Microbial Community Changes in Silkworms Suspected of Septicemia and Identification of Serratia sp.

Diseases that occur in silkworms include soft rot, hardening disease, digestive diseases, and sepsis. However, research on the causes of bacterial diseases occurring in silkworms and the resulting changes in the microbial community is lacking. Therefore, we examined the morphological characteristics of sepsis and changes in the microbial community between silkworms that exhibit a unique odor and healthy silkworms; thus, we established a relationship between disease-causing microorganisms and sepsis. After producing a 16S rRNA amplicon library for samples showing sepsis, we obtained information on the microbial community present in silkworms using next-generation sequencing. Compared to that in healthy silkworms, in silkworms with sepsis, the abundance of the Firmicutes phylum was significantly reduced, while that of Proteobacteria was increased. Serratia sp. was dominant in silkworms with sepsis. After bacterial isolation, identification, and reinfection through the oral cavity, we confirmed this organism as the disease-causing agent; its mortality rate was 1.8 times higher than that caused by Serratia marcescens. In summary, we identified a new causative bacterium of silkworm sepsis through microbial community analysis and confirmed that the microbial community balance was disrupted by the aberrant proliferation of certain bacteria.

Customized Integrating-Sphere System for Absolute Color Measurement of Silk Cocoon with Corrugated Microstructure.

Silk fiber, recognized as a versatile bioresource, holds wide-ranging significance in agriculture and the textile industry. During the breeding of silkworms to yield new varieties, optical sensing techniques have been employed to distinguish the colors of silk cocoons, aiming to assess their improved suitability across diverse industries. Despite visual comparison retaining its primary role in differentiating colors among a range of silk fibers, the presence of uneven surface texture leads to color distortion and inconsistent color perception at varying viewing angles. As a result, these distorted and inconsistent visual assessments contribute to unnecessary fiber wastage within the textile industry. To solve these issues, we have devised an optical system employing an integrating sphere to deliver consistent and uniform illumination from all orientations. Utilizing a ColorChecker, we calibrated the RGB values of silk cocoon images taken within the integrating sphere setup. This process accurately extracts the authentic RGB values of the silk cocoons. Our study not only helps in unraveling the intricate color of silk cocoons but also presents a unique approach applicable to various specimens with uneven surface textures.

Molecular diagnosis of 5 silkworm strains endemic to South Korea using single-nucleotide polymorphisms selected from whole-genome sequences.

The domesticated silkworm, Bombyx mori Linnaeus (Lepidoptera: Bombycidae), often poses a challenge in strain identification due to similarities in morphology and genetic background. In South Korea, around 40 silkworm strains are classified as premium, including 5 endemic tri-molting strains: Goryeosammyeon, Sammyeonhonghoeback, Hansammyeon, Sun7ho, and Sandongsammyeon. These strains have potential for breeding programs in response to emerging industry demands, necessitating a reliable strain identification method. In this study, we established a molecular diagnosis approach for these 5 strains. We selected 2-4 single-nucleotide polymorphisms (SNPs) for each strain from whole-genome sequences of 39 strains, encompassing 37 previously studied and 2 newly added. These SNPs were utilized to construct decision trees for each endemic strain identification. The SNPs can be used to distinguish each target strain from the 38 nontarget strains by the tetra-primer amplification refractory mutation system-polymerase chain reaction, with the exception of HMS which needs the addition of PCR-restriction fragment length polymorphism method at the final step. This decision tree-based method using genomic SNPs, coupled with the 2 typing methods, produced consistent and accurate results, providing 100% accuracy. Additionally, the significant number of remaining SNPs identified in this study could be valuable for future diagnosis of the other strains.

Use of a Silkworm (Bombyx mori) Larvae By-Product for the Treatment of Atopic Dermatitis: Inhibition of NF-κB Nuclear Translocation and MAPK Signaling.

Atopic dermatitis (AD) is a long-lasting inflammatory skin disease that contributes to the global health burden and impacts 10-20% of the world's population. In this study, we determined the anti-AD effect of a by-product of silkworm (Bombyx mori) larval powder, strain Yeonnokjam (SLPY), as a sustainable, natural source for the development of therapeutic agents for AD. HaCaT cells were used to assess the in vitro anti-inflammatory activity of SLPY, and a 1-chloro-2,4-dinitrobenzene (DNCB)-induced mouse model was used to study the in vivo anti-AD effects. SLPY treatment downregulated the expression of the inflammatory cytokines TNF-α, IL1ß, IL-8, and Cox-2 in stimulated HaCaT cells. Similarly, the topical application of SLPY in DNCB-treated mice downregulated the expression of inflammatory cytokines and proteins while ameliorating the clinical features of AD. Further, SLPY treatment inhibited the nuclear translocation of NF-κb p65, thereby supporting the efficacy of SLPY in the treatment of AD.

