Pupae Research Articles

Cocoon-Spinning Behavior and 20-Hydroxyecdysone Regulation of Fibroin Genes in Plutella xylostella.

The diamondback moth Plutella xylostella is a serious pest of crucifers. It has high reproductive potential and is resistant to many insecticides. Typically, the last-instar larvae of P. xylostella, before pupation, move to the lower or outer plant leaves to make a loose silk cocoon and pupate inside for adult formation. To better understand this pivotal stage we studied the cocoon-spinning behavior of P. xylostella and measured three successive phases by video-recording, namely the selection of a pupation site, spinning a loose cocoon and padding the scaffold cocoon. Subsequently, we cloned three fibroin genes related to cocoon production, i.e., fibroin light chain (Fib-L), fibroin heavy chain (Fib-H), and glycoprotein P25. A spatio-temporal study of these three fibroin genes confirmed a high expression in the silk glands during the final larval instar silk-producing stage. In parallel, we did an exogenous treatment of the insect molting hormone 20-hydroxyecdysone (20E), and this suppressed fibroin gene expression, reduced the normal time needed for cocoon spinning, and we also observed a looser cocoon structure under the scanning electron microscope. Hence, we demonstrated that the expression levels of key genes related to the synthesis of 20E [the three Halloween genes Spook (Spo), Shadow (Sad), and Shade (Shd)] decreased significantly during spinning, the expression of the 20E receptor (EcR and USP) was significantly lower during spinning than before spinning, and that the expression levels of CYP18-A1 related to 20E degradation were significantly up-regulated during spinning. The significance of the cocoon and the effects of 20E on the cocoon-spinning behavior of P. xylostella are discussed.

Antibacterial Mechanism of Silkworm Seroins.

Seroin 1 and seroin 2 are abundant in silkworm cocoon silk and show strong antibacterial activities, and thus are thought to protect cocoon silk from damage by bacteria. In this study, we characterized the expression pattern of silkworm seroin 3, and found that seroin 3 is synthesized in the female ovary and secreted into egg to play its roles. After being infected, seroin 1, 2, and 3 were significantly up-regulated in the silkworm. We synthesized the full-length protein of seroin 1, 2, and 3 and their N/C-terminal domain (seroin-N/C), and compared the antimicrobial activities in vitro. All three seroins showed higher antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. Seroin 2 showed better antibacterial effect than seroin 1 and 3, whereas seroin 1/2/3-N was better than seroin 1/2/3-C. We found that seroin 2-C has stronger peptidoglycan binding ability than seroin 2-N per the ELISA test. The binding sites of seroin 2 with bacteria were blocked by peptidoglycan, which resulted in the loss of the antibacterial activity of seroin 2. Collectively, these findings suggest that seroin 1 and 2 play antibacterial roles in cocoon silk, whereas seroin 3 functions in the eggs. The three silkworm seroins have the same antibacterial mechanism, that is, binding to bacterial peptidoglycan by the C-terminal domain and inhibiting bacterial growth by the N-terminal domain.

Efficiency of some plants powder mix as a dietary supplement for silkworm (Bombyx mori)

Bombyx mori L. is one of the most economically important insects because of its production of silk. In this study, we investigated the effect of four plants powder mix on larvae weight, silk gland weight, cocoon parameters and fecundity of moths in two hybrids (local and imported) of silkworm. The most desirable effects (increased final larvae weight by over 8.93%, silk gland weight by over 37.45%, cocoon shell weight by over 39.84% and No. of eggs by over 28.93%) was obtained by treatment of mulberry leaves with (M2) in local hybrid. Meanwhile in imported hybrid the most desirable effects (increased final larvae weight by over 3.75%, silk gland weight by over 51.29%. cocoon shell weight by over 22.17% and No. of eggs by over 55.87%) was got by treatment of mulberry leaves with (M3) and feed it to the larvae. According to the results, It can enrich the nutrient quality of mulberry leaves provided to the silkworm by adding such plant materials, which lead to raising the efficiency of larvae productivity, increasing the quantity and quality of cocoons, and then improving the production of silk.

