Silk Spinning Research Articles

Spider Silk: Rapid, Bottom‐Up Self‐Assembly of MaSp1 into Hierarchically Structured Fibers Through Biomimetic Processing

AbstractSpider dragline silk's exceptional mechanical performance, coupled with its benign production pathway, have inspired the design of diverse smart materials. Remarkably, relatively little is known about the self‐assembly process of major ampullate spidroin 1 (MaSp1) – the main protein constituent of dragline fiber – during its rapid conversion from soluble precursor protein into hierarchically structured mature silk fibers. Herein, the biomimetic potential of MaSp1 is explored using a new recombinant platform with full domain representation. MaSp1 is found to undergo efficient liquid‐liquid phase separation (LLPS) in response to kosmotropic ions, with notably higher propensity compared to MaSp2, and with MaSp1 exhibiting a more complex solubility profile relative to levels of sodium chloride. To further the understanding of silk spinning, the rapid assembly of mesoscale structures is monitored in real‐time in response to ion and pH gradients, revealing a progression from LLPS droplets toward aligned hierarchical fibers. Furthermore, using this biomimetic system, insoluble macroscopic MaSp1 fibers can be readily generated, wherein the application of mechanical deformation triggers the emergence of β‐sheet secondary structures. The insights gained from this study can have broader application in efforts to mimic the formation of biomaterials with spatially organized features across multiple length scales.

Overview and Evolution of Insect Fibroin Heavy Chain (FibH).

The FibH gene, crucial for silk spinning in insects, encodes a protein that significantly influences silk fiber mechanics. Due to its large size and repetitive sequences, limited known sequences of insect FibH impede comprehensive understanding. Here, we analyzed 114 complete FibH gene sequences from Lepidoptera (71 moths, 24 butterflies) and 13 Trichoptera, revealing single-copy FibH in most species, with 2-3 copies in Hesperinae and Heteropterinae (subfamily of skippers). All FibH genes are structured with two exons and one intron (39-45 bp), with the second exon being notably longer. Moths exhibit higher GC content in FibH compared to butterflies and Trichoptera. The FibH composition varies among species, with moths and butterflies favoring Ala, Gly, Ser, Pro, Gln, and Asn, while Trichoptera FibH is enriched in Gly, Ser, and Arg, and has less Ala. Unique to Trichoptera FibH are Tyr, Val, Arg, and Trp, whereas Lepidoptera FibH is marked by polyAla (polyalanine), polySer (polyserine), and the hexapeptide GAGSGA. A phylogenetic analysis suggests that Lepidoptera FibH evolved from Trichoptera, with skipper FibH evolving from Papilionoidea. This study substantially expands the FibH repertoire, providing a foundation for the development of artificial silk.

Effect of Phosphate on the Molecular Properties, Interactions, and Assembly of Engineered Spider Silk Proteins.

Phosphate plays a vital role in spider silk spinning and has been utilized in numerous artificial silk spinning attempts to replicate the remarkable mechanical properties of natural silk fiber. Its application in artificial processes has, however, yielded varying outcomes. It is thus necessary to investigate the origins and mechanisms behind these differences. By using recombinant silk protein SC-ADF3 derived from the garden spider Araneus diadematus, here, we describe its conformational changes under various conditions, elucidating the effect of phosphate on SC-ADF3 silk protein properties and interactions. Our results demonstrate that elevated phosphate levels induce the irreversible conformational conversion of SC-ADF3 from random coils to β-sheet structures, leading to decreased protein solubility over time. Furthermore, exposure of SC-ADF3 to phosphate stiffens already formed structures and reduces the ability to form new interactions. Our findings offer insights into the underlying mechanism through which phosphate-induced β-sheet structures in ADF3-related silk proteins impede fiber formation in the subsequent phases. From a broader perspective, our studies emphasize the significance of silk protein conformation for functional material formation, highlighting that the formation of β-sheet structures at the initial stages of protein assembly will affect the outcome of material forming processes.

