Interactions between fibroin and sericin proteins from Antheraea pernyi and Bombyx mori silk fibers.

PubMed

Du, Shan; Zhang, Jin; Zhou, Wei T; Li, Quan X; Greene, George W; Zhu, Hai J; Li, Jing L; Wang, Xun G

2016-09-15

Silkworm silk fibers are core-shell composites of fibroin and sericin proteins. Studying the interactions between fibroin and sericin is essential for understanding the properties of these composites. It is observed that compared to the domestic silk cocoon Bombyx mori (B. mori), the adhesion between fibroin and sericin from the wild silk cocoon, Antheraea pernyi (A. pernyi), is significantly stronger with a higher degree of heterogeneity. The adsorption of A. pernyi sericin on its fibroin is almost twice the value for B. mori sericin on fibroin, both showing a monolayer Langmuir adsorption. (1)H NMR and FTIR studies demonstrate on a molecular level the stronger interactions and the more intensive complex formation between A. pernyi fibroin and sericin, facilitated by the hydrogen bonding between glycine and serine. The findings of this study may help the design of composites with superior interfacial adhesion between different components. Copyright © 2016 Elsevier Inc. All rights reserved.

Traffic Aware Strategic Aircrew Requests (TASAR) Analysis and Development

NASA Technical Reports Server (NTRS)

Woods, Sharon E.

2016-01-01

This document is the final report and deliverable 30 of Contract No. NNL12AA06C, the Traffic Aware Strategic Aircrew Requests (TASAR) contract awarded via the NASA Research Announcement (NRA). It documents the accomplishments of the contract, the evolution of its role in the overall TASAR project, and lessons learned from its execution.

Traffic Aware Strategic Aircrew Requests (TASAR)

NASA Technical Reports Server (NTRS)

Ballin, Mark G.; Wing, David J.

2012-01-01

Under Instrument Flight Rules, pilots are not permitted to make changes to their approved trajectory without first receiving permission from Air Traffic Control (ATC). Referred to as "user requests," trajectory change requests from aircrews are often denied or deferred by controllers because they have awareness of traffic and airspace constraints not currently available to flight crews. With the introduction of Automatic Dependent Surveillance-Broadcast (ADS-B) and other information services, a rich traffic, weather, and airspace information environment is becoming available on the flight deck. Automation developed by NASA uses this information to aid flight crews in the identification and formulation of optimal conflict-free trajectory requests. The concept of Traffic Aware Strategic Aircrew Requests (TASAR) combines ADS-B and airborne automation to enable user-optimal in-flight trajectory replanning and to increase the likelihood of ATC approval for the resulting trajectory change request. TASAR may improve flight efficiency or other user-desired attributes of the flight while not impacting and potentially benefiting the air traffic controller. This paper describes the TASAR concept of operations, its enabling automation technology which is currently under development, and NASA s plans for concept assessment and maturation.

Comparative genomics of parasitic silkworm microsporidia reveal an association between genome expansion and host adaptation

PubMed Central

2013-01-01

Background Microsporidian Nosema bombycis has received much attention because the pébrine disease of domesticated silkworms results in great economic losses in the silkworm industry. So far, no effective treatment could be found for pébrine. Compared to other known Nosema parasites, N. bombycis can unusually parasitize a broad range of hosts. To gain some insights into the underlying genetic mechanism of pathological ability and host range expansion in this parasite, a comparative genomic approach is conducted. The genome of two Nosema parasites, N. bombycis and N. antheraeae (an obligatory parasite to undomesticated silkworms Antheraea pernyi), were sequenced and compared with their distantly related species, N. ceranae (an obligatory parasite to honey bees). Results Our comparative genomics analysis show that the N. bombycis genome has greatly expanded due to the following three molecular mechanisms: 1) the proliferation of host-derived transposable elements, 2) the acquisition of many horizontally transferred genes from bacteria, and 3) the production of abundnant gene duplications. To our knowledge, duplicated genes derived not only from small-scale events (e.g., tandem duplications) but also from large-scale events (e.g., segmental duplications) have never been seen so abundant in any reported microsporidia genomes. Our relative dating analysis further indicated that these duplication events have arisen recently over very short evolutionary time. Furthermore, several duplicated genes involving in the cytotoxic metabolic pathway were found to undergo positive selection, suggestive of the role of duplicated genes on the adaptive evolution of pathogenic ability. Conclusions Genome expansion is rarely considered as the evolutionary outcome acting on those highly reduced and compact parasitic microsporidian genomes. This study, for the first time, demonstrates that the parasitic genomes can expand, instead of shrink, through several common molecular mechanisms

Nucleopolyhedroviruses (NPV) induce the expression of small heat shock protein 25.4 in Antheraea pernyi.

PubMed

Zhang, Congfen; Zhu, Baojian; Dai, Li Shang; Liu, Chaoliang; Luo, Xuegang

2016-10-15

Nucleopolyhedroviruses (NPVs) is one group of Baculoviruses. The infection of NPV in silkworm is often lethal. To investigate the effective measures to stop the infection of NPV, we cloned cDNA encoding small heat shock protein 25.4 in Antheraea pernyi (Ap-HSP25.4). The translated amino acid sequence consisted of 223 residues with a calculated molecular mass of 25.4kDa and an isoelectronic point (pI) of 4.93. Quantitative real-time PCR was used to investigate the expression patterns and distribution profiles of Ap-sHSP25.4 before and after challenged with NPV. We found that the inhibitors of eicosanoid synthesis could suppress the transcription of Ap-sHSP25.4 in the fat body in a dose dependent manner. And arachidonic acid induced the expression of Ap-sHSP25.4. Thus, we concluded that sHSPs may be promising candidates to boost insect immunity in practice. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of Operational Hazards and Safety Requirements for Traffic Aware Strategic Aircrew Requests (TASAR)

NASA Technical Reports Server (NTRS)

Koczo, Stefan, Jr.

2013-01-01

Safety analyses of the Traffic Aware Strategic Aircrew Requests (TASAR) Electronic Flight Bag (EFB) application are provided to establish its Failure Effects Classification which affects certification and operational approval requirements. TASAR was developed by NASA Langley Research Center to offer flight path improvement opportunities to the pilot during flight for operational benefits (e.g., reduced fuel, flight time). TASAR, using own-ship and network-enabled information concerning the flight and its environment, including weather and Air Traffic Control (ATC) system constraints, provides recommended improvements to the flight trajectory that the pilot can choose to request via Change Requests to ATC for revised clearance. This study reviews the Change Request process of requesting updates to the current clearance, examines the intended function of TASAR, and utilizes two safety assessment methods to establish the Failure Effects Classification of TASAR. Considerable attention has been given in this report to the identification of operational hazards potentially associated with TASAR.

An Operational Safety and Certification Assessment of a TASAR EFB Application

NASA Technical Reports Server (NTRS)

Koczo, Stefan; Wing, David

2013-01-01

This paper presents an overview of a Traffic Aware Strategic Aircrew Requests (TASAR) Electronic Flight Bag application intended to inform the pilot of trajectory improvement opportunities while en route that result in operational benefits. The results of safety analyses and a detailed review of Federal Aviation Administration (FAA) regulatory documents that establish certification and operational approval requirements are presented for TASAR. The safety analyses indicate that TASAR has a likely Failure Effects Classification of “No Effect,” and at most, is no worse than “Minor Effect.” Based on this safety assessment and the detailed review of FAA regulatory documents that determine certification and operational approval requirements, this study concludes that TASAR can be implemented in the flight deck as a Type B software application hosted on a Class 2 Portable Electronic Device (PED) Electronic Flight Bag (EFB). This implementation approach would provide a relatively low-cost path to certification and operational approval for both retrofit and forward fit implementation, while at the same time facilitating the business case for early ADS-B IN equipage. A preliminary review by FAA certification and operational approvers of the analyses presented here confirmed that the conclusions are appropriate and that TASAR will be considered a Type B application.

Study on impact of parasite (Nosema species) on characters of tropical tasar silkworm Anthereae mylitta drury.

PubMed

Velide, Lakshmi; Bhagavanulu, M V K; Rao, A Purushotham

2013-01-01

Infection of the pebrine disease has been found to be highly virulent and harm the cocoon yield as well as characters of silkworm Anthereae mylitta. Therefore, an attempt was made to evaluate the impact of parasite Nosema species on the ecorace (Sukinda) of A. mylitta in respect of transovarial transmitted (T1), secondary infection (T2) and healthy silkworm (T3). In comparison to T3, the number of larval mortality was 16 and 11 in T1 and T2 respectively; whereas as number of pupal mortality was 6 and 5 in T1 and T2 respectivelyThe larval weight, number of moths emerged, number of eggs laid and percent hatchability were reduced in T1 and T2 in comparison to T3.The infected layings were high in T1 (51%) and T2 (42%) as against T3 (0%). Similarly, the infected moths were 34% in T1 and 15% in T2 as against 0 percent in T3. All the characteristics parameters of cocoon were reduced in T1 and T2 against T3. The study explains that there was no significant variation between T1 and T2 on different parameters of larva, pupa and cocoon characters.

Cloning and sequence analysis of the Antheraea pernyi nucleopolyhedrovirus gp64 gene.

PubMed

Wang, Wenbing; Zhu, Shanying; Wang, Liqun; Yu, Feng; Shen, Weide

2005-12-01

Frequent outbreaks of the purulence disease of Chinese oak silkworm are reported in Middle and Northeast China. The disease is produced by the pathogen Antheraea pernyi nucleopolyhedrovirus (AnpeNPV). To obtain molecular information of the virus, the polyhedra of AnpeNPV were purified and characterized. The genomic DNA of AnpeNPV was extracted and digested with HindIII. The genome size of AnpeNPV is estimated at 128 kb. Based on the analysis of DNA fragments digested with HindIII, 23 fragments were bigger than 564 bp. A genomic library was generated using HindIII and the positive clones were sequenced and analysed. The gp64 gene, encoding the baculovirus envelope protein GP64, was found in an insert. The nucleotide sequence analysis indicated that the AnpeNPV gp64 gene consists of a 1,530 nucleotide open reading frame (ORF), encoding a protein of 509 amino acids. Of the eight gp64 homologues, the AnpeNPV gp64 ORF shared the most sequence similarity with the gp64 gene of Anticarsia gemmatalis NPV, but not Bombyx mori NPV. The upstream region of the AnpeNPV gp64 ORF encoded the conserved transcriptional elements for early and late stage of the viral infection cycle. These results indicated that AnpeNPV belongs to group I NPV and was far removed in molecular phylogeny from the BmNPV.

Strain Rate and Anisotropic Microstructure Dependent Mechanical Behaviors of Silkworm Cocoon Shells

PubMed Central

Xu, Jun; Zhang, Wen; Gao, Xiang; Meng, Wanlin; Guan, Juan

2016-01-01

Silkworm cocoons are multi-layered composite structures comprised of high strength silk fiber and sericin, and their mechanical properties have been naturally selected to protect pupas during metamorphosis from various types of external attacks. The present study attempts to gain a comprehensive understanding of the mechanical properties of cocoon shell materials from wild silkworm species Antheraea pernyi under dynamic loading rates. Five dynamic strain rates from 0.00625 s-1 to 12.5 s-1 are tested to show the strain rate sensitivity of the cocoon shell material. In the meantime, the anisotropy of the cocoon shell is considered and the cocoon shell specimens are cut along 0°, 45° and 90° orientation to the short axis of cocoons. Typical mechanical properties including Young’s modulus, yield strength, ultimate strength and ultimate strain are extracted and analyzed from the stress-strain curves. Furthermore, the fracture morphologies of the cocoon shell specimens are observed under scanning electron microscopy to help understand the relationship between the mechanical properties and the microstructures of the cocoon material. A discussion on the dynamic strain rate effect on the mechanical properties of cocoon shell material is followed by fitting our experimental results to two previous models, and the effect could be well explained. We also compare natural and dried cocoon materials for the dynamic strain rate effect and interestingly the dried cocoon shells show better overall mechanical properties. This study provides a different perspective on the mechanical properties of cocoon material as a composite material, and provides some insight for bio-inspired engineering materials. PMID:26939063

Enhanced osteogenic potential of human mesenchymal stem cells on electrospun nanofibrous scaffolds prepared from eri-tasar silk fibroin.

PubMed

Panda, Niladri Nath; Biswas, Amit; Pramanik, Krishna; Jonnalagadda, Sriramakamal

2015-07-01

This study evaluated the mechanical properties and osteogenic potential of a silk fibroin scaffold prepared from a 70:30 blend of Eri (Philosamia ricini) and Tasar (Antheraea mylitta) silk, respectively (ET scaffolds). An electrospinning process was used to prepare uniformly blended, fibrous scaffolds of nanoscale dimensions, as confirmed by scanning and transmission electron microscopy (fiber diameter < 300 nm). Similarly prepared scaffolds derived from gelatin and Bombyx mori (BM) silk fibroin were used as controls. Mechanical testing and atomic force microscopy showed that the ET scaffolds had significantly higher tensile strength (1.83 ± 0.13 MPa) and surface roughness (0.44 μm) compared with BM (1.47 ± 0.10 MPa; 0.37 μm) and gelatin scaffolds (0.6 ± 0.07 MPa; 0.28 μm). All scaffolds were exposed to mesenchymal stem cells isolated to human chord blood (hMSCs) for up to 28 days in vitro. Alamar blue and alkaline phosphatase assay showed greater attachment and proliferation for both ET and BM scaffolds compared with gelatin. The ET scaffolds also promoted greater differentiation of the attached hMSCs as evidenced by higher expression of RunX2, osteocalcin, and CD29/CD44 expression. ET scaffolds also showed significantly higher mineralization, as evidenced by glycosaminoglycan assay, alizarin red staining, and elemental analysis of crystalline composites isolated from the scaffolds. © 2014 Wiley Periodicals, Inc.

Molecular characterization of an Apolipophorin-III gene from the Chinese oak silkworm, Antheraea pernyi (Lepidoptera: Saturniidae).

PubMed

Liu, Qiu-Ning; Lin, Kun-Zhang; Yang, Lin-Nan; Dai, Li-Shang; Wang, Lei; Sun, Yu; Qian, Cen; Wei, Guo-Qing; Liu, Dong-Ran; Zhu, Bao-Jian; Liu, Chao-Liang

2015-03-01

Apolipophorin-III (ApoLp-III) acts in lipid transport, lipoprotein metabolism, and innate immunity in insects. In this study, an ApoLp-III gene of Antheraea pernyi pupae (Ap-ApoLp-III) was isolated and characterized. The full-length cDNA of Ap-ApoLp-III is 687 bp, including a 5'-untranslated region (UTR) of 40 bp, 3'-UTR of 86 bp and an open reading frame of 561 bp encoding a polypeptide of 186 amino acids that contains an Apolipophorin-III precursor domain (PF07464). The deduced Ap-apoLp-III protein sequence has 68, 59, and 23% identity with its orthologs of Manduca sexta, Bombyx mori, and Aedes aegypti, respectively. Phylogenetic analysis showed that the Ap-apoLp-III was close to that of Bombycoidea. qPCR analysis revealed that Ap-ApoLp-III expressed during the four developmental stages and in integument, fat body, and ovaries. After six types of microorganism infections, expression levels of the Ap-ApoLp-III gene were upregulated significantly at different time points compared with control. RNA interference (RNAi) of Ap-ApoLp-III showed that the expression of Ap-ApoLp-III was significantly downregulated using qPCR after injection of E. coli. We infer that the Ap-ApoLp-III gene acts in the innate immunity of A. pernyi. © 2014 Wiley Periodicals, Inc.

Flight Test Assessments of Pilot Workload, System Usability, and Situation Awareness of TASAR

NASA Technical Reports Server (NTRS)

Burke, Kelly A.; Haynes, Mark A.

2016-01-01

Traffic Aware Strategic Aircrew Requests (TASAR) is an onboard automation concept intended to identify trajectory optimizations, in terms of fuel and time saving objectives, clear of known traffic, weather, and airspace restrictions prior to the aircrew initiating a route-change request to Air Traffic Control (ATC). The software implementation of the TASAR concept is the Traffic Aware Planner (TAP). TASAR analysis and development is being executed by the NASA Langley Research Center's Crew Systems and Aviation Operations Branch (CSAOB) under the sponsorship of the Airspace Technology Demonstration (ATD) Project of the NASA Airspace Operations and Safety Program (AOSP). The TASAR Flight Trial-2 (FT-2) was conducted in June, 2015 out of the Newport News/Williamsburg International Airport. This flight trial was conducted using a Piaggio Avanti flight test aircraft and consisted of 12 Evaluation Flights with airline commercial pilots participating as the Evaluation Pilots, three destination airports in Atlanta and Jacksonville Air Route Traffic Control Centers, and one pair of flight plans associated with each destination airport. The primary goal of FT-2 was to reduce risk for upcoming operational trials with NASA partner airlines, Alaska Airlines and Virgin America. To accomplish this primary goal, six independent objectives were conducted during FT-2, however, this paper will report only the findings of Objective 5; the assessment of system usability, pilot perceived workload, and the degree of pilot acceptability of the TAP Human Machine Interface (HMI) during flight operations, via the administration of several subjective measures.

Characterization and Expression Analysis of Receptor for Activated C Kinase from Silk-producing Insect Antheraea pernyi.

PubMed

Zhu, Bao-Jian; Yu, Hao; Tian, Sen; Dai, Li-Shang; Sun, Yu; Liu, Chao-Liang

2016-01-01

The receptor for activated C kinase (RACK) is an important scaffold protein with regulatory functions in cells. However, its role in the immune response of Antheraea pernyi to pathogen challenge remains unclear. To investigate the biological functions of RACK in the wild silkworm A. pernyi, cloning was performed and the expression patterns of the RACK gene were analyzed. Sequence analysis revealed that the RACK gene was 1120 bp containing a 960-bp open reading frame. The deduced RACK protein sequence reveals the higher identity with its homologs from other insects. SDS-PAGE and western blot analysis demonstrated successful expression of a 36-kDa recombinant RACK protein in Escherichia coli. The titer of a rabbit-raised antibody against recombinant RACK protein was about 1: 20000, determined by ELISA. Real-time PCR analysis showed that RACK expression was higher in fat bodies than in other examined A. pernyi tissues. The expression of RACK mRNA in fat bodies of fifth larvae of A. pernyi was obviously induced after nucleopolyhedrovirus, E. coli or Beauveria bassiana challenge. However, the expression patterns of RACK were different in response to these pathogens. Our data suggest that RACK may play a role in the innate immune responses of A. pernyi.

Natural Non-Mulberry Silk Nanoparticles for Potential-Controlled Drug Release

PubMed Central

Wang, Juan; Yin, Zhuping; Xue, Xiang; Kundu, Subhas C.; Mo, Xiumei; Lu, Shenzhou

2016-01-01

Natural silk protein nanoparticles are a promising biomaterial for drug delivery due to their pleiotropic properties, including biocompatibility, high bioavailability, and biodegradability. Chinese oak tasar Antheraea pernyi silk fibroin (ApF) nanoparticles are easily obtained using cations as reagents under mild conditions. The mild conditions are potentially advantageous for the encapsulation of sensitive drugs and therapeutic molecules. In the present study, silk fibroin protein nanoparticles are loaded with differently-charged small-molecule drugs, such as doxorubicin hydrochloride, ibuprofen, and ibuprofen-Na, by simple absorption based on electrostatic interactions. The structure, morphology and biocompatibility of the silk nanoparticles in vitro are investigated. In vitro release of the drugs from the nanoparticles depends on charge-charge interactions between the drugs and the nanoparticles. The release behavior of the compounds from the nanoparticles demonstrates that positively-charged molecules are released in a more prolonged or sustained manner. Cell viability studies with L929 demonstrated that the ApF nanoparticles significantly promoted cell growth. The results suggest that Chinese oak tasar Antheraea pernyi silk fibroin nanoparticles can be used as an alternative matrix for drug carrying and controlled release in diverse biomedical applications. PMID:27916946

Comparative transcriptomic analysis of silkwormBmovo-1 and wild type silkworm ovary

PubMed Central

Xue, Renyu; Hu, Xiaolong; Zhu, Liyuan; Cao, Guangli; Huang, Moli; Xue, Gaoxu; Song, Zuowei; Lu, Jiayu; Chen, Xueying; Gong, Chengliang

2015-01-01

The detailed molecular mechanism of Bmovo-1 regulation of ovary size is unclear. To uncover the mechanism of Bmovo-1 regulation of ovarian development and oogenesis using RNA-Seq, we compared the transcriptomes of wild type (WT) and Bmovo-1-overexpressing silkworm (silkworm+Bmovo-1) ovaries. Using a pair-end Illumina Solexa sequencing strategy, 5,296,942 total reads were obtained from silkworm+Bmovo-1 ovaries and 6,306,078 from WT ovaries. The average read length was about 100 bp. Clean read ratios were 98.79% for silkworm+Bmovo-1 and 98.87% for WT silkworm ovaries. Comparative transcriptome analysis showed 123 upregulated and 111 downregulated genes in silkworm+Bmovo-1 ovaries. These differentially expressed genes were enriched in the extracellular and extracellular spaces and involved in metabolism, genetic information processing, environmental information processing, cellular processes and organismal systems. Bmovo-1 overexpression in silkworm ovaries might promote anabolism for ovarian development and oogenesis and oocyte proliferation and transport of nutrients to ovaries by altering nutrient partitioning, which would support ovary development. Excessive consumption of nutrients for ovary development alters nutrient partitioning and deters silk protein synthesis. PMID:26643037

Role of tropomyosin in silkworm allergy.