Comparison Between Some Mulberry Varieties on Silkworm, Bombyxmori L Economic Traits

Five mulberry varieties were used for tests. These varieties were Morus alba Linn. Var. Kaeryang- Bpong (K1), Morus alba Linn. Var. Kokuso-27 (K2), Morus alba Linn. Var. Canava-2 (K3), Morus alba Linn. Var. Suisfen (K4) and Morus alba Linn. Var. Ardnyl (K5). The agronomical parameters recorded, were shoot length, number shoots/tree, number leaves/ shoot, leaf /shoot ratio, weight of 100 leaf, number leaves/100g, leaf yield/tree, leaf yield/fadden and leaf moisture percentage. Young instars duration, fourth instar duration, fifth instar duration, total larval duration, pupation ratio, cocooning percentage, number of cocoons/liters, cocoon weight, cocoon shell weight, pupal weight and cocoon shell ratio, silk productivity, length and weight of silk filament, size of reeled thread (denier) and silk ratio (silk recovery) were registered. Biochemical analysis was done to estimate the total chlorophyll, chlorophyll A and B & ratio of chlorophyll A/B and carotenoids.

Safety Assessment of Silk Protein Ingredients as Used in Cosmetics.

The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the safety of Hydrolyzed Silk and 9 other silk protein ingredients, which function primarily as skin and hair conditioning agents and bulking agents in cosmetic products. The Panel reviewed relevant data relating to the safety of these ingredients and concluded that 8 ingredients are safe in the present practices of use and concentration in cosmetics, as described in this safety assessment, but that the available data are insufficient for determining the safety of 2 silk protein ingredients in cosmetic products, MEA-Hydrolyzed Silk and Silkworm Cocoon Extract.

Easy conversion of nitrogen‐rich silk cocoon biomass to magnetic nitrogen‐doped carbon nanomaterial for supporting of Palladium and its application

In this study, magnetic nitrogen‐doped carbon (MNC) was fabricated through facile carbonization and activation of natural silk cocoons containing nitrogen and then combined with Fe3O4 nanoparticles to create a good support material for palladium. Palladium immobilization on the support resulted in the formation of magnetic nitrogen‐doped carbon‐Pd (MNC‐Pd). The prepared heterogeneous catalyst was well characterized using FT‐IR, TGA, EDX, FE‐SEM, XRD, VSM, and ICP‐OES techniques. Thereafter, the synthesis of biaryl compounds was conducted to investigate the catalyst performance via the reaction of aryl halides and phenylboronic acid. Further, the catalyst could be used and recycled for six consecutive runs without any significant loss in its activity.

Transdermal peptide conjugated to human connective tissue growth factor with enhanced cell proliferation and hyaluronic acid synthesis activities produced by a silkworm silk gland bioreactor.

Human connective tissue growth factor (CTGF) is a secreted cysteine-rich peptide that stimulates cell proliferation, migration, and extracellular matrix production during tissue development, differentiation, angiogenesis, implantation, wound healing, and fibrosis processes, with broad application in the medical and cosmetic medical fields. However, the production of CTGF is currently limited by its low yield and purity in current bioreactors. In this study, two genetically modified silkworm strains were generated harboring artificially designed CTGF-8ht and pepCTGF-8ht genes, respectively, that contain an enhanced His-tag with eight histidine residues with or without a transdermal peptide (pep). Both recombinant CTGF-8ht and pepCTGF-8ht proteins were successfully expressed in the silkworm silk gland and cocoon, and could be easily extracted and purified from the cocoon by single-affinity immunoprecipitation column chromatography, achieving a purity of more than 95%. Moreover, compared with CTGF-8ht protein, pepCTGF-8ht protein exhibited better cell proliferation activity by activating the extracellular signal-regulated kinase (ERK) pathway and enhanced hyaluronic acid synthesis activity by upregulating hyaluronan synthase 3 expression; moreover, the addition of pep significantly improved the transmembrane ability of CTGF-8ht protein. These results should help to promote the application prospects of CTGF and further guide the design and development of protein drugs from silkworm and other bioreactor systems. KEY POINTS : A silkworm bioreactor was optimized to produce connective tissue growth factor (CTGF) The transgene contained an enhanced 8-His-tag and transmembrane peptide (pep) Recombinant CTGF was easily purified with maintained or higher biological activity.

