 1 g of cocoons. Furthermore, the bioactivity assays showed that the purified recombinant PDGF-BB was able to promote the growth, proliferation and migration of NIH/3T3 cells significantly. These results suggest that the silk gland bioreactor can produce active recombinant PDGF-BB as an efficient mitogen and wound healing agent.

Identification of Genes that Control Silk Yield by RNA Sequencing Analysis of Silkworm (Bombyx mori) Strains of Variable Silk Yield.

Silk is an important natural fiber of high economic value, and thus genetic study of the silkworm is a major area of research. Transcriptome analysis can provide guidance for genetic studies of silk yield traits. In this study, we performed a transcriptome comparison using multiple silkworms with different silk yields. A total of 22 common differentially expressed genes (DEGs) were identified in multiple strains and were mainly involved in metabolic pathways. Among these, seven significant common DEGs were verified by quantitative reverse transcription polymerase chain reaction, and the results coincided with the findings generated by RNA sequencing. Association analysis showed that BGIBMGA003330 and BGIBMGA005780 are significantly associated with cocoon shell weight and encode uridine nucleosidase and small heat shock protein, respectively. Functional annotation of these genes suggest that these play a role in silkworm silk gland development or silk protein synthesis. In addition, we performed principal component analysis (PCA) in combination with wild silkworm analysis, which indicates that modern breeding has a stronger selection effect on silk yield traits than domestication, and imply that silkworm breeding induces aggregation of genes related to silk yield.

Comparative Proteomic Analysis of Posterior Silk Glands of Wild and Domesticated Silkworms Reveals Functional Evolution during Domestication.

The wild silkworm Bombyx mandarina was domesticated to produce silk in China approximately 5000 years ago. Silk production is greatly improved in the domesticated silkworm B. mori, but the molecular basis of the functional evolution of silk gland remains elusive. We performed shotgun proteomics with label-free quantification analysis and identified 1012 and 822 proteins from the posterior silk glands (PSGs) of wild silkworms on the third and fifth days of the fifth instar, respectively, with 128 of these differentially expressed. Bioinformatics analysis revealed that, with the development of the PSG, the up-regulated proteins were mainly involved in the ribosome pathway, similar to what we previously reported for B. mori. Additionally, we screened 50 proteins with differential expression between wild and domesticated silkworms that might be involved in domestication at the two stages. Interestingly, the up-regulated proteins in domesticated compared to wild silkworms were enriched in the ribosome pathway, which is closely related to cell size and translation capacity. Together, these results suggest that functional evolution of the PSG during domestication was driven by reinforcing the advantageous pathways to increase the synthesis efficiency of silk proteins in each cell and thereby improve silk yield.