PubMed

Jeong, Kyoung Yong; Han, In-Soo; Lee, June Yong; Park, Kyung Hee; Lee, Jae-Hyun; Park, Jung-Won

2017-05-01

Silkworm pupae are widely consumed in Asian countries and allergic reactions following consumption have been described. However, false‑positive responses in skin prick allergy tests or non‑specific immunoglobulin E (IgE) responses to total extract of silkworm pupa make diagnosis difficult. Although improved allergy diagnosis is required, molecular characterization of silkworm allergens has not been performed to date, except for Bomb m 1, an arginine kinase. This study aimed to evaluate the allergenicity of tropomyosin, a well‑established invertebrate pan‑allergen, from silkworm pupa. The silkworm tropomyosin gene was cloned by reverse transcription and polymerase chain reaction, and the protein was overexpressed in Escherichia coli and purified by affinity chromatography using Nickel‑resin. IgE reactivity of the recombinant protein was examined by ELISA and competitive inhibition analyses. Silkworm pupa tropomyosin shared 73.5‑92.3% amino acid sequence identity with previously identified allergenic tropomyosins. Sera from eight of 15 patients with silkworm allergy (53.3%) exhibited binding of IgE to the recombinant protein. However, recombinant protein was able to inhibit less than 10% of IgE reactivity to silkworm pupa extract. Of the eight sera tested, six that specifically reacted with silkworm tropomyosin also demonstrated IgE reactivity to shrimp and crab. In the present study, specific IgE to silkworm tropomyosin was detected in patients with silkworm allergy, suggesting that it may be useful in diagnosis of allergy to silkworm pupa.

Non-Mulberry and Mulberry Silk Protein Sericins as Potential Media Supplement for Animal Cell Culture.

PubMed

Sahu, Neety; Pal, Shilpa; Sapru, Sunaina; Kundu, Joydip; Talukdar, Sarmistha; Singh, N Ibotambi; Yao, Juming; Kundu, Subhas C

2016-01-01

Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberry Bombyx mori and different non-mulberry sources, namely, tropical tasar, Antheraea mylitta; muga, Antheraea assama; and eri, Samia ricini, as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however, S. ricini sericin seems to promote better growth of cells amongst other non-mulberry sericins.

Non-Mulberry and Mulberry Silk Protein Sericins as Potential Media Supplement for Animal Cell Culture

PubMed Central

Sahu, Neety; Pal, Shilpa; Sapru, Sunaina; Kundu, Joydip; Talukdar, Sarmistha; Singh, N. Ibotambi; Yao, Juming

2016-01-01

Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberry Bombyx mori and different non-mulberry sources, namely, tropical tasar, Antheraea mylitta; muga, Antheraea assama; and eri, Samia ricini, as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however, S. ricini sericin seems to promote better growth of cells amongst other non-mulberry sericins. PMID:27517047

Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties

USDA-ARS?s Scientific Manuscript database

The development of a spider silk manufacturing process is of great interest. piggyBac vectors were used to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk prote...

Comparative methylomics between domesticated and wild silkworms implies possible epigenetic influences on silkworm domestication

PubMed Central

2013-01-01

Background In contrast to wild species, which have typically evolved phenotypes over long periods of natural selection, domesticates rapidly gained human-preferred agronomic traits in a relatively short-time frame via artificial selection. Under domesticated conditions, many traits can be observed that cannot only be due to environmental alteration. In the case of silkworms, aside from genetic divergence, whether epigenetic divergence played a role in domestication is an unanswered question. The silkworm is still an enigma in that it has two DNA methyltransferases (DNMT1 and DNMT2) but their functionality is unknown. Even in particular the functionality of the widely distributed DNMT1 remains unknown in insects in general. Results By embryonic RNA interference, we reveal that knockdown of silkworm Dnmt1 caused decreased hatchability, providing the first direct experimental evidence of functional significance of insect Dnmt1. In the light of this fact and those that DNA methylation is correlated with gene expression in silkworms and some agronomic traits in domesticated organisms are not stable, we comprehensively compare silk gland methylomes of 3 domesticated (Bombyx mori) and 4 wild (Bombyx mandarina) silkworms to identify differentially methylated genes between the two. We observed 2-fold more differentiated methylated cytosinces (mCs) in domesticated silkworms as compared to their wild counterparts, suggesting a trend of increasing DNA methylation during domestication. Further study of more domesticated and wild silkworms narrowed down the domesticates’ epimutations, and we were able to identify a number of differential genes. One such gene showing demethyaltion in domesticates correspondently displays lower gene expression, and more interestingly, has experienced selective sweep. A methylation-increased gene seems to result in higher expression in domesticates and the function of its Drosophila homolog was previously found to be essential for cell volume

Phylogeny and evolutionary history of the silkworm.

PubMed

Sun, Wei; Yu, Hongsong; Shen, Yihong; Banno, Yutaka; Xiang, Zhonghuai; Zhang, Ze

2012-06-01

The silkworm, Bombyx mori, played an important role in the old Silk Road that connected ancient Asia and Europe. However, to date, there have been few studies of the origins and domestication of this species using molecular methods. In this study, DNA sequences of mitochondrial and nuclear loci were used to infer the phylogeny and evolutionary history of the domesticated silkworm and its relatives. All of the phylogenetic analyses indicated a close relationship between the domesticated silkworm and the Chinese wild silkworm. Domestication was estimated to have occurred about 4100 years ago (ya), and the radiation of the different geographic strains of B. mori about 2000 ya. The Chinese wild silkworm and the Japanese wild silkworm split about 23600 ya. These estimates are in good agreement with the fossil evidence and historical records. In addition, we show that the domesticated silkworm experienced a population expansion around 1000 ya. The divergence times and the population dynamics of silkworms presented in this study will be useful for studies of lepidopteran phylogenetics, in the genetic analysis of domestic animals, and for understanding the spread of human civilizations.

Spectral imaging analysis for silkworm gender classification

NASA Astrophysics Data System (ADS)

Sumriddetchkajorn, Sarun; Kamtongdee, Chakkrit; Sa-Ngiamsak, Chiranut

2013-05-01

We examine the effect of different wavelength spectra in the performance of our optical penetration-based silkworm pupa sex identification system. With available low-cost light emitting diodes (LEDs), each emitting different wavelength spectra at 468 nm, 565 nm, 639 nm, 940 nm, and broad white light, we find that the body of the silkworm pupa can block blue and near infrared light while allowing green and red light pass through. In particular, the red light can clearly highlight an important organ called "chitin gland" of the female, leading to high accuracy of silkworm gender identification. In our experiment with 120 silkworm pupae, measured high average 92.8% and lower average 87.5% accuracies in identifying silkworm gender are obtained under red and white light LEDs, respectively.

Overexpression of host plant urease in transgenic silkworms.

PubMed

Jiang, Liang; Huang, Chunlin; Sun, Qiang; Guo, Huizhen; Peng, Zhengwen; Dang, Yinghui; Liu, Weiqiang; Xing, Dongxu; Xu, Guowen; Zhao, Ping; Xia, Qingyou

2015-06-01

Bombyx mori and mulberry constitute a model of insect-host plant interactions. Urease hydrolyzes urea to ammonia and is important for the nitrogen metabolism of silkworms because ammonia is assimilated into silk protein. Silkworms do not synthesize urease and acquire it from mulberry leaves. We synthesized the artificial DNA sequence ureas using the codon bias of B. mori to encode the signal peptide and mulberry urease protein. A transgenic vector that overexpresses ure-as under control of the silkworm midgut-specific P2 promoter was constructed. Transgenic silkworms were created via embryo microinjection. RT-PCR results showed that urease was expressed during the larval stage and qPCR revealed the expression only in the midgut of transgenic lines. Urea concentration in the midgut and hemolymph of transgenic silkworms was significantly lower than in a nontransgenic line when silkworms were fed an artificial diet. Analysis of the daily body weight and food conversion efficiency of the fourth and fifth instar larvae and economic characteristics indicated no differences between transgenic silkworms and the nontransgenic line. These results suggested that overexpression of host plant urease promoted nitrogen metabolism in silkworms.

Silkworm: A Promising Model Organism in Life Science.

PubMed

Meng, Xu; Zhu, Feifei; Chen, Keping

2017-09-01

As an important economic insect, silkworm Bombyx mori (L.) (Lepidoptera: Bombycidae) has numerous advantages in life science, such as low breeding cost, large progeny size, short generation time, and clear genetic background. Additionally, there are rich genetic resources associated with silkworms. The completion of the silkworm genome has further accelerated it to be a modern model organism in life science. Genomic studies showed that some silkworm genes are highly homologous to certain genes related to human hereditary disease and, therefore, are a candidate model for studying human disease. In this article, we provided a review of silkworm as an important model in various research areas, including human disease, screening of antimicrobial agents, environmental safety monitoring, and antitumor studies. In addition, the application potentiality of silkworm model in life sciences was discussed. © The Author 2017. Published by Oxford University Press on behalf of Entomological Society of America.

Survey and Analysis of Microsatellites in the Silkworm, Bombyx mori

PubMed Central

Prasad, M. Dharma; Muthulakshmi, M.; Madhu, M.; Archak, Sunil; Mita, K.; Nagaraju, J.

2005-01-01

We studied microsatellite frequency and distribution in 21.76-Mb random genomic sequences, 0.67-Mb BAC sequences from the Z chromosome, and 6.3-Mb EST sequences of Bombyx mori. We mined microsatellites of ≥15 bases of mononucleotide repeats and ≥5 repeat units of other classes of repeats. We estimated that microsatellites account for 0.31% of the genome of B. mori. Microsatellite tracts of A, AT, and ATT were the most abundant whereas their number drastically decreased as the length of the repeat motif increased. In general, tri- and hexanucleotide repeats were overrepresented in the transcribed sequences except TAA, GTA, and TGA, which were in excess in genomic sequences. The Z chromosome sequences contained shorter repeat types than the rest of the chromosomes in addition to a higher abundance of AT-rich repeats. Our results showed that base composition of the flanking sequence has an influence on the origin and evolution of microsatellites. Transitions/transversions were high in microsatellites of ESTs, whereas the genomic sequence had an equal number of substitutions and indels. The average heterozygosity value for 23 polymorphic microsatellite loci surveyed in 13 diverse silkmoth strains having 2–14 alleles was 0.54. Only 36 (18.2%) of 198 microsatellite loci were polymorphic between the two divergent silkworm populations and 10 (5%) loci revealed null alleles. The microsatellite map generated using these polymorphic markers resulted in 8 linkage groups. B. mori microsatellite loci were the most conserved in its immediate ancestor, B. mandarina, followed by the wild saturniid silkmoth, Antheraea assama. PMID:15371363

Construction of a full-length cDNA Library from Chinese oak silkworm pupa and identification of a KK-42-binding protein gene in relation to pupa-diapause termination.

PubMed

Li, Yu-Ping; Xia, Run-Xi; Wang, Huan; Li, Xi-Sheng; Liu, Yan-Qun; Wei, Zhao-Jun; Lu, Cheng; Xiang, Zhong-Huai

2009-06-24

In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 x 10(5) cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST) analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4%) were known genes but only five from A. pernyi, 37 (21.2%) were known ESTs without function annotation, and 41 (23.4%) were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151) was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination.

Construction of a full-length cDNA Library from Chinese oak silkworm pupa and identification of a KK-42-binding protein gene in relation to pupa-diapause termination

PubMed Central

Li, Yu-Ping; Xia, Run-Xi; Wang, Huan; Li, Xi-Sheng; Liu, Yan-Qun; Wei, Zhao-Jun; Lu, Cheng; Xiang, Zhong-Huai

2009-01-01

In this study we successfully constructed a full-length cDNA library from Chinese oak silkworm, Antheraea pernyi, the most well-known wild silkworm used for silk production and insect food. Total RNA was extracted from a single fresh female pupa at the diapause stage. The titer of the library was 5 × 105 cfu/ml and the proportion of recombinant clones was approximately 95%. Expressed sequence tag (EST) analysis was used to characterize the library. A total of 175 clustered ESTs consisting of 24 contigs and 151 singlets were generated from 250 effective sequences. Of the 175 unigenes, 97 (55.4%) were known genes but only five from A. pernyi, 37 (21.2%) were known ESTs without function annotation, and 41 (23.4%) were novel ESTs. By EST sequencing, a gene coding KK-42-binding protein in A. pernyi (named as ApKK42-BP; GenBank accession no. FJ744151) was identified and characterized. Protein sequence analysis showed that ApKK42-BP was not a membrane protein but an extracellular protein with a signal peptide at position 1-18, and contained two putative conserved domains, abhydro_lipase and abhydrolase_1, suggesting it may be a member of lipase superfamily. Expression analysis based on number of ESTs showed that ApKK42-BP was an abundant gene in the period of diapause stage, suggesting it may also be involved in pupa-diapause termination. PMID:19564928

Demographic history and gene flow during silkworm domestication

PubMed Central

2014-01-01

Background Gene flow plays an important role in domestication history of domesticated species. However, little is known about the demographic history of domesticated silkworm involving gene flow with its wild relative. Results In this study, four model-based evolutionary scenarios to describe the demographic history of B. mori were hypothesized. Using Approximate Bayesian Computation method and DNA sequence data from 29 nuclear loci, we found that the gene flow at bottleneck model is the most likely scenario for silkworm domestication. The starting time of silkworm domestication was estimated to be approximate 7,500 years ago; the time of domestication termination was 3,984 years ago. Using coalescent simulation analysis, we also found that bi-directional gene flow occurred during silkworm domestication. Conclusions Estimates of silkworm domestication time are nearly consistent with the archeological evidence and our previous results. Importantly, we found that the bi-directional gene flow might occur during silkworm domestication. Our findings add a dimension to highlight the important role of gene flow in domestication of crops and animals. PMID:25123546

Silkworm protein: its possibility as an actuator

NASA Astrophysics Data System (ADS)

Jin, Hyoung-Joon; Myung, Seung Jun; Kim, Heung Soo; Jung, Woochul; Kim, Jaehwan

2006-03-01

The possibility of silkworm (Bombyx mori) protein as a base material of biomimetic actuator was investigated in this paper. Silkworm films were prepared from high concentrations of regenerated fibroin in aqueous solution. Films with thickness of about 100 μm were prepared for coating electrodes. The cast silk films were coated by very thin gold electrode on both sides of the film. Tensile test of cast film showed bi-modal trend, which is typical stress-strain relation of polymeric film. As the test of a possible biomimetic actuator, silkworm film actuator provides bending deformations according to the magnitude and frequency of the applied electric filed. Although the present bending deformation of silkworm film actuator is smaller than that of Electro-Active Paper actuator, it provides the possibility of biomimetic actuator.

Effects of High Magneto-Gravitational Environment on Silkworm Embryogenesis

NASA Astrophysics Data System (ADS)

Tian, Zongcheng; Li, Muwang; Qian, Airong; Xu, Huiyun; Wang, Zhe; Di, Shengmeng; Yang, Pengfei; Hu, Lifang; Ding, Chong; Zhang, Wei; Luo, Mingzhi; Han, Jing; Gao, Xiang; Huang, Yongping; Shang, Peng

2010-04-01

The objective of this research was to observe whether silkworm embryos can survive in a high magneto-gravitational environment (HMGE) and what significant phenotype changes can be produced. The hatching rate, hatching time, life span, growth velocity and cocoon weight of silkworm were measured after silkworm embryos were exposed to HMGE (0 g, 12 T; 1 g, 16 T; and 2 g, 12 T) for a period of time. Compared with the control group, 0 g exposure resulted in a lower hatching rate and a shorter life span. Statistically insignificant morphological changes had been observed for larvae growth velocity, incidence of abnormal markings and weight of cocoons. These results suggest that the effect of HMGE on silkworm embryogenesis is not lethal. Bio-effects of silkworm embryogenesis at 0 g in a HMGE were similar with those of space flight. The hatching time, life span and hatching rates of silkworm may be potential phenotype markers related to exposure in a weightless environment.

Supplementation of an Artificial Medium for the Parasitoid Exorista larvarum (Diptera: Tachnidae) With Hemolymph of Hermetia illucens (Diptera: Stratiomyidae) or Antheraea pernyi (Lepidoptera: Saturniidae).

PubMed

Dindo, Maria Luisa; Vandicke, Jonas; Marchetti, Elisa; Spranghers, Thomas; Bonte, Jochem; De Clercq, Patrick

2016-04-01

The effect of supplementing hemolymph of the black soldier fly, Hermetia illucens (L.), or the Chinese oak silkworm, Antheraea pernyi (Guérin-Méneville), to a basic insect-free artificial medium for the tachinid Exorista larvarum (L.) was investigated. The supplementation (20% w/w) was based on the assumption that insect additives may optimize the media for this parasitoid. Egg hatch, pupal and adult yields, and sex ratio did not differ among the enriched and basic media. Preimaginal development was faster on both hemolymph-enriched media than on the basic medium. Despite the shorter development on the medium supplemented with H. illucens hemolymph than on the basic medium, on the two media puparium weights were comparable. The female flies reared on the medium enriched with H. illucens hemolymph did not lay more eggs, but the latter yielded significantly more puparia compared with the control females. Conversely, the medium enriched with A. pernyi hemolymph yielded lower female puparium weights than the basic medium and produced only one ovipositing female out of the five obtained female adults. These results indicate that the in vitro development of E. larvarum improved when the basic artificial medium was enriched with H. illucens hemolymph, whereas the supplementation with A. pernyi hemolymph negatively affected the quality of the in vitro-reared females.

Nucleotide Diversity and Selection Signature in the Domesticated Silkworm, Bombyx mori, and Wild Silkworm, Bombyx mandarina

PubMed Central

Guo, Yi; Shen, Yi-Hong; Sun, Wei; Kishino, Hirohisa; Xiang, Zhong-Huai; Zhang, Ze

2011-01-01

To investigate the patterns of nucleotide diversity in domesticated silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) and its wild relative, Chinese wild silkworm, Bombyx mandarina Moore, we sequenced nine nuclear genes. Neutrality test and coalescent simulation for these genes were performed to look at bottleneck intensity and selection signature; linkage disequilibrium (LD) within and between loci was employed to investigate allele association. As a result, B. mori lost 33–49% of nucleotide diversity relative to wild silkworm, which is similar to the loss levels found in major cultivated crops. Diversity of B. mori is significantly lower than that of B. mandarina measured as πtotal (0.01166 vs. 0.1741) or θW(0.01124 vs. 0.02206). Bottleneck intensity of domesticated silkworm is 1.5 (in terms of k = Nb/d, Nb-bottleneck population size; d-bottleneck duration) with different durations. Gene DefA showed signature of artificial selection by all analysis methods and might experience strong artificial selection in B. mori during domestication. For nine loci, both curves of LD decay rapidly within 200 bp and drop slowly when distance is > 200 bp, although that of B. mori decays slower than B. mandarina at loci investigated. However, LD could not be estimated at DefA in B. mori and at ER in both silkworms. Elevated LD observed in B. mori may be indicator of selection and demographic events. PMID:22239062

Acute oral toxicity test of chemical compounds in silkworms.

PubMed

Usui, Kimihito; Nishida, Satoshi; Sugita, Takuya; Ueki, Takuro; Matsumoto, Yasuhiko; Okumura, Hidenobu; Sekimizu, Kazuhisa

2016-02-01

This study performed an acute oral toxicity test of 59 compounds in silkworms. These compounds are listed in OECD guidelines as standard substances for a cytotoxicity test, and median lethal dose (LD(50)) werecalculated for each compound. Acute oral LD(50) values in mammals are listed in OECD guidelines and acute oral LD(50) values in silkworms were determined in this study. R(2) for the correlation between LD(50) values in mammals and LD(50) values in silkworms was 0.66. In addition, the acute oral toxicity test in silkworms was performed by two different facilities, and test results from the facilities were highly reproducible. These findings suggest that an acute oral toxicity test in silkworms is a useful way to evaluate the toxicity of compounds in mammals.

Preliminary Benefits Assessment of Traffic Aware Strategic Aircrew Requests (TASAR)

NASA Technical Reports Server (NTRS)

Henderson, Jeff; Idris, Husni; Wing, David J.

2012-01-01

While en route, aircrews submit trajectory change requests to air traffic control (ATC) to better meet their objectives including reduced delays, reduced fuel burn, and passenger comfort. Aircrew requests are currently made with limited to no information on surrounding traffic. Consequently, these requests are uninformed about a key ATC objective, ensuring traffic separation, and therefore less likely to be accepted than requests informed by surrounding traffic and that avoids creating conflicts. This paper studies the benefits of providing aircrews with on-board decision support to generate optimized trajectory requests that are probed and cleared of known separation violations prior to issuing the request to ATC. These informed requests are referred to as traffic aware strategic aircrew requests (TASAR) and leverage traffic surveillance information available through Automatic Dependent Surveillance Broadcast (ADS-B) In capability. Preliminary fast-time simulation results show increased benefits with longer stage lengths since beneficial trajectory changes can be applied over a longer distance. Also, larger benefits were experienced between large hub airports as compared to other airport sizes. On average, an aircraft equipped with TASAR reduced its travel time by about one to four minutes per operation and fuel burn by about 50 to 550 lbs per operation depending on the objective of the aircrew (time, fuel, or weighted combination of time and fuel), class of airspace user, and aircraft type. These preliminary results are based on analysis of approximately one week of traffic in July 2012 and additional analysis is planned on a larger data set to confirm these initial findings.

[Identification of novel therapeutically effective antibiotics using silkworm infection model].

