Silk protein of the larval caddisfly, Sienopsyche marmorata (trichoptera: stenopsychidae)(trichoptera: stenopsychidae)

Stenopsyche marmorata is one of the common caddisfly species inrivers and streamsin Japan which is distributed in East Palaearctic Region･ The larvae spin Silk fiber between stones on the bottom of Bowing water, the silk proteinofS･ marmorata would be one of the best sources of natural biopolymersthat could be used as materials for aquatic industrial purposes･ In order to analyze the basic characteristics towards further applications, we measured physical properties, thermal behaviorsand molecular confわrmation of the silk protein of larval S marmorata･ The wet weight of the larval body andthe silkgland of SOrata Were 543･5土5419and 44･7土17･9 mg, respectively, The water content of the liquid silk protein in the silkglands of the living larvae was 73.4土2.2%. The elongation atthe breaking point of the solid silk proteingland in dry state was 2･2%, whilethat of sampleinwet state was･ 6012%, suggestlngthe silk protein glands become soft and stretchable in wet conditions. FT-IR spectra of the silk protein film showed a major absorption band at 1650 cm-1 (Amide I band), which is attributed to the random coil molecular conformation. The silk fiber showed a major endothemiCpeak at 32loC onthe differential scanning calorimetry (DSC) curve, which position is slightly higher than the endothemic peak for the silk film, Suggesting an increaseinthe thermal stabilityfbr the silk fiber from caddisfly,Article信州大学山地水環境教育研究センター研究報告 6: 1-8(2010)departmental bulletin pape

Flight density of aquatic insect fauna over river water surface in the middle reaches of the Shinano River, mainly among Caddisflies (Trichoptera).

We focused on the relative number of flying adult caddisflies on the river surface captured by sticky board traps with the aim of elucidating differences in the distribution pattern of caddisfly larvae along the slope in the middle reaches of the Shinano River. The individual number of adult caddisflies caught increased from April and decreased from October. Even in the same middle reaches of a river, there was a large difference geographically in the species captured. Hydroptila sp. was caught mainly downstream of the Taishyobashi Bridge, Psychomyia acutipennis (Ulmer 1908) in the vicinity of the Taishyobashi Bridge, and Stenopsyche marmorata Navás 1920 upstream of the Awasabashi Bridge. It is known that the slope of the Shinano River bed suddenly becomes less and the flow rate slower in the area from the Taishyobashi Bridge to the Awasabashi Bridge, and it was shown that the species composition and number of aquatic insects caught changes with the change in the slope of the river bed.ArticleZoosymposia.10:203-213(2016)journal articl

Nanocomposite of silk fibroin nanofiber and montmorillonite: Fabrication and morphology

The purpose of our research is creating a new nanocomposite material. Generally silk fibroin (SF) is regarded as a promising base material for biomedical uses. The incorporation of montmorillonite (MMT) into SF fibers would improve physical properties of the SF fibers. We investigated a new method of combining electospun SF with MMT. Specifically, electrospun silk nanofibers were treated with methanol and dipped in a MMT suspension. We could obtain a nanosheet composite of silk nanofibers and MMT. Their ultrastructures were successfully visualized by high resolution transmission electron microscopy. This compound was comprised of individual silk nanofibers surrounded by thin layers of MMT, each with a thickness of about 1.2 nm. This structure was confirmed by elemental analysis. We also performed IR, NMR and X-ray diffraction analyses in conjunction with morphological data. Conclusively we obtained a new composite of silk nanofiber and MMT, which has never been reported. Using this unique nanocomposite biological tests of its application for a scaffold for tissue engineering are under way.ArticleINTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES. 57:124-128 (2013)journal articl

Molecular cloning, gene expression analysis, and recombinant protein expression of novel silk proteins from larvae of a retreat-maker caddisfly, Stenopsyche marmorata

Retreat-maker larvae of Stenopsyche marmorata, one of the major caddisfly species in Japan, produce silk threads and adhesives to build food capture nets and protective nests in water. Research on these underwater adhesive silk proteins potentially leads to the development of new functional biofiber materials. Recently, we identified four major S. marmorata silk proteins (Smsps), Smsp-1, Smsp-2, Smsp-3, and Smsp-4 from silk glands of S. marmorata larvae. In this study, we cloned full-length cDNAs of Smsp-2, Smsp-3, and Smsp-4 from the cDNA library of the S. marmorata silk glands to reveal the primary sequences of Smsps. Homology search results of the deduced amino acid sequences indicate that Smsp-2 and Smsp-4 are novel proteins. The Smsp-2 sequence [167 amino acids (aa)] has an array of GYD-rich repeat motifs and two (SX)(4)E motifs. The Smsp-4 sequence (132 aa) contains a number of GW-rich repeat motifs and three (SX)(4)E motifs. The Smsp-3 sequence (248 aa) exhibits high homology with fibroin light chain of other caddisflies. Gene expression analysis of Smsps by real-time PCR suggested that the gene expression of Smsp-1 and Smsp-3 was relatively stable throughout the year, whereas that of Smsp-2 and Smsp-4 varied seasonally. Furthermore, Smsps recombinant protein expression was successfully performed in Escherichia coli. The study provides new molecular insights into caddisfly aquatic silk and its potential for future applications. (C) 2015 Elsevier Inc. All rights reserved.ArticleBIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 464(3):814-819 (2015)journal articl