Fiber Length of Eri Silk Cocoons and Influence of Staple Cutting during Spinning Preparatory Process

ABSTRACT The range of single fiber length in both white and brick red eri cocoons is very high due to inactivity of Filipi’s gland of silkworm along with open structure of cocoons’ shells. This wide range can be reduced by staple cutting process after cocoons’ opening. The average fiber length is improved with more value of staple cut length. Due to staple cutting, short fiber generation occurred and descending trend was noticed with increase of staple cut length. Hauteur value is enhanced with increase of staple cut length and significant difference exists between 100 mm and 200 mm staple. Similarly, higher proportion of longer fiber percentage was revealed with increase of staple length. Since there is restriction of long fiber up to 200 mm for setting of rollers in drafting zone of speed frame and ring frame in case of long staple spinning, length can be set between 100 mm and 150 mm. Due to difference in measurement principle, the average fiber length assessed by BISFA oil plate method and Hauteur and Barbe evaluated by Almeter is not similar. So, expressions have been derived for prediction of Hauteur as well as Barbe from fiber length measured by BISFA oil plate method.

Analysis of Nutritional Compounds and Antioxidant Effect of Freeze-Dried powder of the Honey Bee (Apis mellifera L.) Drone (Pupal stage)

Analysis of Nutritional Compounds and Antioxidant Effect of Freeze-Dried powder of the Honey Bee (Apis mellifera L.) Drone (Pupal stage)

Efficacy of Mulberry Varieties on Young Instars Larvae of Silkworm, Bombyx mori L.

Six mulberry varieties were selected for feeding young silkworm, Bombyx mori L. These varieties were Morus alba Linn. Var. Iljiros (A), Morus Ihou Koidz. Var. Josaengrok (B), Morus alba Linn. Var. Kearyang-Seoban (C), Morus Ihou Koidz. Var. Nosang (D), Morus alba Linn. Var. Afiluniz (E) and Morus alba Linn. Var. Kokuso-27(G). During the 4th and 5th larvae instar of silkworm instar, nutritional were tested, data were collected on the biomass of larvae and cocoons for 6 treatments on ingesta, digesta, excreta, approximate digestibility (AD), reference ratio (RR), consumption index (CI), relative growth rate (RGR), respiration (R) and metabolic rate (MR), efficiency conversion of ingesta (ECI) and digesta (ECD) for larva, cocoon, and shell. Further, the ingesta and digesta required for producing one gram of cocoon and shell (I/g and D/g). Characters of young instars duration (YID), fifth duration (FID), total larval period (TLD), cocooning percentage (CP), pupation ratio (PR), sex ratio (SR), number of cocoons/liter (C/L), fresh cocoon weight (FCW), fresh cocoon shell weight (FCSW), fresh pupa weight (FPW), fresh cocoon shell ratio (FCSR) and silk productivity (SP) for female and male. In addition to length of silk filament (FL), weight of silk filament (FW), size of reeled thread (denier; FS) and silk ratio (SR) were investigated.

A Transparent, Skin-Inspired Composite Film with Outstanding Tear Resistance Based on Flat Silk Cocoon.