PubMed

Hamamoto, Hiroshi; Urai, Makoto; Paudel, Atmika; Horie, Ryo; Murakami, Kazuhisa; Sekimizu, Kazuhisa

2012-01-01

Most antibiotics obtained by in vitro screening with antibacterial activity have inappropriate properties as medicines due to their toxicity and pharmacodynamics in animal bodies. Thus, evaluation of the therapeutic effects of these samples using animal models is essential in the crude stage. Mammals are not suitable for therapeutic evaluation of a large number of samples due to high costs and ethical issues. We propose the use of silkworms (Bombyx mori) as model animals for screening therapeutically effective antibiotics. Silkworms are infected by various pathogenic bacteria and are effectively treated with similar ED(50) values of clinically used antibiotics. Furthermore, the drug metabolism pathways, such as cytochrome P450 and conjugation systems, are similar between silkworms and mammals. Silkworms have many advantages compared with other infection models, such as their 1) low cost, 2) few associated ethical problems, 3) adequate body size for easily handling, and 4) easier separation of organs and hemolymph. These features of the silkworm allow for efficient screening of therapeutically effective antibiotics. In this review, we discuss the advantages of the silkworm model in the early stages of drug development and the screening results of some antibiotics using the silkworm infection model.

Silk Spinning in Silkworms and Spiders

PubMed Central

Andersson, Marlene; Johansson, Jan; Rising, Anna

2016-01-01

Spiders and silkworms spin silks that outcompete the toughness of all natural and manmade fibers. Herein, we compare and contrast the spinning of silk in silkworms and spiders, with the aim of identifying features that are important for fiber formation. Although spiders and silkworms are very distantly related, some features of spinning silk seem to be universal. Both spiders and silkworms produce large silk proteins that are highly repetitive and extremely soluble at high pH, likely due to the globular terminal domains that flank an intermediate repetitive region. The silk proteins are produced and stored at a very high concentration in glands, and then transported along a narrowing tube in which they change conformation in response primarily to a pH gradient generated by carbonic anhydrase and proton pumps, as well as to ions and shear forces. The silk proteins thereby convert from random coil and alpha helical soluble conformations to beta sheet fibers. We suggest that factors that need to be optimized for successful production of artificial silk proteins capable of forming tough fibers include protein solubility, pH sensitivity, and preservation of natively folded proteins throughout the purification and initial spinning processes. PMID:27517908

Silk Spinning in Silkworms and Spiders.

PubMed

Andersson, Marlene; Johansson, Jan; Rising, Anna

2016-08-09

Spiders and silkworms spin silks that outcompete the toughness of all natural and manmade fibers. Herein, we compare and contrast the spinning of silk in silkworms and spiders, with the aim of identifying features that are important for fiber formation. Although spiders and silkworms are very distantly related, some features of spinning silk seem to be universal. Both spiders and silkworms produce large silk proteins that are highly repetitive and extremely soluble at high pH, likely due to the globular terminal domains that flank an intermediate repetitive region. The silk proteins are produced and stored at a very high concentration in glands, and then transported along a narrowing tube in which they change conformation in response primarily to a pH gradient generated by carbonic anhydrase and proton pumps, as well as to ions and shear forces. The silk proteins thereby convert from random coil and alpha helical soluble conformations to beta sheet fibers. We suggest that factors that need to be optimized for successful production of artificial silk proteins capable of forming tough fibers include protein solubility, pH sensitivity, and preservation of natively folded proteins throughout the purification and initial spinning processes.

Fruit body formation on silkworm by Cordyceps militaris

USDA-ARS?s Scientific Manuscript database

Injection inoculation protocols for fruit body formation of Cordyceps militaris were investigated to improve the incidence of infection in the silkworm species Bombyx mori. Injection, with suspensions of C. militaris hyphal bodies into living silkworm pupae, was used to test for fruit body productio...

Feeding scenario of the silkworm Bombyx Mori, L. in the BLSS

NASA Astrophysics Data System (ADS)

Yu, XiaoHui; Liu, Hong; Tong, Ling

A simple subunit of the bioregenerative life support system (BLSS) consisting of the ground-controlled mulberry ( Morus alba L.) and the silkworms was set up on the ground. The mulberry tree could provide nutrient mulberry fruits for astronauts and its leaves as the main feedstuff for the silkworms until their third instar. Astronauts utilized curled lettuce ( Lactuca sativa L.) stem as vegetables and the silkworms over third instar could be fed on 65% of inedible leaves of the lettuce. About 71.4% of protein were detected in the silkworm larval powder; thus, 105 silkworms could satisfy the requirement of one person per day. Besides, 18 kinds of amino acids were determined in the obtained silkworm powder. Moreover, the R-criterion was suggested to estimate and optimize the animal feeding facilities. The scenario of treating the wastes is also proposed in this paper. Our results may be valuable for the establishment of a complex BLSS in the future.

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

PubMed Central

Kim, Seong-Ryul; Kwak, Woori; Kim, Hyaekang; Kim, Kee-Young; Kim, Su-Bae; Choi, Kwang-Ho; Kim, Seong-Wan; Hwang, Jae-Sam; Kim, Minjee; Kim, Iksoo; Goo, Tae-Won

2018-01-01

Abstract 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. PMID:29186418

Analysis of silkworm gut microflora in the Bioregenerative Life Support System

NASA Astrophysics Data System (ADS)

Liang, Xue; Liu, lh64. Hong

2012-07-01

Silkworm (Bombyx mori L) has advantages in the nutritional composition, growth characteristics and other factors, it is regarded as animal protein source for astronauts in the Bioregenerative Life Support System (BLSS).Due to the features of BLSS, silkworm breeding way is different from the conventional one (mulberry leaves throughout five instars): they were fed with mulberry and lettuce leaves during the 1st-3rd instars and 4th -5th instars, respectively. As the lettuce stem can be eaten by astronauts, the leaves not favored by humans can be insect's foodstuff. Therefore, it is necessary to investigate the gut microbial composition, the type of dominant bacteria of silkworm raised with this way and the differences from the conventional breeding method, so as to reduce the mortality rate caused by the foodstuff change and to provide more animal protein for astronauts. In this study, 16srDNA sequencing, phylogenetic analysis and denaturing gradient gel electrophoresis method were used to analyze the silkworm gut microbial flora under two breeding manners. The results show that conventional and BLSS breeding way have six dominant bacteria in common: Clostridium, Enterococcus, Bacteroides, Chryseobacterium, Parabacteroides, Paenibacillus. We also found Escherichia, Janthinobacterium, Sedimentibacter, Streptococcus, Bacillus, Arcobacter, Rothia, Polaribacter and Acinetobacter, Anaerofilum, Rummeliibacillus, Anaeroplasma, Serratia in the ground conventional and BLSS special breeding way, respectively. Changing the foodstuff of silkworm leads to the dynamic alteration of gut microbial. Dominant bacteria of the two breeding ways have diversities from each other. The ground conventional breeding way has more abundant bacteria than the BLSS one. Due to the lettuce leaves have replaced mulberry leaves at the beginning of the silkworm 4th instar, some silkworms can not survive without the bacteria that digest and absorb lettuce leaves. We suggest those dominant bacteria

TASAR Flight Trial 2: Assessment of Air Traffic Controller Acceptability of TASAR Requests

NASA Technical Reports Server (NTRS)

Idris, Husni; Enea, Gabriele

2016-01-01

In support of the Flight Trial (FT-2) of NASA's prototype of the Traffic Aware Strategic Aircrew Requests (TASAR) concept, observations were conducted at the air traffic facilities to identify and assess the main factors that affect the acceptability of pilot requests by air traffic controllers. Two observers shadowed air traffic controllers at the Atlanta (ZTL) and Jacksonville (ZJX) air traffic control centers as the test flight pilot made pre-scripted requests to invoke acceptability issues and then they interviewed the observed and other controllers voluntarily. Fifty controllers were interviewed with experience ranging from one to thirty-five years. All interviewed controllers were enthusiastic about the technology and accounting for sector boundaries in pilot requests, particularly if pilots can be made aware of high workload situations. All interviewed controllers accept more than fifty percent of pilot requests; forty percent of them reject less than ten percent of requests. The most common reason for rejecting requests is conflicting with traffic followed by violating letters of agreement (LOAs) and negatively impacting neighboring sector workload, major arrival and departure flows and flow restrictions. Thirty-six requests were made during the test, eight of which were rejected due to: the aircraft already handed off to another sector, violating LOA, opposing traffic, intruding into an active special use airspace (SUA), intruding into another center, weather, and unfamiliarity with the requested waypoint. Nine requests were accepted with delay mostly because the controller needed to locate unfamiliar waypoints or to coordinate with other controllers.

Highly accurate and fast optical penetration-based silkworm gender separation system

NASA Astrophysics Data System (ADS)

Kamtongdee, Chakkrit; Sumriddetchkajorn, Sarun; Chanhorm, Sataporn

2015-07-01

Based on our research work in the last five years, this paper highlights our innovative optical sensing system that can identify and separate silkworm gender highly suitable for sericulture industry. The key idea relies on our proposed optical penetration concepts and once combined with simple image processing operations leads to high accuracy in identifying of silkworm gender. Inside the system, there are electronic and mechanical parts that assist in controlling the overall system operation, processing the optical signal, and separating the female from male silkworm pupae. With current system performance, we achieve a very highly accurate more than 95% in identifying gender of silkworm pupae with an average system operational speed of 30 silkworm pupae/minute. Three of our systems are already in operation at Thailand's Queen Sirikit Sericulture Centers.

Long-term preservation of eri and ailanthus silkworms using frozen gonads.

PubMed

Fukumori, Hisayoshi; Lee, Jung; Fujii, Tsuguru; Kajiura, Zenta; Banno, Yutaka

2017-08-01

Cryopreservation of eri and ailanthus silkworms using frozen gonads was investigated. First, we evaluated the freeze tolerance of ovary and testis in the eri silkworm, which showed high tolerance. Mating between frozen ovary-transplanted females and frozen testis-transplanted males produced 163.0 eggs, yielding 105.7 larvae per moth. In a second experiment, we tested the use of the eri silkworm as a host insect for gonad transplantation from ailanthus silkworm donors. A high success ratio for laid and hatched eggs was demonstrated for ovary transplantation (97.8 and 51.3 eggs per moth, respectively). For testis transplantation, however, the average number of hatched larvae was low (12.0). Mating between host eri females and males in which both frozen ovary and testis of the ailanthus silkworm had been transplanted produced 6.4 fertilized eggs per host moth. Our success in using cross subspecies cryopreservation between these wild silkworms could lead to the alternative use of hosts between species in other insects. Copyright © 2017 Elsevier Inc. All rights reserved.

Construction of transformed, cultured silkworm cells and transgenic silkworm using the site-specific integrase system from phage φC31.

PubMed

Yin, Yajuan; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2014-10-01

The Streptomyces bacteriophage, φC31, uses a site-specific integrase enzyme to perform efficient recombination. The recombination system uses specific sequences to integrate exogenous DNA from the phage into a host. The sequences are known as the attP site in the phage and the attB site in the host. The system can be used as a genetic manipulation tool. In this study it has been applied to the transformation of cultured BmN cells and the construction of transgenic Bombyx mori individuals. A plasmid, pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express a egfp-zeocin fusion gene, was co-transfected into cultured BmN cells with a helper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the egfp-zeocin fusion gene was driven by an ie-1 promoter, downstream of a φC31 attB site. The helper plasmid encoded the φC31 integrase enzyme, which was flanked by two nuclear localization signals. Expression of the egfp-zeocin fusion gene could be observed in transformed cells. The two plasmids were also transferred into silkworm eggs to obtain transgenic silkworms. Successful integration of the fusion gene was indicated by the detection of green fluorescence, which was emitted by the silkworms. Nucleotide sequence analysis demonstrated that the attB site had been cut, to allow recombination between the attB and endogenous pseudo attP sites in the cultured silkworm cells and silkworm individuals.

Physicochemical Properties of Meat Batter Added with Edible Silkworm Pupae (Bombyx mori) and Transglutaminase.

PubMed

Park, Yoo-Sun; Choi, Yun-Sang; Hwang, Ko-Eun; Kim, Tae-Kyung; Lee, Cheol-Won; Shin, Dong-Min; Han, Sung Gu

2017-01-01

This study was conducted to investigate the physicochemical properties of meat batters prepared with fresh pork meat, back fat, water, and salt and formulated with three different amounts (5%, 10%, and 15%) of silkworm pupae ( Bombyx mori ) powder and transglutaminase (TG). Meat batters formulated with silkworm pupae powder showed significantly higher contents of protein and ash than control batter. Addition of silkworm pupae to batter also showed significantly lower cooking loss than the control. Moreover, meat batter containing 15% silkworm pupae showed no significant difference in redness value compared to the control. In addition, pH, viscosity, hardness, gumminess, and chewiness were improved after the addition of silkworm pupae. Furthermore, meat batter formulated with TG and silkworm pupae showed improved hardness, gumminess, chewiness and viscosity compared to control batter. Addition of 1% TG with 15% silkworm pupae to meat batter resulted in significantly higher pH, textures, and viscosity. Our data suggest that both silkworm pupae and TG can be added to meat batter to improve its physicochemical properties. Therefore, combination of silkworm pupae and TG could be a new nutritional and functional source for meat products.

Physicochemical Properties of Meat Batter Added with Edible Silkworm Pupae (Bombyx mori) and Transglutaminase

PubMed Central

Choi, Yun-Sang

2017-01-01

This study was conducted to investigate the physicochemical properties of meat batters prepared with fresh pork meat, back fat, water, and salt and formulated with three different amounts (5%, 10%, and 15%) of silkworm pupae (Bombyx mori) powder and transglutaminase (TG). Meat batters formulated with silkworm pupae powder showed significantly higher contents of protein and ash than control batter. Addition of silkworm pupae to batter also showed significantly lower cooking loss than the control. Moreover, meat batter containing 15% silkworm pupae showed no significant difference in redness value compared to the control. In addition, pH, viscosity, hardness, gumminess, and chewiness were improved after the addition of silkworm pupae. Furthermore, meat batter formulated with TG and silkworm pupae showed improved hardness, gumminess, chewiness and viscosity compared to control batter. Addition of 1% TG with 15% silkworm pupae to meat batter resulted in significantly higher pH, textures, and viscosity. Our data suggest that both silkworm pupae and TG can be added to meat batter to improve its physicochemical properties. Therefore, combination of silkworm pupae and TG could be a new nutritional and functional source for meat products. PMID:28747820

Nutrient composition and respiration characteristics of silkworms in the Bioregenerative Life Support System

NASA Astrophysics Data System (ADS)

Tong, Ling; Yu, Xiaohui; Liu, Hong

As the appropriate space animal candidate, silkworm(Bombyx Mori L.) can supply animal food for taikonauts and consume inedible parts of plants in Bioregenerative Life Support Sys-tem(BLSS). Due to the features of BLSS, the silkworm breeding method in the system differ-ent from the conventional one is feeding the silkworm in the first three developing stages with mulberry leaves and with lettuce leaves in the latter two developing stages. Therefore, it is nec-essary to investigate the biochemical components and respiration characteristics of silkworms raised with this method to supply data bases for the inclusion of silkworms in the system to conduct system experiments. The nutrient compositions of silkworm powder (SP) which are the grinded and freeze-dried silkworm on the 3rd day in the fifth developing stage containing protein, fat, vitamins, minerals and fatty acids were determined with international standard analyzing methods in this study. The results showed that SP was rich in protein and amino acids. There were twelve kinds of essential vitamins, nine kinds of minerals and twelve kinds of fatty acids in SP. In contrast, SP had much better nutrient components than snail, fish, chicken, beef and pork as animal food for crew members. Moreover, 359 kCal can be generated per 100g of SP (dry weight). The respirations of silkworm during its whole growing process under two main physiological statuses which were eating and non-eating leaves were studied. According to the results measured by the animal respiration measuring system, there were much difference among the respirations of silkworms under the two main physiological statuses. The amounts of O2 inhaled and CO2 exhaled by the silkworms when they were eating leaves were more than those under the non-eating status. Even under the same status, the respiration characteristics of silkworms in five different developing stages were also different from one an-other. The respiratory quotients of silkworms under two

Effect of feeding silkworm on growth performance and feed efficiency of snakehead (Channa striata)

NASA Astrophysics Data System (ADS)

Firmani, U.; Lono

2018-04-01

The snakehead, Chana striata is a carnivorous freshwater fish and widely distributed in Asia. High demand of this fish has been triggering many aquaculturist to culture C. stiata. Feed was the important factor for fish growth. Silkworm has high protein content, low fat and can be used as natural feed for finfish. This study investigate the silkworm feed in C. striata. The treatment of this research were A (100 % pellet); B (100 % silkworm); C (combination of 75 % pellet and 25 % silkworm); D (combination of 50 % pellet and 50 % silkworm); and E (combination of 25 % pellet and 75 % silkworm). The variables measured in this study were relatif growth, specific growth rate, feed efficiency, feed conversion ratio, and survival rate. The result show that silkworm gave the high growth performance, feed efficiency and survival rate of the snakehead (Channa striata) compared with the control.

Accumulation of 1-deoxynojirimycin in silkworm, Bombyx mori L.

PubMed

Yin, Hao; Shi, Xin-qin; Sun, Bo; Ye, Jing-jing; Duan, Zu-an; Zhou, Xiao-ling; Cui, Wei-zheng; Wu, Xiao-feng

2010-04-01

1-deoxynojirimycin (1-DNJ) contents in the silkworm, Bombyx mori, at different developmental stages and tissues were investigated by using reverse-phase high-performance liquid chromatography. The 1-DNJ contents of silkworm larvae change significantly with their developmental stages. The male larvae showed higher accumulation efficiency of 1-DNJ than the females and also a significant variation was observed among the silkworm strains. The present results show that tissue distribution of 1-DNJ was significantly higher in blood, digestive juice, and alimentary canal, but no 1-DNJ was observed in the silkgland. Moreover, 1-DNJ was not found in silkworms fed with artificial diet that does not contain mulberry leaf powder. This proves that silkworms obtain 1-DNJ from mulberry leaves; they could not synthesize 1-DNJ by themselves. The accumulation and excretion of 1-DNJ change periodically during the larval stage. There was no 1-DNJ in the newly-hatched larvae and 1-DNJ was mainly accumulated during the early and middle stages of every instar, while excreted at later stages of larval development. Further, it is possible to extract 1-DNJ from the larval feces and it is optimal to develop the 1-DNJ related products for diabetic auxiliary therapy.

Hippo pathway regulates somatic development and cell proliferation of silkworm.

PubMed

Li, Niannian; Tong, Xiaoling; Zeng, Jie; Meng, Gang; Sun, Fuze; Hu, Hai; Song, Jiangbo; Lu, Cheng; Dai, Fangyin

2018-03-01

Hippo signaling pathway (signaling pathway Hippo, hereinafter referred to as the Hippo pathway) was the earliest found in Drosophila (Schneck [1]), which can regulate the development of tissues and organs, even some components of the pathway were identified as tumor suppressor [2]. The pathway was more concerned in fruit flies and mice (Schneck [1]), but little attention has been given in silkworm, an important economic and lepidopteran model insect. In this manuscript, we identified major Hippo pathway related genes (Hippo, Salvador, Warts, Mats, Yorkie) in silkworm and named BmHpo, BmSav, BmWts, BmMats, BmYki. The domain organization of these genes was highly conserved in silkworm and other organisms suggesting that they could use similar protein-protein interactions to construct the Hippo kinase cascades. The expression profiles of these genes in silkworm during embryonic, larval, wandering, pupal and adult stages were analyzed, 14 tissues/organs of the day 3, 5th instar larvae (L5D3) as well. Experimental results showed that the expression of Hippo pathway had some influence on the development of silkworm. In order to find out the mechanism of Hippo pathway affecting silkworm development, BmHpo and BmYki were up-regulated and de-regulated in the cell line of Bombyx mori-BmN-SWU1(NS), and the changes of cell proliferation activity and cell cycle were detected. The distribution of BmYki was detected by immunofluorescence technique. This study provides insights into the genes of Hippo pathway which have a certain effect on somatic development and cell proliferation in silkworm. Copyright © 2018 Elsevier Inc. All rights reserved.

Transgenic breeding of anti-Bombyx mori L. nuclear polyhedrosis virus silkworm Bombyx mori.

PubMed

Yang, Huijuan; Fan, Wei; Wei, Hao; Zhang, Jinwei; Zhou, Zhonghua; Li, Jianying; Lin, Jianrong; Ding, Nong; Zhong, Boxiong

2008-10-01

Silkworm strains resistant to Bombyx mori L. nuclear polyhedrosis virus were obtained through transgenic experiments. piggyBac transposon with an A3 promoter were randomly inserted into the silkworm, driving the enhanced green fluorescent protein (EGFP) reporter gene into the silkworm genome. Polymerase chain reaction results verified the insertion of the extraneous EGFP gene, and fluorescence microscopy showed that the EGFP was expressed in the midgut tissue. The morbidity ratio of the nuclear polyhedrosis decreased from 90% in the original silkworm strain to 66.7% in the transgenic silkworm strain. Compared with the resistance to the Bombyx mori L. nuclear polyhedrosis virus in the Qiufeng strain, which is commonly used in the production, there was an increase of 33 centesimal points in the transgenic silkworms. The antivirotic character in the Chunhua x Qiuyue strain, which was bred from a different transgenic family, was about 10 centesimal points higher than that in the Qiufeng x Baiyu, another crossbreed used in production. Our results indicated a good application value of the transposon-inserted mutation in the breeding of anti-BmNPV silkworm strain.

SilkPathDB: a comprehensive resource for the study of silkworm pathogens.