Structural Characteristics and Properties of Cocoon and Regenerated Silk Fibroin from Different Silkworm Strains.

Silk has attracted the attention of researchers as a biomedical and cosmetic material because of its good biocompatibility and cytocompatibility. Silk is produced from the cocoons of silkworms, which have various strains. In this study, silkworm cocoons and silk fibroins (SFs) were obtained from ten silkworm strains, and their structural characteristics and properties were examined. The morphological structure of the cocoons depended on the silkworm strains. The degumming ratio of silk ranged from 22.8% to 28% depending on the silkworm strains. The highest and lowest solution viscosities of SF were shown by 9671 and 9153, respectively, showing a 12-fold difference. The silkworm strains of 9671, KJ5, and I-NOVI showed a two-fold higher work of ruptures for the regenerated SF film than 181 and 2203, indicating that the silkworm strains considerably influence the mechanical properties of the regenerated SF film. Regardless of the silkworm strain, all silkworm cocoons showed good cell viability, making them suitable candidates for advanced functional biomaterials.

Edible Matrix Code with Photogenic Silk Proteins.

Counterfeit medicines are a healthcare security problem, posing not only a direct threat to patient safety and public health but also causing heavy economic losses. Current anticounterfeiting methods are limited due to the toxicity of the constituent materials and the focus of secondary packaging level protections. We introduce an edible, imperceptible, and scalable matrix code of information representation and data storage for pharmaceutical products. This matrix code is digestible as it is composed of silk fibroin genetically encoded with fluorescent proteins produced by ecofriendly, sustainable silkworm farming. Three distinct fluorescence emission colors are incorporated into a multidimensional parameter space with a variable encoding capacity in a format of matrix arrays. This code is smartphone-readable to extract a digitized security key augmented by a deep neural network for overcoming fabrication imperfections and a cryptographic hash function for enhanced security. The biocompatibility, photostability, thermal stability, long-term reliability, and low bit error ratio of the code support the immediate feasibility for dosage-level anticounterfeit measures and authentication features. The edible code affixed to each medicine can serve as serialization, track and trace, and authentication at the dosage level, empowering every patient to play a role in combating illicit pharmaceuticals.

Biocompatible fluorescent silk fibroin bioink for digital light processing 3D printing.

Chemically modified silk fibroin (SF) bioink has been used for three-dimensional (3D) bioprinting in tissue engineering because of its biocompatibility and printability. Also, fluorescent silk fibroin (FSF) from transgenic silkworms has been recently applied in biomedicine because of its fluorescence property. However, the fabrication of fluorescent hydrogel from FSF has not been elucidated. In this study, we showed the fabrication of a digital light processing (DLP) printable bioink from a chemically modified FSF. This bioink was fabricated by covalent conjugation of FSF and glycidyl methacrylate (GMA) and can be printed into various structures, such as the brain, ear, hand, lung, and internal organs. The physical properties of glycidyl methacrylated fluorescent silk fibroin (FSGMA) hydrogel was like the glycidyl methacrylated non-fluorescent silk fibroin (SGMA) hydrogel. The FSGMA hydrogel significantly retains its fluorescence property and has excellent biocompatibility. All these properties make FSGMA hydrogel a potent tool in encapsulated cell tracking and observing the scaffolds' degradation in vivo. This study suggested that our 3D DLP printable FSF bioink could play a promising role in the biomedical field.

Whole-genome sequences of 37 breeding line Bombyx mori strains and their phenotypes established since 1960s.

Bombyx mori is a key insect in the sericulture industry and one of the very important economic animals that are responsible for not only the livelihood of many farmers internationally but also expended biomedical use. The National Institute of Agricultural Sciences of the Rural Development Administration of Korea (NIAS, RDA, Korea) has been collecting silkworm resources with various phenotypic traits from the 1960s and established breeding lines for using them as genetic resources. And these breeding line strains have been used to develop suitable F1 hybrid strains for specific use. In this study, we report the whole-genome sequences of 37 breeding line B. mori strains established over the past 60 years, along with the description of their phenotypic characteristics with photos of developmental stages. In addition, we report the example phenotypic characteristics of the F1-hybrid strain using these breeding line strains. We hope this data will be used as valuable resources to the related research community for studying B. mori and similar other insects.