Flexible and transparent substrates play a fundamental role as a mechanical support in advanced electronic devices. However, commonly used polymer films, such as polydimethylsiloxane, show low tear resistance because of their crack sensitivity. Herein, inspired by the excellent mechanical robustness of the skin and its fibrous structure, an epoxy-resin-based composite with a flat silk cocoon as a reinforcing fiber network is fabricated. With only 1 wt% of silk fiber, the tensile strength and modulus of the as-prepared composite film are considerably increased by 300% and 612% compared to those of pure resin, while still maintaining flexibility and transparency. More importantly, the composite shows remarkable tear resistance: without fracture after ≈30 000 tensile cycles. The potential application of such transparent composite films as mechanically robust substrates for flexible electronics is also demonstrated. In addition, this study represents a bioinspired strategy to construct high-performance functional composite materials.

The beta-1, 4-N-acetylglucosaminidase 1gene, selected by domestication and breeding, is involved in cocoon construction of Bombyx mori.

Holometabolous insects have distinct larval, pupal, and adult stages. The pupal stage is typically immobile and can be subject to predation, but cocoon offers pupal protection for many insect species. The cocoon provides a space in which the pupa to adult metamorphosis occurs. It also protects the pupa from weather, predators and parasitoids. Silk protein is a precursor of the silk used in cocoon construction. We used the silkworm as a model species to identify genes affecting silk protein synthesis and cocoon construction. We used quantitative genetic analysis to demonstrate that β-1,4-N-acetylglucosaminidase 1 (BmGlcNase1) is associated with synthesis of sericin, the main composite of cocoon. BmGlcNase1 has an expression pattern coupled with silk gland development and cocoon shell weight (CSW) variation, and CSW is an index of the ability to synthesize silk protein. Up-regulated expression of BmGlcNase1 increased sericin content by 13.9% and 22.5% while down-regulation reduced sericin content by 41.2% and 27.3% in the cocoons of females and males, respectively. Genomic sequencing revealed that sequence variation upstream of the BmGlcNase1 transcriptional start site (TSS) is associated with the expression of BmGlcNase1 and CSW. Selective pressure analysis showed that GlcNase1 was differentially selected in insects with and without cocoons (ω1 = 0.044 vs. ω2 = 0.154). This indicates that this gene has a conserved function in the cocooning process of insects. BmGlcNase1 appears to be involved in sericin synthesis and silkworm cocooning.

Silk sericin as a biomaterial for tissue engineering: a review

Silk sericin is a natural polymer produced by silkworm Bombyx mori; which has the function of covering the fibroin filaments so that they remain linked together as well as maintaining the structural integrity of the cocoon. Despite its properties and benefits, sericin is currently discarded during textile production as it is considered a side product of silk cocoons during the degumming process. Among the most important biological properties of sericin are good oxygen permeability, moisture-regulating capacity, resistance to UV radiation, ability to promotes cell growth, biocompatibility and mitogenic effect. All these characteristics make sericin a promising polymer to be used in biomedical applications, especially in tissue engineering and regenerative medicine. Some studies report that sericin favors the biological nature of keratinocytes and fibroblasts, potentializing it as a biomaterial for the repair of epithelial tissue, mainly utilized as wound dressing. Given the importance that sericin can have in the field of bioengineering, and that its use can favor the sericulture agroindustry, this manuscript reviews recent studies of this protein for its application in tissue engineering, specifically for cutaneous regeneration. Highlights Research on SS represents a promising and developing field. SS decreases cell adhesion and favors the proliferation of keratinocytes and fibroblasts. SS has been shown to have a mitogenic effect in some mammalian cells. SS can be crosslinked, copolymerized, and mixed with other polymers.

Fuzzy multi-criteria group decision making approach for grading of mulberry silk cocoons

A multi-criteria group decision making method has been applied for grading of silk cocoons which employs the proficiency of three experts as decision makers to alleviate the fuzziness underlying in silk cocoon parameters. Fuzzy-TOPSIS approach of multi-criteria group decision making method has been used in this study. Four quality parameters of silk cocoon, viz. good cocoon percentage, cocoon weight, cocoon size and compactness are regarded as criteria. The 10 cocoon lots are ranked based on Fuzzy-TOPSIS method. The ranking of cocoon lots attained by this method shows a significant agreement with the ranks devised by the sericulture experts.