PubMed

Li, Tian; Pan, Guo-Qing; Vossbrinck, Charles R; Xu, Jin-Shan; Li, Chun-Feng; Chen, Jie; Long, Meng-Xian; Yang, Ming; Xu, Xiao-Fei; Xu, Chen; Debrunner-Vossbrinck, Bettina A; Zhou, Ze-Yang

2017-01-01

Silkworm pathogens have been heavily impeding the development of sericultural industry and play important roles in lepidopteran ecology, and some of which are used as biological insecticides. Rapid advances in studies on the omics of silkworm pathogens have produced a large amount of data, which need to be brought together centrally in a coherent and systematic manner. This will facilitate the reuse of these data for further analysis. We have collected genomic data for 86 silkworm pathogens from 4 taxa (fungi, microsporidia, bacteria and viruses) and from 4 lepidopteran hosts, and developed the open-access Silkworm Pathogen Database (SilkPathDB) to make this information readily available. The implementation of SilkPathDB involves integrating Drupal and GBrowse as a graphic interface for a Chado relational database which houses all of the datasets involved. The genomes have been assembled and annotated for comparative purposes and allow the search and analysis of homologous sequences, transposable elements, protein subcellular locations, including secreted proteins, and gene ontology. We believe that the SilkPathDB will aid researchers in the identification of silkworm parasites, understanding the mechanisms of silkworm infections, and the developmental ecology of silkworm parasites (gene expression) and their hosts. http://silkpathdb.swu.edu.cn. © The Author(s) 2017. Published by Oxford University Press.

SilkPathDB: a comprehensive resource for the study of silkworm pathogens

PubMed Central

Pan, Guo-Qing; Vossbrinck, Charles R.; Xu, Jin-Shan; Li, Chun-Feng; Chen, Jie; Long, Meng-Xian; Yang, Ming; Xu, Xiao-Fei; Xu, Chen; Debrunner-Vossbrinck, Bettina A.

2017-01-01

Silkworm pathogens have been heavily impeding the development of sericultural industry and play important roles in lepidopteran ecology, and some of which are used as biological insecticides. Rapid advances in studies on the omics of silkworm pathogens have produced a large amount of data, which need to be brought together centrally in a coherent and systematic manner. This will facilitate the reuse of these data for further analysis. We have collected genomic data for 86 silkworm pathogens from 4 taxa (fungi, microsporidia, bacteria and viruses) and from 4 lepidopteran hosts, and developed the open-access Silkworm Pathogen Database (SilkPathDB) to make this information readily available. The implementation of SilkPathDB involves integrating Drupal and GBrowse as a graphic interface for a Chado relational database which houses all of the datasets involved. The genomes have been assembled and annotated for comparative purposes and allow the search and analysis of homologous sequences, transposable elements, protein subcellular locations, including secreted proteins, and gene ontology. We believe that the SilkPathDB will aid researchers in the identification of silkworm parasites, understanding the mechanisms of silkworm infections, and the developmental ecology of silkworm parasites (gene expression) and their hosts. Database URL: http://silkpathdb.swu.edu.cn PMID:28365723

Nutrigenetic screening strains of the mulberry silkworm, Bombyx mori, for nutritional efficiency.

PubMed

Ramesha, Chinnaswamy; Lakshmi, Hothur; Kumari, Savarapu Sugnana; Anuradha, Chevva M; Kumar, Chitta Suresh

2012-01-01

The activity of sericulture is declining due the reduction of mulberry production area in sericulture practicing countries lead to adverse effects on silkworm rearing and cocoon production. Screening for nutrigenetic traits in silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) is an essential prerequisite for better understanding and development of nutritionally efficient breeds/hybrids, which show less food consumption with higher efficiency conversion. The aim of this study was to identify nutritionally efficient polyvoltine silkworm strains using the germplasm breeds RMW(2), RMW(3), RMW(4), RMG(3), RMG(1), RMG(4), RMG(5), RMG(6) and APM(1) as the control. The 1(st) day of 5(th) stage silkworm larvae of polyvoltine strains were subjected to standard gravimetric analysis until spinning for three consecutive generations covering 3 different seasons on 19 nutrigenetic traits. Highly significant (p ≤ 0.001) differences were found among all nutrigenetic traits of polyvoltine silkworm strains in the experimental groups. The nutritionally efficient polvoltine silkworm strains were resulted by utilizing nutrition consumption index and efficiency of conversion of ingesta/cocoon traits as the index. Higher nutritional efficiency conversions were found in the polyvoltine silkworm strains on efficiency of conversion of ingesta to cocoon and shell than control. Comparatively smaller consumption index, respiration, metabolic rate with superior relative growth rate, and quantum of food ingesta and digesta requisite per gram of cocoon and shell were found; the lowest amount was in new polyvoltine strains compared to the control. Furthermore, based on the overall nutrigenetic traits utilized as index or 'biomarkers', three polyvoltine silkworm strains (RMG(4), RMW(2), and RMW(3)) were identified as having the potential for nutrition efficiency conversion. The data from the present study advances our knowledge for the development of nutritionally efficient silkworm breeds

Nutrigenetic screening strains of the mulberry silkworm, Bombyx mori, for nutritional efficiency.

PubMed

Chinnaswamy, Ramesha; Lakshmi, Hothur; Kumari, Savarapu S; Anuradha, Chebba M; Kumar, Chitta S

2012-01-01

The activity of sericulture is declining due the reduction of mulberry production area in sericulture practicing countries lead to adverse effects on silkworm rearing and cocoon production. Screening for nutrigenetic traits in silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) is an essential prerequisite for better understanding and development of nutritionally efficient breeds/hybrids, which show less food consumption with higher efficiency conversion. The aim of this study was to identify nutritionally efficient polyvoltine silkworm strains using the germplasm breeds RMW(2), RMW(3), RMW(4), RMG(3), RMG(1), RMG(4), RMG(5), RMG(6) and APM(1) as the control. The 1(st) day of 5(th) stage silkworm larvae of polyvoltine strains were subjected to standard gravimetric analysis until spinning for three consecutive generations covering three different seasons on 19 nutrigenetic traits. Highly significant (p ≤ 0.001) differences were found among all nutrigenetic traits of polyvoltine silkworm strains in the experimental groups. The nutritionally efficient polvoltine silkworm strains were resulted by utilizing nutrition consumption index and efficiency of conversion of ingesta/cocoon traits as the index. Higher nutritional efficiency conversions were found in the polyvoltine silkworm strains on efficiency of conversion of ingesta to cocoon and shell than control. Comparatively smaller consumption index, respiration, metabolic rate with superior relative growth rate, and quantum of food ingesta and digesta requisite per gram of cocoon and shell were shown; the lowest amount was in new polyvoltine strains compared to the control. Furthermore, based on the overall nutrigenetic traits utilized as index or 'biomarkers', three polyvoltine silkworm strains (RMG(4), RMW(2), and RMW(3)) were identified as having the potential for nutrition efficiency conversion. The data from the present study advances our knowledge for the development of nutritionally efficient silkworm breeds

Nutrigenetic Screening Strains of the Mulberry Silkworm, Bombyx mori, for Nutritional Efficiency

PubMed Central

Chinnaswamy, Ramesha; Lakshmi, Hothur; Kumari, Savarapu S.; Anuradha, Chebba M.; Kumar, Chitta S.

2012-01-01

The activity of sericulture is declining due the reduction of mulberry production area in sericulture practicing countries lead to adverse effects on silkworm rearing and cocoon production. Screening for nutrigenetic traits in silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) is an essential prerequisite for better understanding and development of nutritionally efficient breeds/hybrids, which show less food consumption with higher efficiency conversion. The aim of this study was to identify nutritionally efficient polyvoltine silkworm strains using the germplasm breeds RMW2, RMW3, RMW4, RMG3, RMG1, RMG4, RMG5, RMG6 and APM1 as the control. The 1st day of 5th stage silkworm larvae of polyvoltine strains were subjected to standard gravimetric analysis until spinning for three consecutive generations covering three different seasons on 19 nutrigenetic traits. Highly significant (p ≤ 0.001) differences were found among all nutrigenetic traits of polyvoltine silkworm strains in the experimental groups. The nutritionally efficient polvoltine silkworm strains were resulted by utilizing nutrition consumption index and efficiency of conversion of ingesta/cocoon traits as the index. Higher nutritional efficiency conversions were found in the polyvoltine silkworm strains on efficiency of conversion of ingesta to cocoon and shell than control. Comparatively smaller consumption index, respiration, metabolic rate with superior relative growth rate, and quantum of food ingesta and digesta requisite per gram of cocoon and shell were shown; the lowest amount was in new polyvoltine strains compared to the control. Furthermore, based on the overall nutrigenetic traits utilized as index or ‘biomarkers’, three polyvoltine silkworm strains (RMG4, RMW2, and RMW3) were identified as having the potential for nutrition efficiency conversion. The data from the present study advances our knowledge for the development of nutritionally efficient silkworm breeds/hybrids and their

Nutrigenetic Screening Strains of the Mulberry Silkworm, Bombyx mori, for Nutritional Efficiency

PubMed Central

Ramesha, Chinnaswamy; Lakshmi, Hothur; Kumari, Savarapu Sugnana; Anuradha, Chevva M.; Kumar, Chitta Suresh

2012-01-01

The activity of sericulture is declining due the reduction of mulberry production area in sericulture practicing countries lead to adverse effects on silkworm rearing and cocoon production. Screening for nutrigenetic traits in silkworm, Bombyx mori L. (Lepidoptera: Bombycidae) is an essential prerequisite for better understanding and development of nutritionally efficient breeds/hybrids, which show less food consumption with higher efficiency conversion. The aim of this study was to identify nutritionally efficient polyvoltine silkworm strains using the germplasm breeds RMW2, RMW3, RMW4, RMG3, RMG1, RMG4, RMG5, RMG6 and APM1 as the control. The 1st day of 5th stage silkworm larvae of polyvoltine strains were subjected to standard gravimetric analysis until spinning for three consecutive generations covering 3 different seasons on 19 nutrigenetic traits. Highly significant (p ≤ 0.001) differences were found among all nutrigenetic traits of polyvoltine silkworm strains in the experimental groups. The nutritionally efficient polvoltine silkworm strains were resulted by utilizing nutrition consumption index and efficiency of conversion of ingesta/cocoon traits as the index. Higher nutritional efficiency conversions were found in the polyvoltine silkworm strains on efficiency of conversion of ingesta to cocoon and shell than control. Comparatively smaller consumption index, respiration, metabolic rate with superior relative growth rate, and quantum of food ingesta and digesta requisite per gram of cocoon and shell were found; the lowest amount was in new polyvoltine strains compared to the control. Furthermore, based on the overall nutrigenetic traits utilized as index or ‘biomarkers’, three polyvoltine silkworm strains (RMG4, RMW2, and RMW3) were identified as having the potential for nutrition efficiency conversion. The data from the present study advances our knowledge for the development of nutritionally efficient silkworm breeds/hybrids and their effective

Enhancement of the Gelation Properties of Surimi from Yellowtail Seabream (Parargyrops edita, Sparidae) with Chinese Oak Silkworm Pupa, Antheraea pernyi.

PubMed

Zhu, Jialin; Fan, Daming; Zhao, Jianxin; Zhang, Hao; Huang, Jianlian; Zhou, Wenguo; Zhang, Wenhai; Chen, Wei

2016-02-01

In this study, the textural properties and micromechanism of yellowtail seabream (Parargyrops edita, Sparidae) surimi, with and without Chinese oak silkworm pupa homogenate (SPH), were investigated at different levels. The fresh, freeze-dried, and oven-dried SPH all showed a gel-enhancing ability in suwari (40/90 °C) and modori (67/90 °C) gels, in a concentration-dependent manner. Though the drying treatments can improve the storability of SPH, compared with fresh, the effect of the active substance was weakened. Suwari and modori gels added with 5%(w/w, whole product) fresh SPH had the increase in breaking force and deformation by 37.39% and 47.98%, and 85.14% and 78.49%, respectively, compared with the control gel (without SPH addition). The major myofibrillar protein, especially myosin heavy chain (MHC), was better retained by the addition of SPH. Compared the control group, a finer, denser, and more ordered 3-dimensional gel network microstructure was obtained, and different Df (Fractal dimension) was analyzed by using the box count method. This was found in all samples from 2.838 to 2.864 for suwari gels and 2.795 to 2.857 for modori gels, respectively. Therefore, the modori of yellowtail seabream surimi, linked with endogenous proteases, could be retarded in the presence of SPH, leading to an increase in gel strength. © 2015 Institute of Food Technologists®

Serial analysis of gene expression in the silkworm, Bombyx mori.

PubMed

Huang, Jianhua; Miao, Xuexia; Jin, Weirong; Couble, Pierre; Mita, Kasuei; Zhang, Yong; Liu, Wenbin; Zhuang, Leijun; Shen, Yan; Keime, Celine; Gandrillon, Olivier; Brouilly, Patrick; Briolay, Jerome; Zhao, Guoping; Huang, Yongping

2005-08-01

The silkworm Bombyx mori is one of the most economically important insects and serves as a model for Lepidoptera insects. We used serial analysis of gene expression (SAGE) to derive profiles of expressed genes during the developmental life cycle of the silkworm and to create a reference for understanding silkworm metamorphosis. We generated four SAGE libraries, one from each of the four developmental stages of the silkworm. In total we obtained 257,964 SAGE tags, of which 39,485 were unique tags. Sorted by copy number, 14.1% of the unique tags were detected at a median to high level (five or more copies), 24.2% at lower levels (two to four copies), and 61.7% as single copies. Using a basic local alignment search tool on the EST database, 35% of the tags matched known silkworm expressed sequence tags. SAGE demonstrated that a number of the genes were up- or down-regulated during the four developmental phases of the egg, larva, pupa, and adult. Furthermore, we found that the generation of longer cDNA fragments from SAGE tags constituted the most efficient method of gene identification, which facilitated the analysis of a large number of unknown genes.

Biomimetic Nucleation of Hydroxyapatite Crystals Mediated by Antheraea pernyi Silk Sericin Promotes Osteogenic Differentiation of Human Bone Marrow Derived Mesenchymal Stem Cells

PubMed Central

2015-01-01

Biomacromolecules have been used as templates to grow hydroxyapatite crystals (HAps) by biomineralization to fabricate mineralized materials for potential application in bone tissue engineering. Silk sericin is a protein with features desirable as a biomaterial, such as increased hydrophilicity and biodegradation. Mineralization of the silk sericin from Antheraea pernyi (A. pernyi) silkworm has rarely been reported. Here, for the first time, nucleation of HAps on A. pernyi silk sericin (AS) was attempted through a wet precipitation method and consequently the cell viability and osteogenic differentiation of BMSCs on mineralized AS were investigated. It was found that AS mediated the nucleation of HAps in the form of nanoneedles while self-assembling into β-sheet conformation, leading to the formation of a biomineralized protein based biomaterial. The cell viability assay of BMSCs showed that the mineralization of AS stimulated cell adhesion and proliferation, showing that the resultant AS biomaterial is biocompatible. The differentiation assay confirmed that the mineralized AS significantly promoted the osteogenic differentiation of BMSCs when compared to nonmineralized AS as well as other types of sericin (B. mori sericin), suggesting that the resultant mineralized AS biomaterial has potential in promoting bone formation. This result represented the first work proving the osteogenic differentiation of BMSCs directed by silk sericin. Therefore, the biomineralization of A. pernyi silk sericin coupled with seeding BMSCs on the resultant mineralized biomaterials is a useful strategy to develop the potential application of this unexplored silk sericin in the field of bone tissue engineering. This study lays the foundation for the use of A. pernyi silk sericin as a potential scaffold for tissue engineering. PMID:24666022

Biomimetic nucleation of hydroxyapatite crystals mediated by Antheraea pernyi silk sericin promotes osteogenic differentiation of human bone marrow derived mesenchymal stem cells.

PubMed

Yang, Mingying; Shuai, Yajun; Zhang, Can; Chen, Yuyin; Zhu, Liangjun; Mao, Chuanbin; OuYang, Hongwei

2014-04-14

Biomacromolecules have been used as templates to grow hydroxyapatite crystals (HAps) by biomineralization to fabricate mineralized materials for potential application in bone tissue engineering. Silk sericin is a protein with features desirable as a biomaterial, such as increased hydrophilicity and biodegradation. Mineralization of the silk sericin from Antheraea pernyi (A. pernyi) silkworm has rarely been reported. Here, for the first time, nucleation of HAps on A. pernyi silk sericin (AS) was attempted through a wet precipitation method and consequently the cell viability and osteogenic differentiation of BMSCs on mineralized AS were investigated. It was found that AS mediated the nucleation of HAps in the form of nanoneedles while self-assembling into β-sheet conformation, leading to the formation of a biomineralized protein based biomaterial. The cell viability assay of BMSCs showed that the mineralization of AS stimulated cell adhesion and proliferation, showing that the resultant AS biomaterial is biocompatible. The differentiation assay confirmed that the mineralized AS significantly promoted the osteogenic differentiation of BMSCs when compared to nonmineralized AS as well as other types of sericin (B. mori sericin), suggesting that the resultant mineralized AS biomaterial has potential in promoting bone formation. This result represented the first work proving the osteogenic differentiation of BMSCs directed by silk sericin. Therefore, the biomineralization of A. pernyi silk sericin coupled with seeding BMSCs on the resultant mineralized biomaterials is a useful strategy to develop the potential application of this unexplored silk sericin in the field of bone tissue engineering. This study lays the foundation for the use of A. pernyi silk sericin as a potential scaffold for tissue engineering.

[Effects of silkworm pupa oil on serum lipids level and platelet function in rats].

PubMed

Yang, Xuefeng; Huang, Lianzhen; Hu, Jianping; Li, Tao

2002-08-01

To observe the effects of silkworm pupa oil on serum lipids level and platelet function in rats, according to serum TG, TC level, 40 male Wistar rats are divided into four groups (normal control group, high fat control group, silkworm pupa oil group and silkworm pupa oil + VE group). The rats are fed different diets and six weeks later, serum lipids level and platelet function are measured. The results show that (1) Compared with high fat control group, serum TC, TG, LDL-C level, AI value, Platelet aggregability, plasma TXB2 level and T/P ratio decrease significantly while HDL-C level and 6-k-PGF1 level increase in silkworm pupa oil group; (2) Serum TC, LDL-C level, T/P ratio and platelet aggregability are significantly lower in silkworm pupa oil + VE group than in silkworm pupa oil group. It is suggested that silkworm pupa oil rich in alpha-linolenic acid can reduce serum lipids level and inhibit platelet aggregation, which is more effective with the supplementation with VE.

Genetic diversity among silkworm (Bombyx mori L., Lep., Bombycidae) germplasms revealed by microsatellites.

PubMed

Li, Muwang; Shen, Li; Xu, Anying; Miao, Xuexia; Hou, Chengxiang; Sun, Pingjiang; Zhang, Yuehua; Huang, Yongping

2005-10-01

To determine genetic relationships among strains of silkworm, Bombyx mori L., 31 strains with different origins, number of generations per year, number of molts per generation, and morphological characters were studied using simple sequence repeat (SSR) markers. Twenty-six primer pairs flanking microsatellite sequences in the silkworm genome were assayed. All were polymorphic and unambiguously separated silkworm strains from each other. A total of 188 alleles were detected with a mean value of 7.2 alleles/locus (range 2-17). The average heterozygosity value for each SSR locus ranged from 0 to 0.60, and the highest one was 0.96 (Fl0516 in 4013). The mean polymorphism index content (PIC) was 0.66 (range 0.12-0.89). Unweighted pair group method with arithmetic means (UPGMA) cluster analysis of Nei's genetic distance grouped silkworm strains based on their origin. Seven major ecotypic silkworm groups were analyzed. Principal components analysis (PCA) for SSR data support their UPGMA clustering. The results indicated that SSR markers are an efficient tool for fingerprinting cultivars and conducting genetic-diversity studies in the silkworm.

Silkworm silk-based materials and devices generated using bio-nanotechnology.

PubMed

Huang, Wenwen; Ling, Shengjie; Li, Chunmei; Omenetto, Fiorenzo G; Kaplan, David L

2018-06-25

Silks are natural fibrous protein polymers that are spun by silkworms and spiders. Among silk variants, there has been increasing interest devoted to the silkworm silk of B. mori, due to its availability in large quantities along with its unique material properties. Silk fibroin can be extracted from the cocoons of the B. mori silkworm and combined synergistically with other biomaterials to form biopolymer composites. With the development of recombinant DNA technology, silks can also be rationally designed and synthesized via genetic control. Silk proteins can be processed in aqueous environments into various material formats including films, sponges, electrospun mats and hydrogels. The versatility and sustainability of silk-based materials provides an impressive toolbox for tailoring materials to meet specific applications via eco-friendly approaches. Historically, silkworm silk has been used by the textile industry for thousands of years due to its excellent physical properties, such as lightweight, high mechanical strength, flexibility, and luster. Recently, due to these properties, along with its biocompatibility, biodegradability and non-immunogenicity, silkworm silk has become a candidate for biomedical utility. Further, the FDA has approved silk medical devices for sutures and as a support structure during reconstructive surgery. With increasing needs for implantable and degradable devices, silkworm silk has attracted interest for electronics, photonics for implantable yet degradable medical devices, along with a broader range of utility in different device applications. This Tutorial review summarizes and highlights recent advances in the use of silk-based materials in bio-nanotechnology, with a focus on the fabrication and functionalization methods for in vitro and in vivo applications in the field of tissue engineering, degradable devices and controlled release systems.

Metabolomics differences between silkworms (Bombyx mori) reared on fresh mulberry (Morus) leaves or artificial diets.