Phylogeographic Relationships among Bombyx mandarina (Lepidoptera: Bombycidae) Populations and Their Relationships to B. mori Inferred from Mitochondrial Genomes.

We report 37 mitochondrial genome (mitogenome) sequences of Bombyx mori strains (Lepidoptera: Bombycidae) and four of B. mandarina individuals, each preserved and collected, respectively, in South Korea. These mitogenome sequences combined with 45 public data showed a substantial genetic reduction in B. mori strains compared to the presumed ancestor B. mandarina, with the highest diversity detected in the Chinese origin B. mori. Chinese B. mandarina were divided into northern and southern groups, concordant to the Qinling-Huaihe line, and the northern group was placed as an immediate progenitor of monophyletic B. mori strains in phylogenetic analyses, as has previously been detected. However, one individual that was in close proximity to the south Qinling-Huaihe line was exceptional, belonging to the northern group. The enigmatic South Korean population of B. mandarina, which has often been regarded as a closer genetic group to Japan, was most similar to the northern Chinese group, evidencing substantial gene flow between the two regions. Although a substantial genetic divergence is present between B. mandarina in southern China and Japan, a highly supported sister relationship between the two regional populations may suggest the potential origin of Japanese B. mandarina from southern China instead of the Korean peninsula.

Complete mitochondrial genome of the highly fecund Bombyx mori linnaeus, 1758 (Lepidoptera: Bombycidae) strain Jam 146.

To meet the increasing demands of the society in the current era, new strains of the domesticated silkworm Bombyx mori Linnaeus, 1758 (Lepidoptera: Bombycidae) are being continuously bred. Consequently, cataloging the genetic information of pure lines is essential. The strain Jam 146, whose larvae have atypical pale, crescent-shaped body markings, is an important breeding resource due to its excellent fecundity. In this study, we sequenced the mitochondrial genome (mitogenome) of this strain using next-generation sequencing. The complete genome of this strain has a gene arrangement typical of Lepidoptera. The length of the Jam 146 mitogenome (15,661 bp) is well within the range reported in other B. mori strains, i.e. between 15,629 (Baiyun strain, China) and 15,676 bp (Hukpyobeom strain, South Korea). However, the total length of protein-coding genes, 3,733 codons in Jam 146 and two other silkworm strains previously reported from South Korea, is 13 codons longer than that in other B. mori strains. Phylogenetic analysis of 22 silkworm strains from nine countries showed that the Jam 146 strain forms a strong cluster with three other strains from China, Japan, and South Korea, suggesting that after their split from a common ancestor, the evolutionary divergence among the silkworm strains in these countries has been limited.

Bombyx mori kynurenine 3-monooxygenase gene editing and insect molecular breeding using the clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 system.

Genome editing by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas)9, a third-generation gene scissors, and molecular breeding at the genome level are attracting considerable attention as future breeding techniques. In the present study, genetic and phenotypic analyses were conducted to examine the molecular breeding of Bombyx mori through CRISPR/Cas9-mediated editing of the kynurenine 3-monooxygenase (KMO) gene. The synthesized guide RNAs (gRNAs) were analyzed using T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA, and Cas9 complexes were microinjected into silkworm embryos. After microinjection, the hatching rate and the incidence of mutation were determined as 18.1% and 60%, respectively. Gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed; however, certain embryos and moths produced through sib-mating had significant differences compared to the wild-type. In successive generations, a distinct phenotypic change was also observed by continuous mating. Thus, although there are limitations in the phenotypic expression in breeding through the induction of deletion mutations, as in the present study, the process is believed to yield successful results within a shorter period compared to traditional breeding and is safer than transgenic technology.

Photoelectric Silk via Genetic Encoding and Bioassisted Plasmonics.

Genetically encoded photoelectric silk that can convert photons to electrons (light to electricity) over a wide visible range in a self-power mode is reported. As silk is a versatile host material with electrical conductivity, biocompatibility, and processability, a photoelectric protein is genetically fused with silk by silkworm transgenesis. Specifically, mKate2, which is conventionally known as a far-red fluorescent protein, is used as a photoelectric protein. Characterization of the electrochemical and optical properties of mKate2 silk allows designing a photoelectric measurement system. A series of in situ photocurrent experiments support the sensitive and stable performance of photoelectric conversion. In addition, as a plasmonic nanomaterial with a broad spectral resonance, titanium nitride (TiN) nanoparticles are biologically hybridized into the silk glands, taking full advantage of the silkworms' open circulatory system as well as the absorption band of mKate2 silk. This biological hybridization via direct feeding of TiN nanoparticles further enhances the overall photoelectric conversion ability of mKate2 silk. It is envisioned that the biologically derived photoelectric protein, its ecofriendly scalable production by transgenic silkworms, and the bioassisted plasmonic hybridization can potentially broaden the biomaterial choices for developing next-generation biosensing, retina prosthesis, and neurostimulation applications.