Mechanical, Thermal, Void Fraction and Water Absorption of Silane Surface Modified Silk Fiber Reinforced Epoxy Composites

ABSTRACT In this research, the silk fibers that were successfully extracted from silk cocoons using Na2CO3 were treated with silane coupling agent (3-aminopropyl)trimethoxysilane. The silane treated and untreated silk fiber reinforced epoxy composites were fabricated using the hand lay-up technique. The effects of fiber loading and the presence of silane coupling agent on the mechanical, morphological, thermal, void fraction, and water absorption properties of the silk fiber reinforced epoxy composites were studied. Fourier transform infrared (FTIR) analysis confirmed presence of silane group on the surface of fiber. The void fraction and water absorption increased with increasing fiber loading for both untreated and silane-treated composites. However, the silane-treated composites displayed lower increment of both void fraction and water absorption in comparison to the untreated samples suggesting better properties. The impact, flexural and tensile properties of the treated composites exhibited better property enhancement compared to the untreated samples with optimum fiber loading was observed at 30 wt%. The presence of this coupling agent also increased the thermal properties of the composites in general. The morphological studies revealed good interfacial compatibility between fiber and matrix in the presence of silane coupling agent which consistent with the enhancement observed for impact properties.

Correlating the secondary protein structure of natural spider silk with its guiding properties for Schwann cells.

The successful reconstruction of supercritical peripheral nerve injuries remains a major challenge in modern medicine. Progress in tissue engineering has enabled the development of nerve guidance conduits as an alternative to autologous nerve transplantation and the enrichment of conduits with fibrous materials or hydrogels has shown great potential in bridging nerve defects. The application of the dragline silk of spider genus Nephila as a filament for nerve guidance conduits has led to promising results. However, the use of spider silk has been phenomenological so far and the reasons for its success are still not identified. This renders a targeted tuning of synthetic fibrous luminal fillings such as recombinant silk out of reach. In this work the existing research was extended and in addition to dragline, the cocoon silk of Nephila edulis, as well as the connecting and attaching silk of Avicularia avicularia were investigated. Scanning electron microscopy revealed a difference in size and morphology of the spider silks. However, in vitro experiments indicated that Schwann cells adhere to the four fibers, independent of these two attributes. Raman spectroscopy in native state and aqueous environment demonstrated similar secondary protein structures for dragline, cocoon, and connecting silk. In contrast, the attaching silk showed a significant lower conformation of β-sheets, crucial for the stiffness of the silk. This was in line with the in vitro experiments, where the flexible attaching silk fibers adhered to each other when placed in liquid. This resulted in their inability to guide Schwann cells, leading to the generation of cell agglomerations. This direct comparison demonstrated the crucial role of β-sheets conformation for the guidance properties of natural spider silk, providing essential insights into the necessary material properties for the integration of fibrous luminal fillings in nerve guidance conduits.

Silk cocoon membrane-based immunosensing assay for red blood cell antigen typing

Accurate and fast identification of blood groups is a critical step in the blood transfusion procedure. In this study, we reported a rapid and reliable immunosensing assay for ABO and RhD blood group typing based on natural free-standing silk cocoon membranes. Natural silk cocoon membranes were punched into discs (SMDs) and were treated with heat and detergent to increase wettability. Monoclonal antibodies (mAbs) against silk proteins were successfully prepared through animal immunization. The SMDs were functionalized by affinity binding with specific silk mAbs and crosslinking antibodies. Blood grouping antibodies were coupled with functional SMDs to form the immunosensor for blood typing. Only three pipetting steps were required to perform the assay, without the need for any external analytical equipment. Results could be obtained and distinguished by the naked eye within 30 s, making this immunosensor suitable for point-of-care testing. A positive result was obtained when the SMD showed a visualization red spot after washing, no red spot was observed if there was no reaction. We compared our sensing results with those from the gold-standard tube test on 112 real blood samples. The accuracy was 100 %. Due to the availability of the cocoon membranes-based immunosensor and the facile fabrication process, the SMART can be scaled up easily for mass production.