In-situ observation of silk nanofibril assembly via graphene plasmonic infrared sensor

Silk nanofibrils (SNFs), the fundamental building blocks of silk fibers, endow them with exceptional properties. However, the intricate mechanism governing SNF assembly, a process involving both protein conformational transitions and protein molecule conjunctions, remains elusive. This lack of understanding has hindered the development of artificial silk spinning techniques. In this study, we address this challenge by employing a graphene plasmonic infrared sensor in conjunction with multi-scale molecular dynamics (MD). This unique approach allows us to probe the secondary structure of nanoscale assembly intermediates (0.8–6.2 nm) and their morphological evolution. It also provides insights into the dynamics of silk fibroin (SF) over extended molecular timeframes. Our novel findings reveal that amorphous SFs undergo a conformational transition towards β-sheet-rich oligomers on graphene. These oligomers then connect to evolve into SNFs. These insights provide a comprehensive picture of SNF assembly, paving the way for advancements in biomimetic silk spinning.

Conspecific cues mediate habitat selection and reproductive performance in a haplodiploid spider mite

Abstract Selection of a suitable habitat by animals before settlement is critical for their survival and reproduction. In silk-spinning arthropods like spider mites, denser webs offer protection from predation and serve as a dispersal mode. Settling in habitats with the presence of conspecifics and silk webs can benefit the habitat-searching females. Silk and conspecifics usually coexist, but their distinct effects on female colonization have received little attention. In this study, we used a haplodiploid spider mite, Tetranychus ludeni Zacher (Acari: Tetranychidae), to examine the impact of conspecific cues, including cues from ovipositing conspecifics and silk, on habitat selection and subsequent reproductive performance of females. Results show that females significantly preferred habitats with cues from neighboring conspecifics and silk and neighboring conspecifics induced additive effect to that of silk on habitat selection. Conspecific cues did not boost female reproduction but facilitated females laying larger eggs that were more likely to be fertilized and to develop into daughters. When given a choice between silk-covered and clean habitats, females preferred silk-covered habitats, laid a similar number of eggs with similar size, but produced more daughters, suggesting that T. ludeni females can adjust the size threshold for fertilization in response to the current social environment. Knowledge of this study improves our understanding of spider mite habitat selection and post-settlement reproductive performance behaviors.

The ratio of fibroin to sericin in the middle silk gland of Bombyx mori and its correlation with the extensional behavior of the silk dope.

Understanding how native silk spinning occurs is crucial for designing artificial spinning systems. One often overlooked factor in Bombyx mori is the secretion of sericin proteins. Herein, we investigate the variation in amino acid content at different locations in the middle silk gland (MSG) of B. mori. This variation corresponds to an increase in sericin content when moving towards the anterior region of the MSG, while the posterior region predominantly contains fibroin. We estimate the mass ratio of sericin to fibroin to be ~25/75 wt% in the anterior MSG, depending on the fitting method. Then, we demonstrate that the improvement in the extensional behavior of the silk dope in the MSG correlates with the increase in sericin content. The addition of sericin may decrease the viscosity of the silk dope, a factor associated with an increase in the spinnability of silk. We further discuss whether this effect could also result from other known physicochemical changes within the MSG.

Comparative anatomy of the spinneret musculature in cribellate and ecribellate spiders (Araneae).

Silk production is a prominent characteristic of spiders. The silk is extruded through spigots located on the spinnerets, which are single- to multimembered paired appendages at the end of the abdomen. Most extant spiders have three pairs of spinnerets, and in between either a cribellum (spinning plate) or a colulus (defunct vestigial organ), dividing these spiders into cribellate and ecribellate species. Previous research has shown that cribellate and ecribellate spiders differ not only in the composition of their spinning apparatus but also in the movements of their spinnerets during silk spinning. The objective of this study was to determine whether the differences in spinneret movements are solely due to variations in spinneret shape or whether they are based on differences in muscular anatomy. This was accomplished by analyzing microcomputed tomography scans of the posterior abdomen of each three cribellate and ecribellate species. It was found that the number of muscles did not generally differ between cribellate and ecribellate species, but varied considerably between the species within each of these two groups. Muscle thickness, particularly of the posterior median spinneret, varied slightly between groups, with cribellate spiders exhibiting more robust muscles, possibly to aid in the combing process during cribellar thread production. Interestingly, the vestigial colulus still possesses muscles, that can be homologized with those of the cribellum. This exploration into spinneret anatomy using microcomputed tomography data reveals that despite being small appendages, the spider spinnerets are equipped with a complex musculature that enables them to perform fine-scaled maneuvers to construct different fiber-based materials.