PubMed

Dong, Hui-Ling; Zhang, Sheng-Xiang; Tao, Hui; Chen, Zhuo-Hua; Li, Xue; Qiu, Jian-Feng; Cui, Wen-Zhao; Sima, Yang-Hu; Cui, Wei-Zheng; Xu, Shi-Qing

2017-09-08

Silkworms (Bombyx mori) reared on artificial diets have great potential applications in sericulture. However, the mechanisms underlying the enhancement of metabolic utilization by altering silkworm nutrition are unclear. The aim of this study was to investigate the mechanisms responsible for the poor development and low silk protein synthesis efficiency of silkworms fed artificial diets. After multi-generational selection of the ingestive behavior of silkworms to artificial diets, we obtained two strains, one of which developed well and another in which almost all its larvae starved to death on the artificial diets. Subsequently, we analyzed the metabolomics of larval hemolymph by gas chromatography/liquid chromatography-mass spectrometry, and the results showed that vitamins were in critically short supply, whereas the nitrogen metabolic end product of urea and uric acid were enriched substantially, in the hemolymph of the silkworms reared on the artificial diets. Meanwhile, amino acid metabolic disorders, as well as downregulation of carbohydrate metabolism, energy metabolism, and lipid metabolism, co-occurred. Furthermore, 10 male-dominant metabolites and 27 diet-related metabolites that differed between male and female silkworms were identified. These findings provide important insights into the regulation of silkworm metabolism and silk protein synthesis when silkworms adapt to an artificial diet.

Improvement of light penetration based silkworm gender identification with confined regions of interest

NASA Astrophysics Data System (ADS)

Kamtongdee, Chakkrit; Sumriddetchkajorn, Sarun; Sa-ngiamsak, Chiranut

2013-06-01

Based on our previous work on light penetration-based silkworm gender identification, we find that unwanted optical noises scattering from the surrounding area near the silkworm pupa and the transparent support are sometimes analyzed and misinterpreted leading to incorrect silkworm gender identification. To alleviate this issue, we place a small rectangular hole on a transparent support so that it not only helps the user precisely place the silkworm pupa but also functions as a region of interest (ROI) for blocking unwanted optical noises and for roughly locating the abdomen region in the image for ease of image processing. Apart from the external ROI, we also assign a smaller ROI inside the image in order to remove strong scattering light from all edges of the external ROI and at the same time speed up our image processing operations. With only the external ROI in function, our experiment shows a measured 86% total accuracy in identifying gender of 120 silkworm pupae with a measured average processing time of 38 ms. Combining the external ROI and the image ROI together revamps the total accuracy in identifying the silkworm gender to 95% with a measured faster 18 ms processing time.

Milled non-mulberry silk fibroin microparticles as biomaterial for biomedical applications.

PubMed

Bhardwaj, Nandana; Rajkhowa, Rangam; Wang, Xungai; Devi, Dipali

2015-11-01

Silk fibroin has been widely employed in various forms as biomaterials for biomedical applications due to its superb biocompatibility and tunable degradation and mechanical properties. Herein, silk fibroin microparticles of non-mulberry silkworm species (Antheraea assamensis, Antheraea mylitta and Philosamia ricini) were fabricated via a top-down approach using a combination of wet-milling and spray drying techniques. Microparticles of mulberry silkworm (Bombyx mori) were also utilized for comparative studies. The fabricated microparticles were physico-chemically characterized for size, stability, morphology, chemical composition and thermal properties. The silk fibroin microparticles of all species were porous (∼5μm in size) and showed nearly spherical morphology with rough surface as revealed from dynamic light scattering and microscopic studies. Non-mulberry silk microparticles maintained the typical silk-II structure with β-sheet secondary conformation with higher thermal stability. Additionally, non-mulberry silk fibroin microparticles supported enhanced cell adhesion, spreading and viability of mouse fibroblasts than mulberry silk fibroin microparticles (p<0.001) as evidenced from fluorescence microscopy and cytotoxicity studies. Furthermore, in vitro drug release from the microparticles showed a significantly sustained release over 3 weeks. Taken together, this study demonstrates promising attributes of non-mulberry silk fibroin microparticles as a potential drug delivery vehicle/micro carrier for diverse biomedical applications. Copyright © 2015 Elsevier B.V. All rights reserved.

Optical penetration-based silkworm pupa gender sensor structure.

PubMed

Sumriddetchkajorn, Sarun; Kamtongdee, Chakkrit

2012-02-01

This paper proposes and experimentally demonstrates for what is believed to be the first time a highly sought-after optical structure for highly-accurate identification of the silkworm pupa gender. The key idea is to exploit a long wavelength optical beam in the red or near infrared spectrum that can effectively and safely penetrate the body of a silkworm pupa. Later on, simple image processing operations via image thresholding, blob filtering, and image inversion processes are applied in order to eliminate the unwanted image noises and at the same time highlight the gender gland. Experimental proof of concept using three 636 nm wavelength light emitting diodes, a two-dimensional web camera, an 8 bit microcontroller board, and a notebook computer shows a very high 95.6% total accuracy in identifying the gender of 45 silkworm pupae with a measured fast identification time of 96.6 ms. Other key features include low cost, low component counts, and ease of implementation and control.

Metabolism, transformation and dynamic changes of alkaloids in silkworm during feeding mulberry leaves.

PubMed

Zhang, Liwen; Bai, Yongliang; Su, Shulan; Ouyang, Zhen; Liu, Li; Pan, Gang; Qian, Dawei; Duan, Jinao

2018-06-04

Metabolism, transformation and dynamic changes of DNJ, 2-O-α-D-Gal-DNJ, fagomine, isofagomine and 4-O-β-d-Glc-fagomine from mulberry leaves in silkworms at different instars were observed. UPLC-Q/TOF-MS and UPLC-TQ/MS methods were adopted for qualitative and quantitative analysis respectively. Three species mulberry leaves were used to feed the silkworm as controls. By analyzing and comparing the content changes of DNJ, fagomine and their derivatives in silkworms and silkworm excrement at different instar, we revealed the dynamic changes, confirmed the enrichment effect of the polyhydroxy alkaloids by silkworm, and inferred the conversion process behind this effect. The experimental results indicated that DNJ and its derivatives turned into some intermediate substances in the metabolic process, and finally they converted back and the content increased. Fagomine and its derivatives interconverted into each other in the process, 4-O-β-d-Glc-fagomine transformed into fagomine, while fagomine transformed into isofagomine.

Initial ground experiments of silkworm cultures living on different feedstock for provision of high quality animal protein for human in space

NASA Astrophysics Data System (ADS)

Yang, Yunan; Tang, Liman; Tong, Ling; Liu, Yang; Liu, Hong; Li, Xiaomin

2010-09-01

Silkworm could be an alternative to provide edible animal protein in Controlled Ecological Life Support System (CELSS) for long-term manned space missions. Silkworms can consume non-edible plant residue and convert plant nutrients to high quality edible animal protein for astronauts. The preliminary investigation of silkworm culture was carried out in earth environment. The silkworms were fed with artificial silkworm diet and the leaves of stem lettuce ( Lactuca sativa L. var. angustana Irish) separately and the nutritional structure of silkworm was investigated and compared, The culture experiments showed that: (1) Stem lettuce leaves could be used as food of silkworm. The protein content of silkworm fed with lettuce leaves can reach 70% of dry mass. (2) The protein content of silkworm powder produced by the fifth instar silkworm larvae was 70%, which was similar to the protein content of silkworm pupae. The powder of the fifth instar silkworm larvae can be utilized by astronaut. (3) The biotransformation rate of silkworm larvae between the third instar and the fifth instar could reach above 70%. The biotransformation cycle of silkworm was determined as 24 days. (4) Using the stem lettuce leaves to raise silkworm, the coarse fiber content of silkworm excrements reached about 33%. The requirements of space silkworm culture equipment, feeding approaches and feeding conditions were also preliminarily designed and calculated. It is estimated that 2.2 m 3 of culture space could satisfy daily animal protein demand for seven astronauts.

Microbial shifts of the silkworm larval gut in response to lettuce leaf feeding.

PubMed

Liang, Xue; Fu, Yuming; Tong, Ling; Liu, Hong

2014-04-01

Silkworm (Bombyx mori L.) larvae were used as an ideal animal protein source for astronauts in the bioregenerative life support system (BLSS). Here, we compared the difference in bacterial communities of the silkworm larval gut between the BLSS rearing way (BRW) and the traditional rearing way (TRW) through culture-dependent approach, 16S rRNA gene analysis, and denaturing gradient gel electrophoresis (DGGE). The culture-dependent approach revealed that the numbers of gut bacteria of silkworm in the BRW significantly decreased compared with that of the TRW. The analysis of clone libraries showed that the gut microbiota in the BRW was significantly less diverse than that in the TRW. Acinetobacter and Bacteroides were dominant populations in the BRW, and Bacillus and Arcobacter dominated in the TRW. DGGE profiles confirmed the difference of silkworm gut bacterial community between two rearing ways. These results demonstrate that gut bacteria change from the BRW contributes to the decrease of silkworm physiological activity. This study increases our understanding of the change of silkworm gut microbiota in response to lettuce leaf feeding in the BRW. We could use the dominant populations to make probiotic products for nutrient absorption and disease prevention in the BLSS to improve gut microecology, as well as the yield and quality of animal protein.

Genetic diversity, molecular phylogeny and selection evidence of the silkworm mitochondria implicated by complete resequencing of 41 genomes

PubMed Central

2010-01-01

Background Mitochondria are a valuable resource for studying the evolutionary process and deducing phylogeny. A few mitochondria genomes have been sequenced, but a comprehensive picture of the domestication event for silkworm mitochondria remains to be established. In this study, we integrate the extant data, and perform a whole genome resequencing of Japanese wild silkworm to obtain breakthrough results in silkworm mitochondrial (mt) population, and finally use these to deduce a more comprehensive phylogeny of the Bombycidae. Results We identified 347 single nucleotide polymorphisms (SNPs) in the mt genome, but found no past recombination event to have occurred in the silkworm progenitor. A phylogeny inferred from these whole genome SNPs resulted in a well-classified tree, confirming that the domesticated silkworm, Bombyx mori, most recently diverged from the Chinese wild silkworm, rather than from the Japanese wild silkworm. We showed that the population sizes of the domesticated and Chinese wild silkworms both experience neither expansion nor contraction. We also discovered that one mt gene, named cytochrome b, shows a strong signal of positive selection in the domesticated clade. This gene is related to energy metabolism, and may have played an important role during silkworm domestication. Conclusions We present a comparative analysis on 41 mt genomes of B. mori and B. mandarina from China and Japan. With these, we obtain a much clearer picture of the evolution history of the silkworm. The data and analyses presented here aid our understanding of the silkworm in general, and provide a crucial insight into silkworm phylogeny. PMID:20334646

Vertebrate estrogen regulates the development of female characteristics in silkworm, Bombyx mori.

PubMed

Shen, Guanwang; Lin, Ying; Yang, Congwen; Xing, Runmiao; Zhang, Haiyan; Chen, Enxiang; Han, Chaoshan; Liu, Hongling; Zhang, Weiwei; Xia, Qingyou

2015-01-01

The vertebrate estrogens include 17-β-estradiol (E2), which has an analog in silkworm ovaries. In this study, the Bombyx mori vitellogenin gene (BmVg) was used as a biomarker to analyze the function of the E2 in silkworm. In most oviparous animals, Vg has female-specific expression. However, BmVg expression was also detected in B. mori males. Stage specific fluctuation of BmVg expression was similar in males and females, but expression levels in males were lower than in females. E2 treatment by injection or feeding of male larvae in the final instar stage induced and stimulated male BmVg transcription and protein synthesis. When silkworm ovary primordia were transplanted into males, BmVg was induced in male fat bodies. Transplanted ovaries primordia were also able to develop into ovaries and produce mature eggs. When females were treated with E2 promoted BmVg/BmVn protein accumulation in hemolymph, ovaries and eggs. However, BmVg transcription was decreased in female fat bodies. An E2 analog was identified in the hemolymph of day 3 wandering silkworms using high-performance liquid chromatography. Estradiol titers from fifth late-instar larvae to pupal stage were determined by enzyme-linked immunosorbent assay. The results suggested that silkworms synthesized a vertebrate E2 analog. This study found that E2 promoted the synthesis of BmVg, a female typical protein in silkworms. Copyright © 2014 Elsevier Inc. All rights reserved.

Calcium Oxalate Accumulation in Malpighian Tubules of Silkworm (Bombyx mori)

NASA Astrophysics Data System (ADS)

Wyman, Aaron J.; Webb, Mary Alice

2007-04-01

Silkworm provides an ideal model system for study of calcium oxalate crystallization in kidney-like organs, called Malpighian tubules. During their growth and development, silkworm larvae accumulate massive amounts of calcium oxalate crystals in their Malpighian tubules with no apparent harm to the organism. This manuscript reports studies of crystal structure in the tubules along with analyses identifying molecular constituents of tubule exudate.

Transformation of Neomycin Resistance Gene (neo(R)) into Silkworm (Bombyx mori.L.).

PubMed

Chen, Xiu; Zhao, Yun; Zhang, Feng; Peng, Wei-Ping; Feng, Xiao-Li; Huang, Jun-Ting; Lu, Chang-De

1999-01-01

Neomycin resistance gene (neo(R)) flanked by 5' and 3' regions of fibroin H-chain gene of silkworm (Bombyx mori.L.) was transferred into eggs of silkworm by gene gun in the early period of fertilization. The larvae were fed with an artificial diet containing neomycin in early 24 hours post transfettion, and some of them survived. The neo(R) encoding sequence in G(2) generation derived from the survivals was detected by Southern blotting. The results indicated that neo(R) could be used as a selective marker for studies on transgenic silkworm.

Cadmium transfer and detoxification mechanisms in a soil-mulberry-silkworm system: phytoremediation potential.

PubMed

Zhou, Lingyun; Zhao, Ye; Wang, Shuifeng

2015-11-01

Phytoremediation has been proven to be an environmentally sound alternative for the recovery of contaminated soils, and the economic profit that comes along with the process might stimulate its field use. This study investigated cadmium (Cd) transfer and detoxification mechanisms in a soil-mulberry-silkworm system to estimate the suitability of the mulberry and silkworm as an alternative method for the remediation of Cd-polluted soil; it also explored the underlying mechanisms regulating the trophic transfer of Cd. The results show that both the mulberry and silkworm have high Cd tolerance. The transfer factor suggests that the mulberry has high potential for Cd extraction from polluted soil. The subcellular distribution and chemical forms of Cd in mulberry leaves show that cell wall deposition and vacuolar compartmentalization play important role in Cd tolerance. In the presence of increasing Cd concentrations in silkworm food, detoxification mechanisms (excretion and homeostasis) were activated so that excess Cd was excreted in fecal balls, and metallothionein levels in the mid-gut, the posterior of the silk gland, and the fat body of silkworms were enhanced. And, the Cd concentrations in silk are at a low level, ranging from 0.02 to 0.21 mg kg(-1). Therefore, these mechanisms of detoxification can regulate Cd trophic transfer, and mulberry planting and silkworm breeding has high phytoremediation potential for Cd-contaminated soil.

Evaluation of innate immune stimulating activity of polysaccharides using a silkworm (Bombyx mori) muscle contraction assay.

PubMed

Fujiyuki, T; Hamamoto, H; Ishii, K; Urai, M; Kataoka, K; Takeda, T; Shibata, S; Sekimizu, K

2012-04-01

In silkworm larvae, the mature form of paralytic peptide (PP), an insect cytokine, is produced from pro-PP in association with activation of innate immune responses, resulting in slow muscle contraction. We utilized this reaction, muscle contraction in silkworms coupled with innate immunity stimulation, to quantitatively measure the innate immune stimulating activity of various natural polysaccharides. β-Glucan of Gyrophora esculenta (GE-3), fucoidan from sporophyll of Undaria pinnatifida, and curldan induced silkworm muscle contraction. We further demonstrated that GE-3 had therapeutic effects on silkworms infected by baculovirus. Based on these findings, we propose that the silkworm muscle contraction assay is useful for screening substances that stimulate innate immunity before evaluating therapeutic effectiveness in mammals.

Characterization of anti-CD20 monoclonal antibody produced by transgenic silkworms (Bombyx mori).

PubMed

Tada, Minoru; Tatematsu, Ken-ichiro; Ishii-Watabe, Akiko; Harazono, Akira; Takakura, Daisuke; Hashii, Noritaka; Sezutsu, Hideki; Kawasaki, Nana

2015-01-01

In response to the successful use of monoclonal antibodies (mAbs) in the treatment of various diseases, systems for expressing recombinant mAbs using transgenic animals or plants have been widely developed. The silkworm (Bombyx mori) is a highly domesticated insect that has recently been used for the production of recombinant proteins. Because of their cost-effective breeding and relatively easy production scale-up, transgenic silkworms show great promise as a novel production system for mAbs. In this study, we established a transgenic silkworm stably expressing a human-mouse chimeric anti-CD20 mAb having the same amino acid sequence as rituximab, and compared its characteristics with rituximab produced by Chinese hamster ovary (CHO) cells (MabThera®). The anti-CD20 mAb produced in the transgenic silkworm showed a similar antigen-binding property, but stronger antibody-dependent cell-mediated cytotoxicity (ADCC) and weaker complement-dependent cytotoxicity (CDC) compared to MabThera. Post-translational modification analysis was performed by peptide mapping using liquid chromatography/mass spectrometry. There was a significant difference in the N-glycosylation profile between the CHO- and the silkworm-derived mAbs, but not in other post-translational modifications including oxidation and deamidation. The mass spectra of the N-glycosylated peptide revealed that the observed biological properties were attributable to the characteristic N-glycan structures of the anti-CD20 mAbs produced in the transgenic silkworms, i.e., the lack of the core-fucose and galactose at the non-reducing terminal. These results suggest that the transgenic silkworm may be a promising expression system for the tumor-targeting mAbs with higher ADCC activity.

Ultrafast Screening of a Novel, Moderately Hydrophilic Angiotensin-Converting-Enzyme-Inhibitory Peptide, RYL, from Silkworm Pupa Using an Fe-Doped-Silkworm-Excrement-Derived Biocarbon: Waste Conversion by Waste.

PubMed

Liu, Long; Wei, Yanan; Chang, Qing; Sun, Huaju; Chai, Kungang; Huang, Zuqiang; Zhao, Zhenxia; Zhao, Zhongxing

2017-12-27

A novel, moderately hydrophilic peptide (RYL) with high ACE-inhibitory activity was screened ultrafast via a concept of waste conversion using waste. This novel peptide was screened from silkworm pupa using an Fe-doped porous biocarbon (FL/Z-SE) derived from silkworm excrement. FL/Z-SE possessed magnetic properties and specific selection for peptides due to Fe's dual functions. The selected RYL, which has moderate hydrophilicity (LogP = -0.22), exhibited a comparatively high ACE-inhibitory activity (IC 50 = 3.31 ± 0.11 μM). The inhibitory kinetics and docking-simulation results show that, as a competitive ACE inhibitor, RYL formed five hydrogen bonds with the ACE residues in the S1 and S2 pockets. In this work, both the screening carbon material and the selected ACE-inhibitory peptide were derived from agricultural waste (silkworm excrement and pupa), which offers a new way of thinking about the development of advanced uses of the silkworm byproducts and wastes.

[Optimization of Extraction Technology for Sericin from Silkworm Cocoon with Orthogonal Design].

PubMed

Zhao, Chun-ying; Wang, Yan; Li, Yun-feng; Chen, Zhi-hong

2015-05-01

To optimize the appropriate extracting technology for sericin from Silkworm cocoon. Using sericin extraction rates and sericin content as the indices. The single and orthogonal experiments were used to determine the best conditions. The optimal extraction technology for sericin from Silkworm cocoon was as follows: 1: 30 for the ratio of solid to liquid, 3 h reflux for 2 times of extraction and water temperature at 100 degrees C. The extraction rate of sericin from Silkworm cocoon was 27.1%. The optimal extraction technology is stable, feasible, and can provide reference for further pharmacological study on cocoon sericin.

Anti-Mycobacterium activity of microbial peptides in a silkworm infection model with Mycobacterium smegmatis.

PubMed

Yagi, Akiho; Uchida, Ryuji; Hamamoto, Hiroshi; Sekimizu, Kazuhisa; Kimura, Ken-Ichi; Tomoda, Hiroshi

2017-05-01

An in vivo-mimic silkworm infection model with Mycobacterium smegmatis was established. When silkworms were raised at 37 °C following an injection of M. smegmatis cells (1.25 × 10 7 CFU larva -1  g -1 ) into the silkworm hemolymph, they died within 48 h. Under these conditions, four microbial peptides with anti-M. smegmatis activity, lariatin A, calpinactam, lysocin E and propeptin, exerted therapeutic effects in a dose-dependent manner, and these are also clinically used agents that are active against Mycobacterium tuberculosis. These results indicate that the silkworm infection model with M. smegmatis is practically useful for the screening of therapeutically effective anti-M. tuberculosis antibiotics.

Lead in the soil-mulberry (Morus alba L.)-silkworm (Bombyx mori) food chain: translocation and detoxification.

PubMed

Zhou, Lingyun; Zhao, Ye; Wang, Shuifeng; Han, Shasha; Liu, Jing

2015-06-01

The translocation of lead (Pb) in the soil-mulberry-silkworm food chain and the process of Pb detoxification in the mulberry-silkworm chain were investigated. The amount of Pb in mulberry, silkworm, feces and silk increased in a dose-responsive manner to the Pb contents in the soils. Mulberry roots sequestered most of the Pb, ranging from 230.78 to 1209.25 mg kg(-1). Over 92% of the Pb in the mulberry leaves was deposited in the cell wall, and 95.29-95.57% of the Pb in the mulberry leaves was integrated with oxalic acid, pectates and protein, and it had low bioavailability. The Pb concentrations in the silkworm feces were 4.50-4.64 times higher than those in the leaves. The synthesis of metallothioneins in three tissues of the silkworms was induced to achieve Pb homeostasis under Pb stress. These results indicated the mechanism involved in Pb transfer along the food chain was controlled by the detoxification of Pb in different trophic levels. Planting mulberry and rearing silkworm could be a promising approach for the remediation of Pb-polluted soils due to the Pb tolerance of mulberry and silkworm. Copyright © 2015. Published by Elsevier Ltd.