Identifying parents and generating hybrids with high combining ability for yielding fresh cocoon and raw silk in silkworm (Bombxy mori L.)

ABSTRACT This study was carried out to identify parents and hybrids by determination of general combining ability (GCA), specific combining ability (SCA), and heterosis in pure lines and their hybrids, which were selected from our gene sources, to determine new hybrid combinations that could be alternative or higher in yielding compared with M × N hybrid used in production. Combining ability and heterosis effects were studied for the eight quantitative characters through line × tester mating design. Chinese origin lines [...]

Nitrogen source-mediated cocoon silk-derived N, O-doped porous carbons for high performance symmetric supercapacitor

Rational design and synthesis of high-efficient and cost-effective electrode materials are pivotal for developing high performance supercapacitors. Herein, we synthesized N, O, co-doped hierarchical porous carbon fibers (NPCFs) towards high performance symmetric supercapacitor with N-rich cocoon silk as precursor via an alkali activation and a g-C3N4 mediated strategy. By adjusting the addition of g-C3N4, the N doping content and pore structures can be regulated, and the optimized material NPCF1 (mass ratio, primary char CF: g-C3N4 = 1:1) exhibits high N doping content (6.05%) with domination of active pyridinic-N pyrrolic-N, and more abundant pores as well as higher specific surface area (2643.75 m2 g−1). Meanwhile, NPCF1 as supercapacitor electrode can deliver high specific capacitance of 392 F g−1 at 1 A g−1 and excellent rate performance (222 F g−1 at 20 A g−1) in 6 M KOH. More impressively, NPCF assembled symmetric supercapacitor with 1.0 M neutral Na2SO4 electrolyte can still achieve a high operating voltage of 1.8 V, an outstanding energy density of 20.77 W h kg−1 at a power density of 450 W kg−1. Such universal small molecule nitrogen source-mediated strategy may inspire new progress of synthesizing advanced carbons for energy-related application.

Exploring the theoretical and experimental optimization of high-performance triboelectric nanogenerators using microarchitectured silk cocoon films

Triboelectric nanogenerators (TENGs) developed using eco-friendly natural materials instead of traditional electronic materials are more favorable for biocompatible applications, as well as from a sustainable life-cycle analysis perspective. Microarchitectured silkworm fibroin films with high surface roughness and an outstanding ability to lose electrons are used to design TENGs. An alcohol-annealing treatment is utilized to strengthen the resistance of the silk film (SF) against humidity and aqueous solubility. Herein, for the first time, the distance-dependent electric field theoretical model is employed to optimize the TENG parameters to achieve high output, which shows excellent agreement with the experimental outputs of SF-based TENG. The alcohol-treated microarchitectured SF (AT-MASF) with a polytetrafluoroethylene positive contact exhibits a stable and high electrical output even in harsh environments. These studies can lead us closer to the attractive future vision of realizing biodegradable TENG systems for harness/sensing various biomechanical activities even under real/humid environments. The potential and real-time application of the proposed AT-MASF-based TENG is demonstrated by directly employing its electric power to drive a number of low-power portable electronics and for sensing in human-body centric activities. • Silk film with high surface roughness was proposed by an inexpensive technique for TENGs. • The alcohol-annealing treatment was performed to strengthen such silk film against humidity and aqueous solubility. • The novel DDEF model was adopted to theoretically investigate the optimum triboelectric material opposite to the SF. • The surface roughness effect of silk film on the performance of TENG is also investigated by DDEF model. • The feasibility of AT-MASF-TENG is demonstrated by sensing daily human-activities.