Edible unclonable functions.

Counterfeit medicines are a fundamental security problem. Counterfeiting medication poses a tremendous threat to patient safety, public health, and the economy in developed and less developed countries. Current solutions are often vulnerable due to the limited security levels. We propose that the highest protection against counterfeit medicines would be a combination of a physically unclonable function (PUF) with on-dose authentication. A PUF can provide a digital fingerprint with multiple pairs of input challenges and output responses. On-dose authentication can verify every individual pill without removing the identification tag. Here, we report on-dose PUFs that can be directly attached onto the surface of medicines, be swallowed, and digested. Fluorescent proteins and silk proteins serve as edible photonic biomaterials and the photoluminescent properties provide parametric support of challenge-response pairs. Such edible cryptographic primitives can play an important role in pharmaceutical anti-counterfeiting and other security applications requiring immediate destruction or vanishing features.

Esophageal Perforation after Anterior Cervical Spine Surgery.

STUDY DESIGN: Retrospective case analyses. PURPOSE: To investigate the causes, diagnosis, and management of esophageal perforation, depending on the time of diagnosis. OVERVIEW OF LITERATURE: To date, few studies have addressed these issues. METHODS: A total of seven patients were included in this study. The patients were classified into three groups based on esophageal perforation diagnosis time: intraoperative (diagnosed during surgery), perioperative (diagnosed within 30 days postoperatively), and delayed (diagnosed >30 days postoperatively) groups. RESULTS: In the intraoperative group (N=2), infectious spondylitis was the main cause of esophageal perforation. Anterior plate and screw removal, followed by posterior instrumentation, was performed. The injured esophagus was managed by omentum flap repair in one patient and primary repair in one patient. In the perioperative group (N=2), revision surgery for infection and metal failure were the main causes of esophageal perforation. In both cases, food residue was drained on the third postoperative day. The injured esophagus was managed conservatively. In the delayed group (N=3), chronic irritation caused by metal failure was the main cause of esophageal perforation. In all patients, there was no associated infection. The anterior instrumentation was removed, and the two patients were treated by primary repair, and one patient was treated using sternocleidomastoid muscle flap. One patient in intraoperative group died of sepsis. CONCLUSIONS: The main cause of intraoperative esophageal perforation was esophageal adhesions because of infectious spondylitis. However, perioperative and delayed esophageal perforations were caused by chronic irritation because of metal failure. Anterior plate and screw removal was necessary, and posterior instrumentation and fusion may be considered, depending on the fusion status.

Complete mitochondrial genome of the silkworm strain, Chilseongjam Bombyx mori (Lepidoptera: Bombycidae), with a unique larval body marking.

Recently, a new silkworm strain with a peculiar larval marking and rare cocoon colour was bred in Korea for educational learning and exhibition. In order to obtain the genetic information of the newly bred strain, Chilseongjam Bombyx mori (Lepidoptera: Bombycidae), its complete mitochondrial genome (mitogenome) was sequenced. The mitogenome is 15,660 bp in length, contains a typical set of genes, and has gene arrangement and composition typical of Lepidoptera. However, the Chilseongjam strain mitogenome is 4-36 bp longer than 19 other strains originating from other countries and 16 bp shorter than the whole genome of a Korean Hukpyobeom strain. In particular, the Chilseongjam strain has an intergenic spacer sequence that is shorter than that of the Hukpyobeom strain at the tRNAHis and ND4 junction as it has fewer microsatellite-like AT repeats. Phylogenetic analyses conducted using a total of 21 silkworm strains originating from nine countries revealed a few subgroups with moderate-to-high nodal support (80-94%). The Korean Chilseongjam strain formed a relatively strong subgroup (85%) with a Japanese strain (J106) instead of the Korean Hukpyobeom strain.

Green-Light-Activated Photoreaction via Genetic Hybridization of Far-Red Fluorescent Protein and Silk.