Effect of eIF6 on the development of silk glands and silk protein synthesis of the silkworm, Bombyx mori

The silkworm is a lepidopteran domesticated from the wild silkworm, mostly valued for its efficient synthesis of silk protein. This species' ability to spin silk has supported the 5500-year-old silk industry and the globally known “Silk Road”, making the transformation of mulberry leaves into silk of great concern. Therefore, research on the silk-related genes of silkworms and their regulatory mechanisms has attracted increasing attention. Previous studies have revealed that domestic silk gland cells are endoreduplication cells, and their high-copy genome and special chromatin conformation provide conditions for the high expression of silk proteins. In this study, we systematically investigate the expression pattern of eukaryotic initiation factors (eIFs) and identified the eIF6 as a eukaryotic translation initiation factor involved in the synthesis of silk proteins. We generated an eIF6 gene deletion mutant strain of silkworm using the CRISPR/Cas9 system and investigated the function of eIF6 in silk gland development and silk protein synthesis. The results showed that deletion of eIF6 inhibited the individual development of silkworm larvae, inhibited the development of silk glands, and significantly reduced the cocoon layer ratio. Therefore, we elucidated the function of eIF6 in the development of silk glands and the synthesis of silk proteins, which is important for further elucidation of the developmental process of silk glands and the mechanism underlying the ultra-high expression of silk proteins.

Studies on the Effect of Differential Feeding on the Commercial Characteristics of Mulberry Silkworm (Bombyx mori L) in Wet Summer in West Bengal, India

The study evaluated the effect of feeding of different maturity level of mulberry leaves i.e. tender, medium, mix, mature, over mature leaves and feeding of shoot during late stages on the commercial characteristics of crossbreed (N×M12 (W)) during wet summer in West Bengal for sustainable development in Sericulture Industry. The data was compared with control where conventional leaf feeding method was followed. The most of the larval and cocoon characters were recorded significantly higher in shoot feeding fed batch and Medium leaves fed batches. Significantly higher post cocoon parameters viz. average filament length, nonbreakable filament length; renditta and raw silk recovery percentage etc. were recorded in medium leaf fed batches and shoot rearing batch. Though qualitative analysis of leaf suggested that tender leaf is more nutritious than other maturity level of leaves but due to prevailing of high moisture in the rearing room as well as in rearing bed during wet summer, it becomes difficult for silkworm larvae to release water during late stage and particularly during spinning if silkworms are fed with high moisture containing tender leaves. More important thing is that whatever water released by silkworm larvae particularly during the end of Vth instar and initiation of spinning, fifty percent of it goes for silk spinning. Present investigation indicates that it is better to feed silkworm larvae with shoot and medium leaf during late instar in wet summer. Mature and over mature leaves fed batch showed inferior performance in terms of all the characters due to their poor nutritional composition.

Large Male Caterpillars Are the Primary Builders: Exploring Tent Construction and Foraging Behaviour in Gregarious Pine Processionary Caterpillar.