Vitellogenin from the silkworm, Bombyx mori: an effective anti-bacterial agent.

PubMed

Singh, Nitin Kumar; Pakkianathan, Britto Cathrin; Kumar, Manish; Prasad, Tulika; Kannan, Mani; König, Simone; Krishnan, Muthukalingan

2013-01-01

Silkworm, Bombyx mori, vitellogenin (Vg) was isolated from perivisceral fat body of day 3 of pupa. Both Vg subunits were co-purified as verified by mass spectrometry and immunoblot. Purified Vg responded to specific tests for major posttranslational modifications on native gels indicating its nature as lipo-glyco-phosphoprotein. The Vg fraction had strong antibacterial activity against Gram negative bacterium Escherichia coli and Gram positive bacterium Bacillus subtilis. Microscopic images showed binding of Vg to bacterial cells and their destruction. When infected silkworm larvae were treated with purified Vg they survived the full life cycle in contrast to untreated animals. This result showed that Vg has the ability to inhibit the proliferation of bacteria in the silkworm fluid system without disturbing the regular metabolism of the host.

JAK/STAT signaling pathway-mediated immune response in silkworm (Bombyx mori) challenged by Beauveria bassiana.

PubMed

Geng, Tao; Lv, Ding-Ding; Huang, Yu-Xia; Hou, Cheng-Xiang; Qin, Guang-Xing; Guo, Xi-Jie

2016-12-20

Innate immunity was critical in insects defensive system and able to be induced by Janus kinase/signal transducer and activator of transcription cascade transduction (JAK/STAT) signaling pathway. Currently, it had been identified many JAK/STAT signaling pathway-related genes in silkworm, but little function was known on insect innate immunity. To explore the roles of JAK/STAT pathway in antifungal immune response in silkworm (Bombyx mori) against Beauveria bassiana infection, the expression patterns of B. mori C-type lectin 5 (BmCTL5) and genes encoding 6 components of JAK/STAT signaling pathway in silkworm challenged by B. bassiana were analyzed using quantitative real time PCR. Meanwhile the activation of JAK/STAT signaling pathway by various pathogenic micro-organisms and the affect of JAK/STAT signaling pathway inhibitors on antifungal activity in silkworm hemolymph was also detected. Moreover, RNAi assay of BmCTL5 and the affect on expression levels of signaling factors were also analyzed. We found that JAK/STAT pathway could be obviously activated in silkworm challenged with B. bassiana and had no response to bacteria and B. mori cytoplasmic polyhedrosis virus (BmCPV). However, the temporal expression patterns of JAK/STAT signaling pathway related genes were significantly different. B. mori downstream receptor kinase (BmDRK) might be a positive regulator of JAK/STAT signaling pathway in silkworm against B. bassiana infection. Moreover, antifungal activity assay showed that the suppression of JAK/STAT signaling pathway by inhibitors could significantly inhibit the antifungal activity in hemolymph and resulted in increased sensitivity of silkworm to B. bassiana infection, indicating that JAK/STAT signaling pathway might be involved in the synthesis and secretion of antifungal substances. The results of RNAi assays suggested that BmCTL5 might be one pattern recognition receptors for JAK/STAT signaling pathway in silkworm. These findings yield insights for better

Genome-wide identification and characterization of Fox genes in the silkworm, Bombyx mori.

PubMed

Song, JiangBo; Li, ZhiQuan; Tong, XiaoLing; Chen, Cong; Chen, Min; Meng, Gang; Chen, Peng; Li, ChunLin; Xin, YaQun; Gai, TingTing; Dai, FangYin; Lu, Cheng

2015-09-01

The forkhead box (Fox) transcription factor family has a characteristic of forkhead domain, a winged DNA-binding domain. The Fox genes have been classified into 23 subfamilies, designated FoxA to FoxS, of which the FoxR and FoxS subfamilies are specific to vertebrates. In this review, using whole-genome scanning, we identified 17 distinct Fox genes distributed on 13 chromosomes of the silkworm, Bombyx mori. A phylogenetic tree showed that the silkworm Fox genes could be classified into 13 subfamilies. The FoxK subfamily is specifically absent from the silkworm, although it is present in other lepidopteran insects, including Danaus plexippus and Heliconius melpomene. Microarray data revealed that the Fox genes have distinct expression patterns in the tissues on day 3 of the 5th instar larva. A Gene Ontology analysis suggested that the Fox genes have roles in cellular components, molecular functions, and biological processes, except in pore complex biogenesis. An analysis of the selective pressure on the proteins indicated that most of the amino acid sites in the Fox proteins are undergoing strong purifying selection. Here, we summarize the general characteristics of the Fox genes in the silkworm, which should support further functional studies of the silkworm Fox proteins.

Vitellogenin from the Silkworm, Bombyx mori: An Effective Anti-Bacterial Agent

PubMed Central

Kumar, Manish; Prasad, Tulika; Kannan, Mani; König, Simone

2013-01-01

Silkworm, Bombyx mori, vitellogenin (Vg) was isolated from perivisceral fat body of day 3 of pupa. Both Vg subunits were co-purified as verified by mass spectrometry and immunoblot. Purified Vg responded to specific tests for major posttranslational modifications on native gels indicating its nature as lipo-glyco-phosphoprotein. The Vg fraction had strong antibacterial activity against Gram negative bacterium Escherichia coli and Gram positive bacterium Bacillus subtilis. Microscopic images showed binding of Vg to bacterial cells and their destruction. When infected silkworm larvae were treated with purified Vg they survived the full life cycle in contrast to untreated animals. This result showed that Vg has the ability to inhibit the proliferation of bacteria in the silkworm fluid system without disturbing the regular metabolism of the host. PMID:24058454

Fabrication and characterization of biomaterial film from gland silk of muga and eri silkworms.

PubMed

Dutta, Saranga; Talukdar, Bijit; Bharali, Rupjyoti; Rajkhowa, Rangam; Devi, Dipali

2013-05-01

This study discusses the possibilities of liquid silk (Silk gland silk) of Muga and Eri silk, the indigenous non mulberry silkworms of North Eastern region of India, as potential biomaterials. Silk protein fibroin of Bombyx mori, commonly known as mulberry silkworm, has been extensively studied as a versatile biomaterial. As properties of different silk-based biomaterials vary significantly, it is important to characterize the non mulberry silkworms also in this aspect. Fibroin was extracted from the posterior silk gland of full grown fifth instars larvae, and 2D film was fabricated using standard methods. The films were characterized using SEM, Dynamic contact angle test, FTIR, XRD, DSC, and TGA and compared with respective silk fibers. SEM images of films reveal presence of some globules and filamentous structure. Films of both the silkworms were found to be amorphous with random coil conformation, hydrophobic in nature, and resistant to organic solvents. Non mulberry silk films had higher thermal resistance than mulberry silk. Fibers were thermally more stable than the films. This study provides insight into the new arena of research in application of liquid silk of non mulberry silkworms as biomaterials. Copyright © 2012 Wiley Periodicals, Inc.

Comparative proteomic analysis reveals the suppressive effects of dietary high glucose on the midgut growth of silkworm.

PubMed

Feng, Fan; Chen, Liang; Lian, Chaoqun; Xia, Hengchuan; Zhou, Yang; Yao, Qin; Chen, Keping

2014-08-28

The silkworm, Bombyx mori, is an important model of lepidoptera insect, and it has been used for several models of human diseases. In human being, long-term high-sugar diet can induce the occurrence of diabetes and other related diseases. Interestingly, our experiments revealed the high glucose diet also has a suppressive effect on the development of silkworms. To investigate the molecular mechanism by which high-glucose diet inhibited the midgut growth in silkworms, we employed comparative proteomic analysis to globally identify proteins differentially expressed in normal and high-glucose diet group silkworms. In all, 28 differently proteins were suppressed and 5 proteins induced in high-glucose diet group. Gene ontology analysis showed that most of these differently proteins are mainly involved in metabolic process, catalytic and cellular process. A development related protein, imaginal disk growth factor (IDGF), was further confirmed by western blot exclusively expressing in the normal diet group silkworms. Taken together, our data suggests that IDGF plays a critical role in impairing the development of silkworms by a high-glucose diet. Glucose has been thought to play essential roles in growth and development of silkworm. In this paper, we certified firstly that high-glucose diet can suppress the growth of silkworm, and comparative proteomic was employed to reveal the inhibition mechanism. Moreover, an important regulation related protein (IDGF) was found to involve in this inhibition process. These results will help us get a deeper understanding of the relationship between diet and healthy. Furthermore, IDGF may be the critical protein for reducing the blood sugar in silkworm, and it may be used for screening human hypoglycemic drug. The work has not been submitted elsewhere for publication, in whole or in part, and all the authors have approved the manuscript. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification and characterization of an arginine kinase as a major allergen from silkworm (Bombyx mori) larvae.

PubMed

Liu, Zhigang; Xia, Lixin; Wu, Yulan; Xia, Qingyou; Chen, Jiajie; Roux, Kenneth H

2009-01-01

The silkworm, Bombyx mori, is an important insect in the textile industry and its pupa are used in Chinese cuisine and traditional Chinese medicine. The silk, urine and dander of silkworms is often the cause of allergies in sericulture workers and the pupa has been found to be a food allergen in China. Recent studies have focused on reporting cases of silkworm allergies, but only a few studies have addressed the specific allergens present in the B. mori silkworm. We collected sera from 10 patients with a positive skin prick test to silkworm crude extract (SCE) and analyzed these samples by Western blot and ELISA. The cDNA of arginine kinase from the B. mori silkworm was also cloned and expressed in high yield in Escherichia coli. Allergenicity and cross-allergenicity of the recombinant B. mori arginine kinase (rBmAK) were investigated by ELISA inhibition assay. Collected sera all reacted to a 42-kDa protein in a Western blot with SCE as the antigen. Preincubation of sera with rBmAK eliminated the reactivity of the patients' sera to this 42-kDa band. All patient sera also exhibited positive reactivity to SCE in an ELISA assay. BmAK also demonstrated cross-reactivity with a recombinant AK from cockroach. Arginine kinase from the B. mori silkworm is a major allergen and crossreacts with cockroach AK. Copyright 2009 S. Karger AG, Basel.

Spatiotemporal expression profile of the Pumilio gene in the embryonic development of silkworm.

PubMed

Chen, Liang; You, Zaizhi; Xia, Hengchuan; Tang, Qi; Zhou, Yang; Yao, Qin; Chen, Keping

2014-01-01

We previously identified a pumilio gene in silkworm (Bombyx mori L.), designated BmPUM, which was specifically expressed in the ovary and testis. To further characterize this gene's involvement in silkworm development, we have determined the spatiotemporal expression pattern of BmPUM during all embryonic stages. Real-time polymerase chain reaction (RT-PCR) analysis revealed that BmPUM was expressed in all stages of silkworm embryos and that its transcript levels displayed two distinct peaks. The first was observed at the germ-band formation stage (1 d after oviposition) and dropped to a low level at the gonad formation stage (5 d after oviposition). The second was detected at the stage of bristle follicle occurrence (6 d after oviposition), which was confirmed by Western blot analysis and immunohistochemistry. Nanos (Nos), functioning together with Pum in abdomen formation of Drosophila embryos, was also highly expressed at the beginning (0 h to 1 d after oviposition) of embryogenesis, but its transcript levels were very low after the stage of germ-band formation. These results suggest that BmPUM functions with Bombyx mori nanos (Bm-nanos) at the early stages of silkworm embryonic development, and may then play a role in gonad formation and the occurrence of bristle follicles. Our data thus provide a foundation to uncover the role of BmPUM during silkworm development.

iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus.

PubMed

Gao, Kun; Deng, Xiang-Yuan; Shang, Meng-Ke; Qin, Guang-Xing; Hou, Cheng-Xiang; Guo, Xi-Jie

2017-01-30

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis

Eri silkworm: a source of edible oil with a high content of α-linolenic acid and of significant nutritional value.

PubMed

Longvah, Thingnganing; Manghtya, Korra; Qadri, Syed S Y H

2012-07-01

The study was undertaken to provide value addition to spent eri silkworm as an alternative source of edible oil for the food and feed industry by carrying out a short-term nutritional and toxicological evaluation of eri silkworm pupae oil using Wistar NIN rats. Growth performance of rats fed either sunflower oil (Control) or eri silkworm pupae oil (Experimental) was comparable. Histopathological examination of the various tissues showed no signs of toxicity even after feeding the eri silkworm oil for 18 weeks. Serum cholesterol and triglyceride was significantly reduced (P < 0.05) while high-density lipoprotein cholesterol was significantly increased (P < 0.05) which is attributed to the high α-linolenic acid content of eri silkworm oil. The study showed that eri silkworm pupae oil is safe and nutritionally equivalent to commonly used vegetable oils. Eri silkworm pupae can be harvested to provide a cost effective alternative edible oil that can be used to nutritional advantage in the food and feed industry. Therefore eri silkworm and its host plants offer an excellent example of multiple product crops and of sustainable agricultural practice with excellent opportunity for economic and nutritional benefits. Copyright © 2012 Society of Chemical Industry.

Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori.

PubMed

Jeong, Kyoung Yong; Son, Mina; Lee, June Yong; Park, Kyung Hee; Lee, Jae-Hyun; Park, Jung-Won

2016-01-01

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.

Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori

PubMed Central

Son, Mina; Lee, June Yong

2016-01-01

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm. PMID:26770033

Functions and substrates of NEDDylation during cell cycle in the silkworm, Bombyx mori.

PubMed

Li, Zhiqing; Cui, Qixin; Wang, Xiaoyan; Li, Bingqian; Zhao, Dongchao; Xia, Qingyou; Zhao, Ping

2017-11-01

NEDDylation, a post-translational modification mediated by the conjugation of the ubiquitin-like protein Nedd8 to specific substrates, is an essential biological process that regulates cell cycle progression in eukaryotes. Here, we report the conservation of NEDDylation machinery and NEDDylated proteins in the silkworm, Bombyx mori. We have identified all the components necessary for reversible NEDDylation in the silkworm including Nedd8, E1, E2, E3, and deNEDDylation enzymes. By the approach of RNAi-mediated gene silencing, it was shown that knockdown of BmNedd8 and the conjugating enzymes decreased the global level of NEDDylation, while knockdown of deNEDDylation enzymes increased the prevalence of this modification in cultured silkworm cells. Moreover, the lack of the NEDDylation system caused cell cycle arrest at the G2/M phase and resulted in defects in chromosome congression and segregation. Using the wild-type and mutants of BmNedd8, we identified the specific substrates of BmNedd8, which are involved in the regulation for many cellular processes, including ribosome biogenesis, spliceosome structure, spindle formation, metabolism, and RNA biogenesis. This clearly demonstrates that the NEDDylation system is able to control multiple pathways in the silkworm. Altogether, the information on the functions and substrates of the NEDDylation system presented here could provide a basis for future investigations of protein NEDDylation and its regulatory mechanism on cell cycle progression in the silkworm. Copyright © 2017. Published by Elsevier Ltd.

Evaluation of drug-induced tissue injury by measuring alanine aminotransferase (ALT) activity in silkworm hemolymph

PubMed Central

2012-01-01

Background Our previous studies suggest silkworms can be used as model animals instead of mammals in pharmacologic studies to develop novel therapeutic medicines. We examined the usefulness of the silkworm larvae Bombyx mori as an animal model for evaluating tissue injury induced by various cytotoxic drugs. Drugs that induce hepatotoxic effects in mammals were injected into the silkworm hemocoel, and alanine aminotransferase (ALT) activity was measured in the hemolymph 1 day later. Results Injection of CCl4 into the hemocoel led to an increase in ALT activity. The increase in ALT activity was attenuated by pretreatment with N-acetyl-L-cysteine. Injection of benzoic acid derivatives, ferric sulfate, sodium valproate, tetracycline, amiodarone hydrochloride, methyldopa, ketoconazole, pemoline (Betanamin), N-nitroso-fenfluramine, and D-galactosamine also increased ALT activity. Conclusions These findings indicate that silkworms are useful for evaluating the effects of chemicals that induce tissue injury in mammals. PMID:23137391

Sericin Composition in the Silk of Antheraea yamamai.

PubMed

Zurovec, Michal; Yonemura, Naoyuki; Kludkiewicz, Barbara; Sehnal, František; Kodrik, Dalibor; Vieira, Ligia Cota; Kucerova, Lucie; Strnad, Hynek; Konik, Peter; Sehadova, Hana

2016-05-09

The silks produced by caterpillars consist of fibroin proteins that form two core filaments, and sericin proteins that seal filaments into a fiber and conglutinate fibers in the cocoon. Sericin genes are well-known in Bombyx mori (Bombycidae) but have received little attention in other insects. This paper shows that Antheraea yamamai (Saturniidae) contains five sericin genes very different from the three sericin genes of B. mori. In spite of differences, all known sericins are characterized by short exons 1 and 2 (out of 3-12 exons), expression in the middle silk gland section, presence of repeats with high contents of Ser and charged amino acid residues, and secretion as a sticky silk component soluble in hot water. The B. mori sericins represent tentative phylogenetic lineages (I) BmSer1 and orthologs in Saturniidae, (II) BmSer2, and (III) BmSer3 and related sericins of Saturniidae and of the pyralid Galleria mellonella. The lineage (IV) seems to be limited to Saturniidae. Concerted evolution of the sericin genes was apparently associated with gene amplifications as well as gene loses. Differences in the silk fiber morphology indicate that the cocktail of sericins linking the filaments and coating the fiber is modified during spinning. Silks are composite biomaterials of conserved function in spite of great diversity of their composition.

Transcriptome Analysis of Thermal Parthenogenesis of the Domesticated Silkworm.

PubMed

Liu, Peigang; Wang, Yongqiang; Du, Xin; Yao, Lusong; Li, Fengbo; Meng, Zhiqi

2015-01-01

Thermal induction of parthenogenesis (also known as thermal parthenogenesis) in silkworms is an important technique that has been used in artificial insemination, expansion of hybridization, transgenesis and sericultural production; however, the exact mechanisms of this induction remain unclear. This study aimed to investigate the gene expression profile in silkworms undergoing thermal parthenogenesis using RNA-seq analysis. The transcriptome profiles indicated that in non-induced and induced eggs, the numbers of differentially expressed genes (DEGs) for the parthenogenetic line (PL) and amphigenetic line (AL) were 538 and 545, respectively, as determined by fold-change ≥ 2. Gene ontology (GO) analysis showed that DEGs between two lines were mainly involved in reproduction, formation of chorion, female gamete generation and cell development pathways. Upregulation of many chorion genes in AL suggests that the maturation rate of AL eggs was slower than PL eggs. Some DEGs related to reactive oxygen species removal, DNA repair and heat shock response were differentially expressed between the two lines, such as MPV-17, REV1 and HSP68. These results supported the view that a large fraction of genes are differentially expressed between PL and AL, which offers a new approach to identifying the molecular mechanism of silkworm thermal parthenogenesis.

Transcriptome Analysis of Thermal Parthenogenesis of the Domesticated Silkworm

PubMed Central

Du, Xin; Yao, Lusong; Li, Fengbo; Meng, Zhiqi

2015-01-01

Thermal induction of parthenogenesis (also known as thermal parthenogenesis) in silkworms is an important technique that has been used in artificial insemination, expansion of hybridization, transgenesis and sericultural production; however, the exact mechanisms of this induction remain unclear. This study aimed to investigate the gene expression profile in silkworms undergoing thermal parthenogenesis using RNA-seq analysis. The transcriptome profiles indicated that in non-induced and induced eggs, the numbers of differentially expressed genes (DEGs) for the parthenogenetic line (PL) and amphigenetic line (AL) were 538 and 545, respectively, as determined by fold-change ≥ 2. Gene ontology (GO) analysis showed that DEGs between two lines were mainly involved in reproduction, formation of chorion, female gamete generation and cell development pathways. Upregulation of many chorion genes in AL suggests that the maturation rate of AL eggs was slower than PL eggs. Some DEGs related to reactive oxygen species removal, DNA repair and heat shock response were differentially expressed between the two lines, such as MPV-17, REV1 and HSP68. These results supported the view that a large fraction of genes are differentially expressed between PL and AL, which offers a new approach to identifying the molecular mechanism of silkworm thermal parthenogenesis. PMID:26274803

Sublethal dose of phoxim and Bombyx mori nucleopolyhedrovirus interact to elevate silkworm mortality.