Characterization of silk gland ribosomes from a bivoltine caddisfly, Stenopsyche marmorata: translational suppression of a silk protein in cold conditions

Larval Stenopsyche marmorata constructs food capture nets and fixed retreats underwater using self-produced proteinaceous silk fibers. In the Chikuma River (Nagano Prefecture, Japan) S. marmorata has a bivoltine life cycle; overwintering larvae grow slowly with reduced net spinning activity in winter. We recently reported constant transcript abundance of S. marmorata silk protein 1 (Smsp-1), a core S. marmorata silk fiber component, in all seasons, implying translational suppression in the silk gland during winter. Herein, we prepared and characterized silk gland ribosomes from seasonally collected S. marmorata larvae. Ribosomes from silk glands immediately frozen in liquid nitrogen (LN2) after dissection exhibited comparable translation elongation activity in spring, summer, and autumn. Conversely, silk glands obtained in winter did not contain active ribosomes and Smsp-1. Ribosomes from silk glands immersed in ice-cold physiological saline solution for approximately 4 h were translationally inactive, despite summer collection and Smsp-1 expression. The ribosomal inactivation occurs because of defects in the formation of 80S ribosomes, presumably due to splitting of 60S subunits containing 28S rRNA with central hidden break, in response to cold stress. These results suggest a novel-type ribosome-regulated translation control mechanism.ArticleBiochemical and Biophysical Research Communications.469(2):210-215(2016)journal articl

Stability toward alkaline hydrolysis of B. mori silk fibroin grafted with methacrylamide

Bombyx mori silk fibroin fibers were grafted with methacrylamide (MAA) and characterized by Raman and infrared (IR) vibrational spectroscopy before and after hydrolysis in NaOH 5% to elucidate the possible interactions between the two components and the stability of the fibers toward alkaline hydrolysis. Upon grafting, the fibers underwent conformational rearrangements toward a more unordered state and lost orientation at weight gains higher than 60%. Vibrational spectroscopy disclosed the occurrence of intermolecular interactions (mainly hydrogen bonds) between B. mori silk fibroin and polyMAA in the grafted fibers, and the formation of covalent bonds has been explored. These strong interactions made the grafted fibers as a whole more stable toward alkaline hydrolysis because they prevented the solubilization of the polymer upon hydrolysis and made slower the transformation of its CONH2 groups into COOH and COO− groups. Upon hydrolysis, silk fibroin underwent an enrichment in the β-sheet crystalline domains, because of the preferential removal of the unordered domains, which were more prone to the OH− attack. IR and Raman spectroscopy proved valid techniques to investigate the degradation mechanism and kinetics of grafted silk fibroin fibers and so for designing high-performing silk-based materials. The A731/A1004 Raman intensity ratio was proposed to spectroscopically evaluate the composition of the grafted samples; its value was found to linearly increase with weight gain (R2 = 0.998), envisaging the possibility of using Raman spectroscopy as a routine analytical technique for qualitative and quantitative characterization of grafted industrial samples. Copyright © 2016 John Wiley & Sons, Ltd

Influence of grafting with acrylate compounds on the conformational rearrangements of silk fibroin upon electrospinning and treatment with aqueous methanol

Silk fabrics from Bombyx mori silkworm were grafted with 2-hydroxyethyl methacrylate (HEMA) as well as a binary system of HEMA and 4-hydroxybutyl acrylate (HBA) and then analysed by Raman and infrared (IR) spectroscopy to elucidate the interactions between the components and their possible conformational changes. The samples were then dissolved in trifluoroacetic acid and electrospun; the influence of the grafted polymers on the silk fibroin rearrangements upon these treatments was investigated by vibrational spectroscopy. Upon grafting, the fabrics underwent conformational rearrangements towards a more unordered state, although they kept their prevailing β-sheet conformation; also the polymeric component underwent hydrogen bonding and backbone rearrangements upon interaction with silk fibroin and the occurrence of strong covalent bonds cannot be excluded. By immersing the as-electrospun grafted and pure fibroin nanofibres (prevalently unordered) in aqueous methanol, they partially recovered the β-sheet content observed in the corresponding starting fabrics; the percentage of recovery decreased along the series: pure silk > HEMA-grafted silk > HEMA and HBA-grafted silk. This trend suggests that the presence of the polyHEMA grafted component hinders the silk fibroin recrystallization into β-sheet upon aqueous methanol treatment; moreover, the addition of the more sterically hindered HBA monomer in the grafting system further prevented this process. Copyright © 2016 John Wiley & Sons, Ltd