As a social organism, living in a communal structure is one of the most important physical barriers against environmental elements and natural enemies. Thaumetopoea pityocampa (Notodontidae, Thaumetopoeinae) caterpillars are conifer pests that spend most of their larval stage in winter. Although T. pityocampa holds economic and medical significance, the tent construction and foraging behaviour are poorly understood. We observed the tent construction behaviour in autumn (October and November) when third- and fourth-instar T. pityocampa caterpillars build the 'winter tent' that can withstand winter conditions. Just before sunset, with no rain and temperatures over 12 °C, tent construction was undertaken by early active individuals, primarily larger male caterpillars. Early active caterpillars emerge from the tent first and spin silk on the tent for expansion and strength. Once temperatures dropped below 12 °C and twilight had passed, the early active caterpillars went out to forage and were later joined by the late active caterpillars, which were predominantly smaller females that had remained inside the tent. Foraging behaviour was continuously monitored for the first to fourth larval instars in the field. Foraging was more frequent in younger instars when environmental temperatures were warmer and became continuous and prolonged in later instar caterpillars as temperatures dropped. The final tent structure built by later instar caterpillars had the thickest layer of silk on the southern side of the tent compared to other orientations to receive maximum solar radiation during the winter. Our study provided additional insights into the collective nest building, foraging and social behaviours observed in Lepidoptera, as well as the roles of individuals within non-eusocial insect colonies.

A brief review on the mechanisms and approaches of silk spinning-inspired biofabrication.

Silk spinning, observed in spiders and insects, exhibits a remarkable biological source of inspiration for advanced polymer fabrications. Because of the systems design, silk spinning represents a holistic and circular approach to sustainable polymer fabrication, characterized by renewable resources, ambient and aqueous processing conditions, and fully recyclable "wastes." Also, silk spinning results in structures that are characterized by the combination of monolithic proteinaceous composition and mechanical strength, as well as demonstrate tunable degradation profiles and minimal immunogenicity, thus making it a viable alternative to most synthetic polymers for the development of advanced biomedical devices. However, the fundamental mechanisms of silk spinning remain incompletely understood, thus impeding the efforts to harness the advantageous properties of silk spinning. Here, we present a concise and timely review of several essential features of silk spinning, including the molecular designs of silk proteins and the solvent cues along the spinning apparatus. The solvent cues, including salt ions, pH, and water content, are suggested to direct the hierarchical assembly of silk proteins and thus play a central role in silk spinning. We also discuss several hypotheses on the roles of solvent cues to provide a relatively comprehensive analysis and to identify the current knowledge gap. We then review the state-of-the-art bioinspired fabrications with silk proteins, including fiber spinning and additive approaches/three-dimensional (3D) printing. An emphasis throughout the article is placed on the universal characteristics of silk spinning developed through millions of years of individual evolution pathways in spiders and silkworms. This review serves as a stepping stone for future research endeavors, facilitating the in vitro recapitulation of silk spinning and advancing the field of bioinspired polymer fabrication.

Biomimetic Spun Silk Ionotronic Fibers for Intelligent Discrimination of Motions and Tactile Stimuli.

Innovation in the ionotronics field has significantly accelerated the development of ultraflexible devices and machines. However, it is still challenging to develop efficient ionotronic-based fibers with necessary stretchability, resilience, and conductivity due to inherent conflict in producing spinning dopes with both high polymer and ion concentrations and low viscosities. Inspired by the liquid crystalline spinning of animal silk, this study circumvents the inherent tradeoff in other spinning methods by dry spinning a nematic silk microfibril dope solution. The liquid crystalline texture allows the spinning dope to flow through the spinneret and form free-standing fibers under minimal external forces. The resultant silk-sourced ionotronic fibers (SSIFs) are highly stretchable, tough, resilient, and fatigue-resistant. These mechanical advantages ensure a rapid and recoverable electromechanical response of SSIFs to kinematic deformations. Further, the incorporation of SSIFs into core-shell triboelectric nanogenerator fibers provides outstanding stable and sensitive triboelectric response to precisely and sensitively perceive small pressures. Moreover, by implementing a combination of machine learning and Internet of Things techniques, the SSIFs can sort objects made of different materials. With these structural, processing, performance, and functional merits, the SSIFs prepared herein are expected to be applied in human-machine interfaces.