PubMed

Gu, ZhiYa; Li, FanChi; Hu, JingSheng; Ding, Chao; Wang, Chaoqian; Tian, JiangHai; Xue, Bin; Xu, KaiZun; Shen, WeiDe; Li, Bing

2017-03-01

Silkworm (Bombyx mori) is an economically important insect. It is relatively less resistant to certain chemicals and environment exposures such as pesticides and pathogens. After pesticide exposures, the silkworms are more susceptible to microbial infections. The mechanism underlying the susceptibility might be related to immune response and oxidative stress. A sublethal dose of phoxim combined with Bombyx mori nucleopolyhedrovirus (BmNPV) elevated the silkworm mortality at 96 h. We found a higher content of H 2 O 2 and increased levels of genes related to oxidative stress and immune response after treatment with a sublethal dose of phoxim for 24 h or 48 h. However, such response decreased with longer pesticide treatment. Mortality increased by 44% when B. mori was exposed to combined treatment with BmNPV and phoxim rather than BmNPV alone. The level of examined immune-related and oxidative-stress-related genes significantly decreased in the combined treatment group compared with the BmNPV group. Our results indicated that, with long-term exposure to pesticides such as OPs, even at sublethal dose, the oxidative stress response and immune responses in silkworm were inhibited, which may lead to further immune impairment and accumulation of oxidative stress, resulting in susceptibility to the virus and harm to the silkworm. Our study provided insights for understanding the susceptibility to pathogen after pesticide exposures, which may promote the development of better pesticide controls to avoid significant economic losses. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

Variation of lifespan in multiple strains, and effects of dietary restriction and BmFoxO on lifespan in silkworm, Bombyx mori.

PubMed

Song, Jiangbo; Tang, Dongmei; Li, Zhiquan; Tong, Xiaoling; Zhang, Jianfei; Han, Minjin; Hu, Hai; Lu, Cheng; Dai, Fangyin

2017-01-31

Established animal models have accelerated our understanding of the mechanisms involved in lifespan determination. However, more experimental animals are required to clarify the complex mechanisms behind the phenomena of aging and lifespan. In this study, we reported the variation of lifespan in nine distinct silkworm strains. Lifespan correlated significantly with BmFoxO gene expression in the representative silkworm strains tested (Xiafang, Dazao-N, and N4). In general, the female silkworm was longer lived than the male of the same strain. Dietary restriction extended the silkworm lifespan compared with that of silkworms fed ad libitum. The expression of BmFoxO was significantly elevated in the dietary restriction group on day 3 of the 4th instar and day 3 of the 5th instar, suggesting that BmFoxO contributes to dietary restriction-mediated lifespan extension. The RNA interference and overexpression of the BmFoxO gene significantly shortened and extended the silkworm adulthood, respectively. In conclusion, our findings suggest that the silkworm might serve as a promising experimental animal to explore the complex biological mechanisms of lifespan determination.

Seasonal Variation in Food Consumption, Assimilation, and Conversion Efficiency of Indian Bivoltine Hybrid Silkworm, Bombyx mori

PubMed Central

Rahmathulla, V. K.; Suresh, H. M.

2012-01-01

Food consumption and utilization is influenced by various biotic and abiotic factors. Under different environmental, feeding, and nutritional conditions, and with ingestion of the same amount of mulberry leaves, the silkworm shows significant difference in its ability to digest, absorb, and convert food to body matter. Here, influences of season, temperature, and humidity on food intake, assimilation, and conversion efficiency of the Indian bivoltine hybrid (CSR2 × CSR4) Bombyx mori L. (Lepidoptera: Bombycidae) were studied. The results indicated that food ingestion and assimilation were significantly higher among silkworm batches where optimum temperature and humidity were maintained compared with silkworm batches exposed to natural climatic conditions of the respective season. However, during summer the nutritional efficiency parameters were significantly higher among silkworms reared under natural temperature and humidity conditions when compared with the control. During the winter and rainy season, the nutritional efficiency parameters were significantly higher in control batches, where optimum temperature and humidity were maintained. Ingesta and digesta required to produce one gram of cocoon/shell were also lower in control batches for all seasons except summer. This may be due to the physiological adaptation of silkworms to overcome stress during the summer season. PMID:23414194

Postoperative anti-adhesion ability of a novel carboxymethyl chitosan from silkworm pupa in a rat cecal abrasion model.

PubMed

Zhu, Lin; Zhang, Yu-Qing

2016-04-01

N,O-Carboxymethyl chitosan (NOCC) can prevent postsurgical adhesion formation. Here, we described the preparation of a novel silkworm pupa NOCC and its effects on the prevention of postoperative adhesion in a rat cecal abrasion model. The degree of deacetylation (DDA) of silkworm pupa chitosan was only 49.87 ± 0.86%; regardless, it was used as the raw material to construct the novel silkworm pupa NOCC, which had a weaker crystallinity than the NOCC standard. Sixty male Sprague-Dawley rats were divided into three groups and treated as follows: 0.9% normal saline solution as a negative control, medical anti-adhesion gel as a positive control and the silkworm pupa NOCC anti-adhesion solution. Two and three weeks after surgery, the animals were killed and the adhesion formation was scored. The silkworm pupa NOCC solution significantly decreased the levels of WBC, TNF-α, IL-1β, IL-2, IL-6 and IL-8 but had no effect on IL-4. Additionally, a lower level of TGF-β1 expression was found in the silkworm pupa NOCC group, and significantly less collagen (P<0.01) and fewer inflammatory cells and fibroblasts were detected in the animals of this group. These results suggested that the novel NOCC from silkworm pupa using the method described here have potential applications in the prevention of postoperative intestinal adhesion. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of chemical isotope labeling liquid chromatography mass spectrometry for silkworm hemolymph metabolomics.

PubMed

Shen, Weifeng; Han, Wei; Li, Yunong; Meng, Zhiqi; Cai, Leiming; Li, Liang

2016-10-26

Silkworm (Bombyx mori) is a very useful target insect for evaluation of endocrine disruptor chemicals (EDCs) due to mature breeding techniques, complete endocrine system and broad basic knowledge on developmental biology. Comparative metabolomics of silkworms with and without EDC exposure offers another dimension of studying EDCs. In this work, we report a workflow on metabolomic profiling of silkworm hemolymph based on high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) and demonstrate its application in studying the metabolic changes associated with the pesticide dichlorodiphenyltrichloroethane (DDT) exposure in silkworm. Hemolymph samples were taken from mature silkworms after growing on diet that contained DDT at four different concentrations (1, 0.1, 0.01, 0.001 ppm) as well as on diet without DDT as controls. They were subjected to differential 12 C-/ 13 C-dansyl labeling of the amine/phenol submetabolome, LC-UV quantification of the total amount of labeled metabolites for sample normalization, and LC-MS detection and relative quantification of individual metabolites in comparative samples. The total concentration of labeled metabolites did not show any significant change between four DDT-treatment groups and one control group. Multivariate statistical analysis of the metabolome data set showed that there was a distinct metabolomic separation between the five groups. Out of the 2044 detected peak pairs, 338 and 1471 metabolites have been putatively identified against the HMDB database and the EML library, respectively. 65 metabolites were identified by the dansyl library searching based on the accurate mass and retention time. Among the 65 identified metabolites, 33 positive metabolites had changes of greater than 1.20-fold or less than 0.83-fold in one or more groups with p-value of smaller than 0.05. Several useful biomarkers including serine, methionine, tryptophan, asymmetric dimethylarginine, N

Mechanical Properties of Transgenic Silkworm Silk Under High Strain Rate Tensile Loading

NASA Astrophysics Data System (ADS)

Chu, J.-M.; Claus, B.; Chen, W.

2017-12-01

Studies have shown that transgenic silkworm silk may be capable of having similar properties of spider silk while being mass-producible. In this research, the tensile stress-strain response of transgenic silkworm silk fiber is systematically characterized using a quasi-static load frame and a tension Kolsky bar over a range of strain-rates between 10^{-3} and 700/s. The results show that transgenic silkworm silk tends to have higher overall ultimate stress and failure strain at high strain rate (700/s) compared to quasi-static strain rates, indicating rate sensitivity of the material. The failure strain at the high strain rate is higher than that of spider silk. However, the stress levels are significantly below that of spider silk, and far below that of high-performance fiber. Failure surfaces are examined via scanning electron microscopy and reveal that the failure modes are similar to those of spider silk.

β-Fructofuranosidase Genes of the Silkworm, Bombyx mori

PubMed Central

Daimon, Takaaki; Taguchi, Tomohiro; Meng, Yan; Katsuma, Susumu; Mita, Kazuei; Shimada, Toru

2008-01-01

Mulberry latex contains extremely high concentrations of alkaloidal sugar mimic glycosidase inhibitors, such as 1,4-dideoxy-1,4-imino-d-arabinitol (d-AB1) and 1-deoxynojirimycin (DNJ). Although these compounds do not harm the silkworm, Bombyx mori, a mulberry specialist, they are highly toxic to insects that do not normally feed on mulberry leaves. d-AB1 and DNJ are strong inhibitors of α-glucosidases (EC 3.2.1.20); however, they do not affect the activity ofβ-fructofuranosidases (EC 3.2.1.26). Althoughα-glucosidase genes are found in a wide range of organisms, β-fructofuranosidase genes have not been identified in any animals so far. In this study, we report the identification and characterization of β-fructofuranosidase genes (BmSuc1 and BmSuc2) from B. mori. The BmSuc1 gene was highly expressed in the midgut and silk gland, whereas the expression of BmSuc2 gene was not detected. BmSuc1 encodes a functional β-fructofuranosidase, whose enzymatic activity was not inhibited by DNJ or d-AB1. We also showed that BmSUC1 protein localized within the midgut goblet cell cavities. Collectively, our data clearly demonstrated that BmSuc1 serves as a sugar-digesting enzyme in the silkworm physiology. This anomalous presence of the β-fructofuranosidase gene in the B. mori genome may partly explain why the silkworm can circumvent the mulberry's defense system. PMID:18397891

RNA-Seq Analyses for Two Silkworm Strains Reveals Insight into Their Susceptibility and Resistance to Beauveria bassiana Infection.

PubMed

Xing, Dongxu; Yang, Qiong; Jiang, Liang; Li, Qingrong; Xiao, Yang; Ye, Mingqiang; Xia, Qingyou

2017-02-10

The silkworm Bombyx mori is an economically important species. White muscardine caused by Beauveria bassiana is the main fungal disease in sericulture, and understanding the silkworm responses to B. bassiana infection is of particular interest. Herein, we investigated the molecular mechanisms underlying these responses in two silkworm strains Haoyue (HY, sensitive to B. bassiana ) and Kang 8 (K8, resistant to B. bassiana ) using an RNA-seq approach. For each strain, three biological replicates for immersion treatment, two replicates for injection treatment and three untreated controls were collected to generate 16 libraries for sequencing. Differentially expressed genes (DEGs) between treated samples and untreated controls, and between the two silkworm strains, were identified. DEGs and the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the two strains exhibited an obvious difference. Several genes encoding cuticle proteins, serine proteinase inhibitors (SPI) and antimicrobial peptides (AMP) and the drug metabolism pathway involved in toxin detoxification were considered to be related to the resistance of K8 to B. bassiana. These results revealed insight into the resistance and susceptibility of two silkworm strains against B. bassiana infection and provided a roadmap for silkworm molecular breeding to enhance its resistance to B. bassiana .

RNA-Seq Analyses for Two Silkworm Strains Reveals Insight into Their Susceptibility and Resistance to Beauveria bassiana Infection

PubMed Central

Xing, Dongxu; Yang, Qiong; Jiang, Liang; Li, Qingrong; Xiao, Yang; Ye, Mingqiang; Xia, Qingyou

2017-01-01

The silkworm Bombyx mori is an economically important species. White muscardine caused by Beauveria bassiana is the main fungal disease in sericulture, and understanding the silkworm responses to B. bassiana infection is of particular interest. Herein, we investigated the molecular mechanisms underlying these responses in two silkworm strains Haoyue (HY, sensitive to B. bassiana) and Kang 8 (K8, resistant to B. bassiana) using an RNA-seq approach. For each strain, three biological replicates for immersion treatment, two replicates for injection treatment and three untreated controls were collected to generate 16 libraries for sequencing. Differentially expressed genes (DEGs) between treated samples and untreated controls, and between the two silkworm strains, were identified. DEGs and the enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the two strains exhibited an obvious difference. Several genes encoding cuticle proteins, serine proteinase inhibitors (SPI) and antimicrobial peptides (AMP) and the drug metabolism pathway involved in toxin detoxification were considered to be related to the resistance of K8 to B. bassiana. These results revealed insight into the resistance and susceptibility of two silkworm strains against B. bassiana infection and provided a roadmap for silkworm molecular breeding to enhance its resistance to B. bassiana. PMID:28208575

Alteration of a recombinant protein N-glycan structure in silkworms by partial suppression of N-acetylglucosaminidase gene expression.

PubMed

Kato, Tatsuya; Kikuta, Kotaro; Kanematsu, Ayumi; Kondo, Sachiko; Yagi, Hirokazu; Kato, Koichi; Park, Enoch Y

2017-09-01

To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae. Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man 3 GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins. Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.

A possible mechanism for low affinity of silkworm Na+/K+-ATPase for K.

PubMed

Homareda, Haruo; Otsu, Masahiro; Yamamoto, Sachiko; Ushimaru, Makoto; Ito, Sayaka; Fukutomi, Toshiyuki; Jo, Taeho; Eishi, Yoshinobu; Hara, Yukichi

2017-12-01

The affinity for K + of silkworm nerve Na + /K + -ATPase is markedly lower than that of mammalian Na + /K + -ATPase (Homareda 2010). In order to obtain clues on the molecular basis of the difference in K + affinities, we cloned cDNAs of silkworm (Bombyx mori) nerve Na + /K + -ATPase α and β subunits, and analyzed the deduced amino acid sequences. The molecular masses of the α and β subunits were presumed to be 111.5 kDa with ten transmembrane segments and 37.7 kDa with a single transmembrane segment, respectively. The α subunit showed 75% identity and 93% homology with the pig Na + /K + -ATPase α1 subunit. On the other hand, the amino acid identity of the β subunit with mammalian counterparts was as low as 30%. Cloned α and β cDNAs were co-expressed in cultured silkworm ovary-derived cells, BM-N cells, which lack endogenous Na + /K + -ATPase. Na + /K + -ATPase expressed in the cultured cells showed a low affinity for K + and a high affinity for Na + , characteristic of the silkworm nerve Na + /K + -ATPase. These results suggest that the β subunit is responsible for the affinity for K + of Na + /K + -ATPase.

Pharmacokinetic parameters explain the therapeutic activity of antimicrobial agents in a silkworm infection model.

PubMed

Paudel, Atmika; Panthee, Suresh; Urai, Makoto; Hamamoto, Hiroshi; Ohwada, Tomohiko; Sekimizu, Kazuhisa

2018-01-25

Poor pharmacokinetic parameters are a major reason for the lack of therapeutic activity of some drug candidates. Determining the pharmacokinetic parameters of drug candidates at an early stage of development requires an inexpensive animal model with few associated ethical issues. In this study, we used the silkworm infection model to perform structure-activity relationship studies of an antimicrobial agent, GPI0039, a novel nitrofuran dichloro-benzyl ester, and successfully identified compound 5, a nitrothiophene dichloro-benzyl ester, as a potent antimicrobial agent with superior therapeutic activity in the silkworm infection model. Further, we compared the pharmacokinetic parameters of compound 5 with a nitrothiophene benzyl ester lacking chlorine, compound 7, that exerted similar antimicrobial activity but had less therapeutic activity in silkworms, and examined the metabolism of these antimicrobial agents in human liver fractions in vitro. Compound 5 had appropriate pharmacokinetic parameters, such as an adequate half-life, slow clearance, large area under the curve, low volume of distribution, and long mean residence time, compared with compound 7, and was slowly metabolized by human liver fractions. These findings suggest that the therapeutic effectiveness of an antimicrobial agent in the silkworms reflects appropriate pharmacokinetic properties.

Toyama Kametaro and Vernon Kellogg: silkworm inheritance experiments in Japan, Siam, and the United States, 1900-1912.

PubMed

Onaga, Lisa

2010-01-01

Japanese agricultural scientist Toyama Kametaro's report about the Mendelian inheritance of silkworm cocoon color in Studies on the Hybridology of Insects (1906) spurred changes in Japanese silk production and thrust Toyama and his work into a scholarly exchange with American entomologist Vernon Kellogg. Toyama's work, based on research conducted in Japan and Siam, came under international scrutiny at a time when analyses of inheritance flourished after the "rediscovery" of Mendel's laws of heredity in 1900. The hybrid silkworm studies in Asia attracted the attention of Kellogg, who was concerned with how experimental biology would be used to study the causes of natural selection. He challenged Toyama's conclusions that Mendelism alone could explain the inheritance patterns of silkworm characters such as cocoon color because they had been subject to hundreds of years of artificial selection, or breeding. This examination of the intersection of Japanese sericulture and American entomology probes how practical differences in scientific interests, societal responsibilities, and silkworm materiality were negotiated throughout the processes of legitimating Mendelian genetics on opposite sides of the Pacific. The ways in which Toyama and Kellogg assigned importance to certain silkworm properties show how conflicting intellectual orientations arose in studies of the same organism. Contestation about Mendelism took place not just on a theoretical level, but the debate was fashioned through each scientist's rationale about the categorization of silkworm breeds and races and what counted as "natural". This further mediated the acceptability of the silkworm not as an experimental organism, but as an appropriately "natural" insect with which to demonstrate laws of inheritance. All these shed light on the challenges that came along with the use of agricultural animals to convincingly articulate new biological principles.

Evaluation of pathogenicity of Candida albicans in germination-ready states using a silkworm infection model.

PubMed

Matsumoto, Haruhito; Nagao, Jun-ichi; Cho, Tamaki; Kodama, Jun

2013-01-01

We previously developed an N-acetyl-D-glucosamine (GlcNAc) medium which induces Candida albicans to undergo a yeast-to-hyphal transition through a cAMP-PKA pathway. Microarray analysis demonstrated that 18 genes, including ALS3 that encodes a cell wall adhesion, were upregulated by 30-min incubation of yeast cells at 37°C in the GlcNAc medium. To investigate the differences between morphological transition and morphotype in C. albicans as a consequence of infection, this study utilized a silkworm infection model as an invertebrate mini-host. We prepared 3 different conditions of C. albicans cells in vitro by changing the incubation times in the GlcNAc medium: yeast-form cells at 0 min (Y0 cells), yeast-form cells in germination-ready state at 60 min (Y60 cells), and hyphal cells at 120 min (H120 cells), and compared their pathogenicities. We performed the infection study at various temperatures to find temperature-dependent virulence factors in vivo. Y60 cells in germination-ready state in the GlcNAc medium showed higher pathogenicity in vivo compared to Y0 and H120 cells at 30°C. Y60 cells proliferated in silkworms 24 h post-injection at 30°C, whereas the other 2 cell types did not. In vitro analysis demonstrated that Y60 cells, but not Y0 cells, germinated in the silkworm hemolymph at 30°C. However, Y0 and Y60 cells showed a similar degree of germination in the silkworm hemolymph at 37°C, although no significant difference in silkworm survival after infection with each cell type was observed at 37°C. These results suggested that the germination-ready state induced by the GlcNAc medium contributed to virulence in the silkworm.

High-Toughness Silk Produced by a Transgenic Silkworm Expressing Spider (Araneus ventricosus) Dragline Silk Protein

PubMed Central

Kuwana, Yoshihiko; Sezutsu, Hideki; Nakajima, Ken-ichi; Tamada, Yasushi; Kojima, Katsura

2014-01-01

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4–2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms. PMID:25162624

High-toughness silk produced by a transgenic silkworm expressing spider (Araneus ventricosus) dragline silk protein.

PubMed

Kuwana, Yoshihiko; Sezutsu, Hideki; Nakajima, Ken-ichi; Tamada, Yasushi; Kojima, Katsura

2014-01-01

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4-2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.

From silkworms to bees: Diseases of beneficial insects

USDA-ARS?s Scientific Manuscript database

The diseases of the silkworm (Bombyx mori) and managed bees, including the honey bee (Apis mellifera), bumbles bees (Bombus spp.), the alfalfa leafcutting bee (Megachile rotundata), and mason bees (Osmia spp.) are reviewed, with diagnostic descriptions and a summary of control methods for production...

Polyhydroxylated alkaloids isolated from mulberry trees (Morusalba L.) and silkworms (Bombyx mori L.).

PubMed

Asano, N; Yamashita, T; Yasuda, K; Ikeda, K; Kizu, H; Kameda, Y; Kato, A; Nash, R J; Lee, H S; Ryu, K S

2001-09-01

New polyhydroxylated alkaloids, (2R,3R,4R)-2-hydroxymethyl-3,4-dihydroxypyrrolidine-N-propionamide from the root bark of Morus alba L., and 4-O-alpha-D-galactopyranosyl-calystegine B(2) and 3 beta,6 beta-dihydroxynortropane from the fruits, were isolated by column chromatography using a variety of ion-exchange resins. Fifteen other polyhydroxylated alkaloids were also isolated. 1-Deoxynojirimycin, a potent alpha-glucosidase inhibitor, was concentrated 2.7-fold by silkworms feeding on mulberry leaves. Some alkaloids contained in mulberry leaves were potent inhibitors of mammalian digestive glycosidases but not inhibitors of silkworm midgut glycosidases, suggesting that the silkworm has enzymes specially adapted to enable it to feed on mulberry leaves. The possibility of preventing the onset of diabetes and obesity using natural dietary supplements containing 1-deoxynojirimycin and other alpha-glucosidase inhibitors in high concentration is of great potential interest.

Intestinal microecology associated with fluoride resistance capability of the silkworm (Bombyx mori L.).