Fluorescent proteins often result in phototoxicity and cytotoxicity, in particular because some red fluorescent proteins produce and release reactive oxygen species (ROS). The photogeneration of ROS is considered as a detrimental side effect in cellular imaging or is proactively utilized for ablating cancerous tissue. As ancient textiles or biomaterials, silk produced by silkworms can directly be used as fabrics or be processed into materials and structures to host other functional nanomaterials. It is reported that transgenic fusion of far-red fluorescent protein (mKate2) with silk provides a photosensitizer hybridization platform for photoinducible control of ROS. Taking advantage of green (visible) light activation, native and regenerated mKate2 silk can produce and release superoxide and singlet oxygen, in a comparable manner of visible light-driven plasmonic photocatalysis. Thus, the genetic expression of mKate2 in silk offers immediately exploitable and scalable photocatalyst-like biomaterials. It is further envisioned that mKate2 silk can potentially rule out hazardous concerns associated with foreign semiconductor photocatalytic nanomaterials.

Publisher Correction: Anderson light localization in biological nanostructures of native silk.

The original PDF version of this Article contained errors in Equations 1 and 2. Both equations omitted all Γ terms. This has been corrected in the PDF version of the Article. The HTML version was correct from the time of publication.

Anderson light localization in biological nanostructures of native silk.

Light in biological media is known as freely diffusing because interference is negligible. Here, we show Anderson light localization in quasi-two-dimensional protein nanostructures produced by silkworms (Bombyx mori). For transmission channels in native silk, the light flux is governed by a few localized modes. Relative spatial fluctuations in transmission quantities are proximal to the Anderson regime. The sizes of passive cavities (smaller than a single fibre) and the statistics of modes (decomposed from excitation at the gain-loss equilibrium) differentiate silk from other diffusive structures sharing microscopic morphological similarity. Because the strong reflectivity from Anderson localization is combined with the high emissivity of the biomolecules in infra-red radiation, silk radiates heat more than it absorbs for passive cooling. This collective evidence explains how a silkworm designs a nanoarchitectured optical window of resonant tunnelling in the physically closed structures, while suppressing most of transmission in the visible spectrum and emitting thermal radiation.

Genome sequence of the Japanese oak silk moth, Antheraea yamamai: the first draft genome in the family Saturniidae.

Background: Antheraea yamamai, also known as the Japanese oak silk moth, is a wild species of silk moth. Silk produced by A. yamamai, referred to as tensan silk, shows different characteristics such as thickness, compressive elasticity, and chemical resistance compared with common silk produced from the domesticated silkworm, Bombyx mori. Its unique characteristics have led to its use in many research fields including biotechnology and medical science, and the scientific as well as economic importance of the wild silk moth continues to gradually increase. However, no genomic information for the wild silk moth, including A. yamamai, is currently available. Findings: In order to construct the A. yamamai genome, a total of 147G base pairs using Illumina and Pacbio sequencing platforms were generated, providing 210-fold coverage based on the 700-Mb estimated genome size of A. yamamai. The assembled genome of A. yamamai was 656 Mb (>2 kb) with 3675 scaffolds, and the N50 length of assembly was 739 Kb with a 34.07% GC ratio. Identified repeat elements covered 37.33% of the total genome, and the completeness of the constructed genome assembly was estimated to be 96.7% by Benchmarking Universal Single-Copy Orthologs v2 analysis. A total of 15 481 genes were identified using Evidence Modeler based on the gene prediction results obtained from 3 different methods (ab initio, RNA-seq-based, known-gene-based) and manual curation. Conclusions: Here we present the genome sequence of A. yamamai, the first genome sequence of the wild silk moth. These results provide valuable genomic information, which will help enrich our understanding of the molecular mechanisms relating to not only specific phenotypes such as wild silk itself but also the genomic evolution of Saturniidae.

Visible light biophotosensors using biliverdin from Antheraea yamamai.

We report an endogenous photoelectric biomolecule and demonstrate that such a biomolecule can be used to detect visible light. We identify the green pigment abundantly present in natural silk cocoons of Antheraea yamamai (Japanese oak silkmoth) as biliverdin, using mass spectroscopy and optical spectroscopy. Biliverdin extracted from the green silk cocoons generates photocurrent upon light illumination with distinct colors. We further characterize the basic performance, responsiveness, and stability of the biliverdin-based biophotosensors at a photovoltaic device level using blue, green, orange, and red light illumination. Biliverdin could potentially serve as an optoelectric biomolecule toward the development of next-generation implantable photosensors and artificial photoreceptors.