The Evaluation of the Biological Effects of Melanin by Using Silkworm as a Model Animal.
ABSTRACT: Melanin has been reported to have potential applications in industries such as cosmetics and food due to its anti-UV and antioxidative qualities. However, the corresponding data on its safety evaluation or biological consequences are fairly limited; such data are critical given its widespread use. The effect of different concentrations (1, 2, 3, and 4%) of melanin on growth status (larvae length and weight, cocoon weight, and morphology), the microstructure of the various tissues (fat body, silk gland, and midgut), and silk properties was studied by using the silkworm (bombyx mori) as the model organism. The weight and length of silkworm larvae fed with melanin were lower than the control, indicating that melanin appears to have a negative effect on the growth status of silkworms; however, the histophysiology analysis indicates that the cell morphologies are not changed, the XRD and FTIR spectra indicate that the secondary and crystalline structures of silks are also well preserved, and the thermogravimetric analysis and tensile test indicate that the thermal stability and mechanical properties are well maintained and even improved to some extent. Generally, it indicates that melanin has a certain inhibitory effect on the growth of silkworm larva but causes no harm to the cell microstructures or silk properties; this demonstrates that the safety of melanin as a food addictive should be considered seriously. The increase of thermal stability and mechanical properties shows that melanin may be a good chemical modifier in textile industries.
Project description:Ecdysteroid UDP glucosyltransferase (EGT) is a baculovirus-encoded protein which can hinder the normal molting of insects by inactivating 20-hydroxyecdysone (20E). Here we expressed EGT in the last-instar silkworm larvae using the GAL4/ UAS system. Compared with the control, for the EGT overexpressed silkworm, the hemolymph 20E content was significantly decreased, the feeding and spinning periods of the last-instar silkworm larvae were extended, the cocoon shell ratio was significantly increased, and the transformation from silkworm larvae to pupa was blocked. Increasing EGT expression resulted in the decrease of 20E content in the hemolymph of silkworm larvae, treating the EGT overexpressed male silkworm with 20E decreased the larval weight and cocoon shell ratio, confirming that the increase in the availability of nutrients to the cocoon and an increase in the cocoon shell weight in the hybrid transgenic silkworms is because of the EGT-induced reduction in active 20E content. Furthermore, though the sericin and flavonoid contents were increased in the cocoon of the EGT overexpressing silkworm, the production of silk fibroin didn't change.
Project description:Transgenic silkworm expression systems have been applied for producing various recombinant proteins. Knocking out or downregulating an endogenous silk protein is considered a viable strategy for improving the ability of transgenic expression systems to produce exogenous proteins. Here, we report the expression of human epidermal growth factor (hEGF) in a <i>P25</i> gene knockout silkworm. The <i>hEGF</i> gene regulated by the <i>P25</i> gene promoter was integrated into a silkworm's genome. Five transgenic positive silkworm lineages were generated with different insertion sites on silkworm chromosomes and the ability to synthesize and secrete proteins into cocoons. Then, a cross-strategy was used to produce transgenic silkworms with a <i>P25</i> gene knockout background. The results of the protein analysis showed that the loss of an endogenous P25 protein can increase the hEGF production to about 2.2-fold more than normal silkworms. Compared to those of transgenic silkworms with wild type (non-knockout) background, the morphology and secondary structure of cocoon silks were barely changed in transgenic silkworms with a <i>P25</i> gene knockout background, indicating their similar physical properties of cocoon silks. In conclusion, <i>P25</i> gene knockout silkworms may become an efficient bioreactor for the production of exogenous proteins and a promising tool for producing various protein-containing silk biomaterials.
Project description:Many lepidopteran larvae produce silk feeding shelters and cocoons to protect themselves and the developing pupa. As caterpillars evolved, the quality of the silk, shape of the cocoon, and techniques in forming and leaving the cocoon underwent a number of changes. The silk of <i>Pseudoips prasinana</i> has previously been studied using X-ray analysis and classified in the same category as that of <i>Bombyx mori</i>, suggesting that silks of both species have similar properties despite their considerable phylogenetic distance. In the present study, we examined <i>P. prasinana</i> silk using 'omics' technology, including silk gland RNA sequencing (RNA-seq) and a mass spectrometry-based proteomic analysis of cocoon proteins. We found that although the central repetitive amino acid sequences encoding crystalline domains of fibroin heavy chain molecules are almost identical in both species, the resulting fibers exhibit quite different mechanical properties. Our results suggest that these differences are most probably due to the higher content of fibrohexamerin and fibrohexamerin-like molecules in <i>P. prasinana</i> silk. Furthermore, we show that whilst <i>P. prasinana</i> cocoons are predominantly made of silk similar to that of other Lepidoptera, they also contain a second, minor silk type, which is present only at the escape valve.
Project description:The silk of silkworm consists of fibroin fiber coated by sericins. In addition, some nonprotein components were also identified in the sericin fraction. The presence of nonprotein components in the silk has not been well explained. In the present study, methods based on gas chromatography-mass spectrometry were used to identify the metabolites in the cocoon silk from a wild silkworm and two domestic silkworm strains. In total, 45 metabolites were in the cocoon silk, including organic acids, fatty acids, carbohydrates, amino acids, and hydrocarbons. Comparative analyses revealed that 17 metabolites were significant more in the wild silkworm cocoon than in the domestic silkworm cocoon, including three organic acids, three fatty acids, three aldoses, four sugar alcohols, three hydrocarbons, and pyridine. Of them, citric acid in the wild silkworm cocoon is more than 40 times that in the domestic silkworm cocoon, which may have protective value against microbes. The carbohydrate, lipid, and the long-chain hydrocarbons may act as water repellent to make the pupa survive longer in the dry environment. Many metabolites in the cocoon silk may play roles to improve the silk resistance. Lots of nonprotein components were identified in the silk for the first time, providing useful data for understanding the biological function of the cocoon silk.