Disabling spidroin N-terminal homologs' reverse reaction unveils why its intermolecular disulfide bonds have not evolved for 380 million years

Spiders, ubiquitous predators known for their powerful silks, rely on spidroins that self-assemble from high-concentration solutions stored in silk glands, which are mediated by the NT and CT domains. CT homodimers containing intermolecular disulfide bonds enhance silk performance, promoting spider survival and reproduction. However, no NT capable of forming such disulfide bonds has been identified. Our study reveals that NT homodimers with sulfur substitution can form under alkaline conditions, shedding light on why spiders have not evolved intermolecular disulfide bonds in the NT module during their 380 million years of evolution. This discovery significantly advances our comprehension of spider evolution and silk spinning mechanisms, while also providing novel insights into protein storage, assembly, as well as the mechanisms and therapeutic strategies for neurodegenerative diseases associated with protein aggregation.

Large-scale and cost-effective production of recombinant human serum albumin (rHSA) in transgenic Bombyx mori cocoons

HSA is considered a versatile natural cargo carrier with multiple bio-functions and applications. However, insufficient supply of HSA has limited widespread use. Although various recombinant expression systems had been applied to produce the rHSA to overcome the limited resource, cost-effective and large scale production of rHSA remains a challenge. Herein, we provide a strategy for the large-scale and cost-effective production of rHSA in cocoons of transgenic silkworms, achieving a final 13.54 ± 1.34 g/kg of rHSA yield in cocoons. rHSA was efficiently synthesized and stable over the long-term in the cocoons at room temperature. Artificial control of silk crystal structure during silk spinning significantly facilitated rHSA extraction and purification, with 99.69 ± 0.33 % purity and a productivity of 8.06 ± 0.17 g rHSA from 1 kg cocoons. The rHSA had the same secondary structure to natural HSA, along with effective drug binding capacity, biocompatibility, and bio-safe. The rHSA was successfully evaluated as a potential substitute in serum-free cell culture. These findings suggest the silkworm bioreactor is promising for large-scale and cost-effective production of high quality rHSA to meet the increased worldwide demand.

Bridging Biocondensation and Biomimetic Fibrillation by Regulating Intra- And Interchain Hydrogen Bonds in Artificial Biomacromolecular Condensates.

Utilizing biocondensates as feedstocks can be a state-of-the-art strategy for emulating natural silk spinning. Although current biocondensates can form solid fibers using a biomimetic draw spinning method, the fibrillation is primarily achieved through evaporation of highly concentrated biocondensates rather than structural conversion in natural spinning. Current artificial biocondensates lack biomimetic features of stress-induced fibrillation since they are unable to replicate the structural complexity of native proteins in the dope. Herein, we successfully achieved biomimetic fibrillation at significantly reduced concentrations by constructing artificial biocondensates using naturally derived silk fibroin. The biomimetic features of stress-induced fibrillation in native proteins are replicated in our artificial biocondensates by tailoring multivalent interactions in biocondensation. Our findings unravel the fundamental correlations between biocondensation and stress-induced fibrillation. This work can not only provide a framework for designing artificial biocondensates in biomimetic spinning but also improve the molecular insights into natural spinning.

A transcriptional atlas of the silk gland in Antheraea pernyi revealed by IsoSeq

Silk fibers spun by the silk gland of Antheraea pernyi have many unique properties and are of great value in genetic improvement and non-traditional applications. However, the complete transcriptional landscape and accurate genic annotation of the silk gland are yet to be conducted, which limits related studies on this organ. In this study, isoform sequencing revealed the full-length transcriptome of the A. pernyi silk gland, producing 12,572 high-confidence isoforms from 7,658 genes, among which more than 40 % of genes have not yet been annotated in the reference genome. Moreover, approximately 9 % of isoforms are computationally identified as long non-coding RNAs (lncRNAs). Up to 1,492 alternative splicing (AS) and 3,068 alternative polyadenylation (APA) events were revealed within this transcriptome. In addition, 2,569 putative transcription factors (TFs) belonging to 68 different families were first identified in A. pernyi genome, including 871 TFs in silk gland, and some TF families have undergone expansion or contraction. This study significantly improve our knowledge of the genes expressed in the silk gland of A. pernyi and provide a valuable resource for the in-depth study of silk protein synthesis and spinning, genetic improvement, and non-traditional applications in A. pernyi.