Project description:Sonic properties of spider silks are measured independent of the web using laser vibrometry and ballistic impact providing insights into Nature's design of functionalized high-performance materials. Through comparison to cocoon silk and other industrial fibers, we find that major ampullate silk has the largest wavespeed range of any known material.
Project description:The transport pathway of specific dietary carotenoids from the midgut lumen to the silk gland in the silkworm, Bombyx mori, is a model system for selective carotenoid transport because several genetic mutants with defects in parts of this pathway have been identified that manifest altered cocoon pigmentation. In the wild-type silkworm, which has both genes, Yellow blood (Y) and Yellow cocoon (C), lutein is transferred selectively from the hemolymph lipoprotein to the silk gland cells where it is accumulated into the cocoon. The Y gene encodes an intracellular carotenoid-binding protein (CBP) containing a lipid-binding domain known as the steroidogenic acute regulatory protein-related lipid transfer domain. Positional cloning and transgenic rescue experiments revealed that the C gene encodes Cameo2, a transmembrane protein gene belonging to the CD36 family genes, some of which, such as the mammalian SR-BI and the fruit fly ninaD, are reported as lipoprotein receptors or implicated in carotenoid transport for visual system. In C mutant larvae, Cameo2 expression was strongly repressed in the silk gland in a specific manner, resulting in colorless silk glands and white cocoons. The developmental profile of Cameo2 expression, CBP expression, and lutein pigmentation in the silk gland of the yellow cocoon strain were correlated. We hypothesize that selective delivery of lutein to specific tissue requires the combination of two components: 1) CBP as a carotenoid transporter in cytosol and 2) Cameo2 as a transmembrane receptor on the surface of the cells.
Project description: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.
Project description:Filippi's glands (FGs), formerly also called Lyonet's glands, are accessory secretory structures of the labial (silk) glands of lepidopteran caterpillars, which were implicated to play an important role in the maturation of the silk material and the construction of the cocoon. In our previous study, we have identified several species of giant silk moths that completely lack the FGs. Interestingly, the absence of FGs in these species correlates with the construction of a loose cocoon architecture. We investigated the functions of FGs by their surgical extirpation in the last instar larvae of the silkworm, <i>Bombyx mori.</i> We found that the absence of FGs altered the structure of the resulting cocoon, in which the different layers of silk were separated. In further experiments, we found no effects of the absence of FGs on larval cocoon formation behavior or on changes in cocoon mass or lipid content. Differential proteomic analysis revealed no significant contribution of structural proteins from FGs to silk cocoon material, but we identified several low abundance proteins that may play a role in posttranslational modifications of some silk proteins. Proteomic analysis also revealed a difference in phosphorylation of the N-terminal sequence of fibroin-heavy chain molecule. Thus, FGs appear to affect silk stickiness during spinning by regulating posttranslational modifications. This could also explain the link that exists between the absence of these glands and the formation of loose cocoons in some giant silk moth species.
Project description:Silkworm, <i>Bombyx mori</i> L., research involves studies on improving strains for enhanced sustainability of high-quality silk production. Several of these have investigated the factors affecting growth and development of silkworm larvae and cocoon characteristics that subsequently affect the yield and quality of silk. The gut microbiota has been reported to impact growth and development of silkworms and has been linked, in particular, with absorption and utilization of nutrients and immunity to diseases. The silkworm strains maintained in the Philippines lack sufficient biological data for use in strain improvement. This prompted efforts to augment the data by profiling bacterial communities through high-throughput 16S rRNA gene amplicon sequencing and analysis in four of the local silkworm strains that are bred and maintained in the country. Results of the study showed that the four silkworm strains are abundant in bacteria that belong to the genera <i>Pseudomonas</i>, <i>Sphingomonas</i>, <i>Delftia</i>, <i>Methylobacterium</i> and <i>Acinetobacter</i>. Results also showed that bacterial diversity and evenness increase as larvae mature, which can be correlated to larval development and shifts in the amount and age of mulberry leaves the larvae consume.
Project description:Fluorescent silk is fundamentally important for the development of future tissue engineering scaffolds. Despite great progress in the preparation of a variety of colored silks, fluorescent silk with enhanced mechanical properties has yet to be explored. In this study, we report on the fabrication of intrinsically super-strong fluorescent silk by feeding Bombyx mori silkworm carbon nanodots (CNDs). The CNDs were incorporated into silk fibroin, hindering the conformation transformation, confining crystallization, and inducing orientation of mesophase. The resultant silk exhibited super-strong mechanical properties with breaking strength of 521.9 ± 82.7 MPa and breaking elongation of 19.2 ± 4.3%, improvements of 55.1% and 53.6%, respectively, in comparison with regular silk. The CNDs-reinforced silk displayed intrinsic blue fluorescence when exposed to 405 nm laser and exhibited no cytotoxic effect on cells, suggesting that multi-functional silks would be potentially useful in bioimaging and other applications.