Flow-Induced Protein Chain Deformation, Segmental Orientation, and Phase Separation in Native Silk Feedstock.

The ability of many arthropods to spin silk and its many uses bear testament to its importance in Nature. Despite over a century of research, however, the spinning process is still not fully understood. While it is widely accepted that flow and chain alignment may be involved, the link to protein gelation remains obscure. Using combinations of rheology, polarized light imaging, and infrared spectroscopy to probe different length scales, this work explored flow-induced gelation of native silk feedstock from Bombyx mori larvae. Protein chain deformation, orientation, and microphase separation were observed, culminating in the formation of antiparallel β-sheet structures while the work rate during flow appeared as an important criterion. Moreover, infrared spectroscopy provided direct observations suggesting a loss of protein hydration during flow-induced gelation of fibroin in native silk feedstock, which is consistent with recently reported hypotheses.

The Fractal Network Structure of Silk Fibroin Molecules and Its Effect on Spinning of Silkworm Silk.

Animal silk is usually considered to exist as a solid fiber with a highly ordered structure, formed by the hierarchical assembly starting from a single silk fibroin (SF) chain. However, this study showed that silk protein molecules existed in the form of a fractal network structure in aqueous solution, rather than as a single chain. This type of network was relatively rigid with low fractal dimension. Finite element analysis revealed that this network structure significantly helped in the stable storage of SF prior to the spinning process and in the rapid formation of a β-sheeted nanocrystalline and nematic texture during spinning. Further, the strong but brittle mechanical properties of Bombyx mori silk could also be well-explained through the fractal network model of silk fibroin. The strength was mainly derived from the dual network structure, consisting of nodes and β-sheet cross-links, whereas the brittleness could be attributed to the rigidity of the SF chains between these nodes and cross-links. In summary, this study presents insights from network topology for understanding the spinning process of natural silk and the structure-property relationship in silk materials.

Wild Silkworm Cocoon Waste Conversion into Tough Regenerated Silk Fibers by Solution Spinning.

Wild silkworm silk fibers have garnered attention owing to their softness, natural color, lightweight, and excellent mechanical properties. Because most wild silkworm cocoons obtained are pierced or dirty after the eclosion process, it is difficult to reel the long filament from the pierced cocoons to use as textile materials. Therefore, damaged wild silkworm cocoons are typically removed during the industrial process. Artificial silk spinning has been developed to transform domesticated silkworm silk solutions into regenerated silk fibers. However, regenerated fibers derived from wild silkworm silk have not been reported. Here, we produced regenerated silk fibers using a dry-wet spinning method using a dope solution derived from wild silkworm silk cocoon wastes. These regenerated silk fibers have thick and uniform diameters, unlike native silk fibers, contributing to their usefulness for sterilization and handling in medical applications. Moreover, they exhibited the same level of mechanical strength as their native counterparts. The molecular orientation and crystallinity of the regenerated silk fibers were adjustable by the drawing process, enabling the realization of their various tensile properties. This study promotes the utilization of unused protein resources to produce mechanically stable and tough silk-based fibers.

Recent developments in artificial spider silk and functional gel fibers

AbstractIt is highly desirable to develop fiber materials with high strength and toughness while increasing fiber strength always results in a decrease in toughness. Spider silk is a natural fiber material with an excellent combination of high strength and toughness, which is produced from the spinning dope solution by gelation and drawing spinning process. This encourages people to prepare artificial fibers by mimicking the material, structure, and spinning of natural spider silk. In this review, we first summarized the preparation of artificial spider silk prepared via such a gelation process from different types of materials, including nonrecombinant proteins, recombinant proteins, polypeptides, synthetic polymers, and polymer nanocomposites. In addition, different spinning approaches for spinning artificial spider silk are also summarized. In the third section, some novel application scenarios of the artificial spider silk were summarized, such as artificial muscles, sensing, and smart fibers.