PubMed

Li, Guan-Nan; Xia, Xue-Juan; Tang, Wen-Chao; Zhu, Yong

2016-08-01

The silkworm (Bombyx mori L.) is an ideal model of Lepidoptera. However, the diversity and function of the intestinal microbiota in the gut of silkworm remain largely unknown. Changes in the intestinal microecology in fluoride-resistant strain T6 and fluoride-susceptible strain 734 of the silkworm in response to fluoride exposure were investigated. T6 and 734 were treated with 200 mg/kg fluoride (designated as T6-T and 734-T groups) and deionized water (designated as T6-C and 734-C groups). Culture-dependent approach revealed that the numbers of intestinal bacteria in the 734-T group significantly decreased compared with that in the 734-C group (4.8 ± 0.6 × 10(7) CFU/mL vs. 7.5 ± 0.7 × 10(7) CFU/mL; P < 0.05). Analyses of the intestinal content pH showed that the pH decreased in the 734-T group only. Additionally, SCFA concentrations significantly decreased in both treatment groups compared with the control groups. High-throughput sequencing indicated that the intestinal microbiota in the 734-T group was significantly more diverse than those in the other groups. The bacterial community was composed of two dominant groups (Firmicutes and Proteobacteria). Principal component analyses revealed a significant difference in the composition of the intestinal microbiota in the 734-T group compared with those in the other groups. Thaumarchaeota and Euryarchaeota were more abundant in the 734-T group, but they were less abundant in the other groups. This study enhances our understanding about the diversity and function of silkworm intestinal microbiota in response to fluoride exposure among silkworm strains with diverse resistance.

Development of Novel Antibiotic Lysocin E Identified by Silkworm Infection Model.

PubMed

Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2017-01-01

In this symposium, we reported the identification and mechanistic analysis of a novel antibiotic named lysocin E. Lysocin E was identified by screening for therapeutic effectiveness in a silkworm Staphylococcus aureus infection model. The advantages of the silkworm infection model for screening and purification of antibiotics from the culture supernatant of soil bacteria are: 1) low cost; 2) no ethical issues; 3) convenient for evaluation of the therapeutic effectiveness of antibiotics; and 4) pharmacokinetics similar to those of mammals. Lysocin E has remarkable features compared with known antibiotics such as a novel mechanism of action and target. Here, we summarize our reports presented in this symposium.

Comparative proteomic analysis between the domesticated silkworm (Bombyx mori) reared on fresh mulberry leaves and on artificial diet.

PubMed

Zhou, Zhong-Hua; Yang, Hui-Juan; Chen, Ming; Lou, Cheng-Fu; Zhang, Yao-Zhou; Chen, Ke-Ping; Wang, Yong; Yu, Mei-Lan; Yu, Fang; Li, Jian-Ying; Zhong, Bo-Xiong

2008-12-01

To gain an insight into the effects of different diets on growth and development of the domesticated silkworm at protein level, we employed comparative proteomic approach to investigate the proteomic differences of midgut, hemolymph, fat body and posterior silk gland of the silkworms reared on fresh mulberry leaves and on artificial diet. Seventy-six differentially expressed proteins were identified by MALDI TOF/TOF MS, and among them, 41 proteins were up-regulated, and 35 proteins were downregulated. Database searches, combined with GO analysis and KEGG pathway analysis revealed that some hemolymph proteins such as Nuecin, Gloverin-like proteins, PGRP, P50 and beta/-N-acetylglucosamidase were related to innate immunity of the silkworm, and some proteins identified in silkworm midgut including Myosin 1 light chain, Tropomyosin 1, Profilin, Serpin-2 and GSH-Px were involved in digestion and nutrition absorption. Moreover, two up-regulated enzymes in fat body of larvae reared on artificial diet were identified as V-ATPase subunit B and Arginine kinase which participate in energy metabolism. Furthermore, 6 down-regulated proteins identified in posterior silk gland of silkworm larvae reared on artificial diet including Ribosomal protein SA, EF-2, EF-1gamma, AspAT, ERp57 and PHB were related to silk synthesis. Our results suggested that the different diets could alter the expression of proteins related to immune system, digestion and absorption of nutrient, energy metabolism and silk synthesis poor nutrition and absorption of nutrition in silkworm. The results also confirmed that the poor nutrient absorption, weakened innate immunity, decreased energy metabolism and reduced silk synthesis are the main reasons for low cocoons yield, inferior filament quality, low survival rate of young larvae and insufficient resistance against specific pathogens in the silkworms fed on artificial diet.

Identification and methods for prevention of Enterococcus mundtii infection in silkworm larvae, Bombyx mori, reared on artificial diet.

PubMed

Nwibo, Don Daniel; Matsumoto, Yasuhiko; Sekimizu, Kazuhisa

2015-06-01

Previously, it was reported that Enterococcus mundtii (E. mundtii) was associated with flacherie disease of silkworm larvae reared on artificial diet. In this study, we report that E. mundtii was isolated from diseased silkworm larvae, and validated as a pathogenic bacterium of the animal. When silkworm larva was infected with 1.04 × 10⁶ colony-forming units of E. mundtii via oral administration of diet, half population died within six days, indicating that the bacterium is pathogenic to silkworm. Less severe infection was found to cause anorexia and hamper the development of larvae. This pathogen was found to proliferate in both time- and dose-dependent manner in the gastrointestinal tract of the animal. The bacterium was isolated from powder of artificial diet made from mulberry leaves, and from mulberry leaves growing at a field. Minimum inhibitory concentration determination revealed that this bacterium was susceptible to tested antibiotics. Vancomycin treatment of diet significantly decreased the number of E. mundtii in intestine of silkworm larvae infected with the bacteria, compared to control. Furthermore, autoclaving or gamma ray irradiation of diet was also effective for exclusion of E. mundtii from the diet without the loss of its nutrient capacities. These results suggest that mulberry leaves used in making artificial diet for silkworm larvae is one of the sources of E. mundtii infection; and that antibiotic treatment, autoclaving or gamma ray irradiation of artificial diet can exclude the bacteria.

Mineralization and biocompatibility of Antheraea pernyi (A. pernyi) silk sericin film for potential bone tissue engineering.

PubMed

Yang, Mingying; Mandal, Namita; Shuai, Yajun; Zhou, Guanshan; Min, Sijia; Zhu, Liangjun

2014-01-01

This study aimed to investigate the mineralization of Antheraea pernyi (A. pernyi) silk sericin. Mineralization of A. pernyi sericin was performed by alternative soaking in calcium and phosphate. The inhibition of precipitation of calcium carbonate and von Kossa staining on A. pernyi sericin were tested, and the corresponding results prove that A. pernyi sericin has Ca binding activity. Scanning electron microscope (SEM) observation shows that spherical crystals could be nucleated on the A. pernyi sericin film. These crystals were confirmed to be hydroxyapatite according to FT-IR and XRD spectra, indicating that A. pernyi sericin is capable of mineralization. In addition, cell adhesion and growth activity assay demonstrate that A. pernyi sericin shows excellent biocompatibility for the growth of MG-63 cells.

Silkworm Sericin: Properties and Biomedical Applications.

PubMed

Kunz, Regina Inês; Brancalhão, Rose Meire Costa; Ribeiro, Lucinéia de Fátima Chasko; Natali, Maria Raquel Marçal

2016-01-01

Silk sericin is a natural polymer produced by silkworm, Bombyx mori , which surrounds and keeps together two fibroin filaments in silk thread used in the cocoon. The recovery and reuse of sericin usually discarded by the textile industry not only minimizes environmental issues but also has a high scientific and commercial value. The physicochemical properties of the molecule are responsible for numerous applications in biomedicine and are influenced by the extraction method and silkworm lineage, which can lead to variations in molecular weight and amino acid concentration of sericin. The presence of highly hydrophobic amino acids and its antioxidant potential make it possible for sericin to be applied in the food and cosmetic industry. The moisturizing power allows indications as a therapeutic agent for wound healing, stimulating cell proliferation, protection against ultraviolet radiation, and formulating creams and shampoos. The antioxidant activity associated with low digestibility of sericin that expands the application in the medical field, such as antitumour, antimicrobial and anti-inflammatory agent, anticoagulant, acts in colon health, improving constipation and protects the body from obesity through improved plasma lipid profile. In addition, the properties of sericin allow its application as a culture medium and cryopreservation, in tissue engineering and for drug delivery, demonstrating its effective use, as an important biomaterial.

Silkworm Sericin: Properties and Biomedical Applications

PubMed Central

Ribeiro, Lucinéia de Fátima Chasko

2016-01-01

Silk sericin is a natural polymer produced by silkworm, Bombyx mori, which surrounds and keeps together two fibroin filaments in silk thread used in the cocoon. The recovery and reuse of sericin usually discarded by the textile industry not only minimizes environmental issues but also has a high scientific and commercial value. The physicochemical properties of the molecule are responsible for numerous applications in biomedicine and are influenced by the extraction method and silkworm lineage, which can lead to variations in molecular weight and amino acid concentration of sericin. The presence of highly hydrophobic amino acids and its antioxidant potential make it possible for sericin to be applied in the food and cosmetic industry. The moisturizing power allows indications as a therapeutic agent for wound healing, stimulating cell proliferation, protection against ultraviolet radiation, and formulating creams and shampoos. The antioxidant activity associated with low digestibility of sericin that expands the application in the medical field, such as antitumour, antimicrobial and anti-inflammatory agent, anticoagulant, acts in colon health, improving constipation and protects the body from obesity through improved plasma lipid profile. In addition, the properties of sericin allow its application as a culture medium and cryopreservation, in tissue engineering and for drug delivery, demonstrating its effective use, as an important biomaterial. PMID:27965981

A new arylalkylamine N-acetyltransferase in silkworm (Bombyx mori) affects integument pigmentation.

PubMed

Long, Yaohang; Li, Jiaorong; Zhao, Tianfu; Li, Guannan; Zhu, Yong

2015-04-01

Dopamine is a precursor for melanin synthesis. Arylalkylamine N-acetyltransferase (AANAT) is involved in the melatonin formation in insects because it could catalyze the transformation from dopamine to dopamine-N-acetyldopamine. In this study, we identified a new AANAT gene in the silkworm (Bombyx mori) and assessed its role in the silkworm. The cDNA of this gene encodes 233 amino acids that shares 57 % amino acid identity with the Bm-iAANAT protein. We thus refer to this gene as Bm-iAANAT2. To investigate the role of Bm-iAANAT2, we constructed a transgenic interference system using a 3xp3 promoter to suppress the expression of Bm-iAANAT2 in the silkworm. We observed that melanin deposition occurs in the head and integument in transgenic lines. To verify the melanism pattern, dopamine content and the enzyme activity of AANAT were determined by high-performance liquid chromatography (HPLC). We found that an increase in dopamine levels affects melanism patterns on the heads of transgenic B. mori. A reduction in the enzyme activity of AANAT leads to changes in dopamine levels. We analyzed the expression of the Bm-iAANAT2 genes by qPCR and found that the expression of Bm-iAANAT2 gene is significantly lower in transgenic lines. Our results lead us to conclude that Bm-iAANAT2 is a new arylalkylamine N-acetyltransferase gene in the silkworm and is involved in the metabolism of the dopamine to avoid the generation of melanin.

Female qualities in males: vitellogenin synthesis induced by ovary transplants into the male silkworm, Bombyx mori.

PubMed

Yang, Congwen; Lin, Ying; Shen, Guanwang; Chen, Enxiang; Wang, Yanxia; Luo, Juan; Zhang, Haiyan; Xing, Runmiao; Xia, Qingyou

2014-10-10

Female qualities in males are common in vertebrates but have not been extensively reported in insects. Vitellogenin (Vg) is highly expressed in the female fat body and is generally required for the formation of yolk proteins in the insect egg. Vg upregulation is generally regarded as a female quality in female oviparous animals. In this study, we found that Bombyx mori Vg (BmVg) is especially highly expressed in the female pupa. Downregulation of the BmVg gene in the female pupa by RNA interference (RNAi) interfered with egg formation and embryonic development, showing the importance of BmVg in these processes. So, we used BmVg as a biomarker for female qualities in the silkworm. Hematoxylin-eosin staining and immunofluorescence histochemistry showed that ovary transplants induced BmVg synthesis in the male pupa fat body. Ovaries transplanted into male silkworms produced only a few eggs with deformed yolk granules. These results suggested that the amount of BmVg in the male silkworm was insufficient for eggs to undergo complete embryonic development. After 17-beta-estradiol was used to treat male pupae and male pupal fat bodies, BmVg was upregulated in vivo and in vitro. These findings indicated that the male silkworm has innate female qualities that were induced by a transplanted ovary and 17β-estradiol. However, in silkworms, female qualities in males are not as complete as in females. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization of the complete mitochondrial genome of the giant silkworm moth, Eriogyna pyretorum (Lepidoptera: Saturniidae).

PubMed

Jiang, Shao-Tong; Hong, Gui-Yun; Yu, Miao; Li, Na; Yang, Ying; Liu, Yan-Qun; Wei, Zhao-Jun

2009-05-22

The complete mitochondrial genome (mitogenome) of Eriogyna pyretorum (Lepidoptera: Saturniidae) was determined as being composed of 15,327 base pairs (bp), including 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The arrangement of the PCGs is the same as that found in the other sequenced lepidopteran. The AT skewness for the E. pyretorum mitogenome is slightly negative (-0.031), indicating the occurrence of more Ts than As. The nucleotide composition of the E. pyretorum mitogenome is also biased toward A + T nucleotides (80.82%). All PCGs are initiated by ATN codons, except for cytochrome c oxidase subunit 1 and 2 (cox1 and cox2). Two of the 13 PCGs harbor the incomplete termination codon by T. All tRNA genes have a typical clover-leaf structure of mitochondrial tRNA, with the exception of trnS1(AGN) and trnS2(UCN). Phylogenetic analysis among the available lepidopteran species supports the current morphology-based hypothesis that Bombycoidea, Geometroidea, Notodontidea, Papilionoidea and Pyraloidea are monophyletic. As has been previously suggested, Bombycidae (Bombyx mori and Bombyx mandarina), Sphingoidae (Manduca sexta) and Saturniidae (Antheraea pernyi, Antheraea yamamai, E. pyretorum and Caligula boisduvalii) formed a group.

Characterization of the complete mitochondrial genome of the giant silkworm moth, Eriogyna pyretorum (Lepidoptera: Saturniidae)

PubMed Central

Jiang, Shao-Tong; Hong, Gui-Yun; Yu, Miao; Li, Na; Yang, Ying; Liu, Yan-Qun; Wei, Zhao-Jun

2009-01-01

The complete mitochondrial genome (mitogenome) of Eriogyna pyretorum (Lepidoptera: Saturniidae) was determined as being composed of 15,327 base pairs (bp), including 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The arrangement of the PCGs is the same as that found in the other sequenced lepidopteran. The AT skewness for the E. pyretorum mitogenome is slightly negative (-0.031), indicating the occurrence of more Ts than As. The nucleotide composition of the E. pyretorum mitogenome is also biased toward A + T nucleotides (80.82%). All PCGs are initiated by ATN codons, except for cytochrome c oxidase subunit 1 and 2 (cox1 and cox2). Two of the 13 PCGs harbor the incomplete termination codon by T. All tRNA genes have a typical clover-leaf structure of mitochondrial tRNA, with the exception of trnS1(AGN) and trnS2(UCN). Phylogenetic analysis among the available lepidopteran species supports the current morphology-based hypothesis that Bombycoidea, Geometroidea, Notodontidea, Papilionoidea and Pyraloidea are monophyletic. As has been previously suggested, Bombycidae (Bombyx mori and Bombyx mandarina), Sphingoidae (Manduca sexta) and Saturniidae (Antheraea pernyi, Antheraea yamamai, E. pyretorum and Caligula boisduvalii) formed a group. PMID:19471586

Cloning, expression and phylogenetic analysis of Hemolin, from the Chinese oak silkmoth, Antheraea pernyi.

PubMed

Li, Wenli; Terenius, Olle; Hirai, Makoto; Nilsson, Anders S; Faye, Ingrid

2005-01-01

The Chinese oak silk moth Antheraea pernyi is an important silk producer. To understand microbial resistance of this moth, we cloned Hemolin, encoding a multifunctional immune protein belonging to the immunoglobulin superfamily, and examined the expression in gonads and fat body. The ApHemolin amino acid sequence was compared to other Hemolin sequences in order to predict functional sites. Several sites were conserved; among them a phosphate binding site, which according to 3D structure modelling does not appear in neuroglian, the phylogenetically closest related protein. In addition, two conserved KDG sequences in the C-C' loop of immunoglobulin domains 1 and 3, give rise to gamma-turns, which is a common motif in the C'-C'' loop of the hypervariable region L2 in vertebrate immunoglobulins. The comparisons also show variable regions of specific interest for future studies of hemolin and its interaction with microbial entities.

Transcriptional profiling of midgut immunity response and degeneration in the wandering silkworm, Bombyx mori.

PubMed

Xu, Qiuyun; Lu, Anrui; Xiao, Guohua; Yang, Bing; Zhang, Jie; Li, Xuquan; Guan, Jingmin; Shao, Qimiao; Beerntsen, Brenda T; Zhang, Peng; Wang, Chengshu; Ling, Erjun

2012-01-01

Lepidoptera insects have a novel development process comprising several metamorphic stages during their life cycle compared with vertebrate animals. Unlike most Lepidoptera insects that live on nectar during the adult stage, the Bombyx mori silkworm adults do not eat anything and die after egg-laying. In addition, the midguts of Lepidoptera insects produce antimicrobial proteins during the wandering stage when the larval tissues undergo numerous changes. The exact mechanisms responsible for these phenomena remain unclear. We used the silkworm as a model and performed genome-wide transcriptional profiling of the midgut between the feeding stage and the wandering stage. Many genes concerned with metabolism, digestion, and ion and small molecule transportation were down-regulated during the wandering stage, indicating that the wandering stage midgut loses its normal functions. Microarray profiling, qRT-PCR and western blot proved the production of antimicrobial proteins (peptides) in the midgut during the wandering stage. Different genes of the immune deficiency (Imd) pathway were up-regulated during the wandering stage. However, some key genes belonging to the Toll pathway showed no change in their transcription levels. Unlike butterfly (Pachliopta aristolochiae), the midgut of silkworm moth has a layer of cells, indicating that the development of midgut since the wandering stage is not usual. Cell division in the midgut was observed only for a short time during the wandering stage. However, there was extensive cell apoptosis before pupation. The imbalance of cell division and apoptosis probably drives the continuous degeneration of the midgut in the silkworm since the wandering stage. This study provided an insight into the mechanism of the degeneration of the silkworm midgut and the production of innate immunity-related proteins during the wandering stage. The imbalance of cell division and apoptosis induces irreversible degeneration of the midgut. The Imd pathway

Developmental Changes for the Hemolymph Metabolome of Silkworm (Bombyx moriL.)

PubMed Central

Zhou, Lihong; Li, Huihui; Hao, Fuhua; Li, Ning; Liu, Xin; Wang, Guoliang; Wang, Yulan; Tang, Huiru

2015-01-01

Silkworm (Bombyx mori) is a lepidopteran-holometabolic model organism. To understand its developmental biochemistry, we characterized the larval hemolymph metabonome from the third instar to prepupa stage using 1H NMR spectroscopy whilst hemolymph fatty acid composition using GC-FID/MS. We unambiguously assigned more than 60 metabolites, among which tyrosine-o-β-glucuronide, mesaconate, homocarnosine, and picolinate were reported for the first time from the silkworm hemolymph. Phosphorylcholine was the most abundant metabolite in all developmental stages with exception for the periods before the third and fourth molting. We also found obvious developmental dependence for the hemolymph metabonome involving multiple pathways including protein biosyntheses, glycolysis, TCA cycle, the metabolisms of choline amino acids, fatty acids, purines, and pyrimidines. Most hemolymph amino acids had two elevations during the feeding period of the fourth instar and prepupa stage. Trehalose was the major blood sugar before day 8 of the fifth instar, whereas glucose became the major blood sugar after spinning. C16:0, C18:0 and its unsaturated forms were dominant fatty acids in hemolymph. The developmental changes of hemolymph metabonome were associated with dietary nutrient intakes, biosyntheses of cell membrane, pigments, proteins, and energy metabolism. These findings offered essential biochemistry information in terms of the dynamic metabolic changes during silkworm development. PMID:25825269

Developmental Changes for the Hemolymph Metabolome of Silkworm (Bombyx mori L.).

PubMed

Zhou, Lihong; Li, Huihui; Hao, Fuhua; Li, Ning; Liu, Xin; Wang, Guoliang; Wang, Yulan; Tang, Huiru

2015-05-01

Silkworm (Bombyx mori) is a lepidopteran-holometabolic model organism. To understand its developmental biochemistry, we characterized the larval hemolymph metabonome from the third instar to prepupa stage using (1)H NMR spectroscopy whilst hemolymph fatty acid composition using GC-FID/MS. We unambiguously assigned more than 60 metabolites, among which tyrosine-o-β-glucuronide, mesaconate, homocarnosine, and picolinate were reported for the first time from the silkworm hemolymph. Phosphorylcholine was the most abundant metabolite in all developmental stages with exception for the periods before the third and fourth molting. We also found obvious developmental dependence for the hemolymph metabonome involving multiple pathways including protein biosyntheses, glycolysis, TCA cycle, the metabolisms of choline amino acids, fatty acids, purines, and pyrimidines. Most hemolymph amino acids had two elevations during the feeding period of the fourth instar and prepupa stage. Trehalose was the major blood sugar before day 8 of the fifth instar, whereas glucose became the major blood sugar after spinning. C16:0, C18:0 and its unsaturated forms were dominant fatty acids in hemolymph. The developmental changes of hemolymph metabonome were associated with dietary nutrient intakes, biosyntheses of cell membrane, pigments, proteins, and energy metabolism. These findings offered essential biochemistry information in terms of the dynamic metabolic changes during silkworm development.

Sterol composition in larvae of the silkworm, Bombyx mori.

PubMed

Nagata, Shinji; Nagasawa, Hiromichi

2011-01-01

Sterols in silkworm larvae were analyzed. Cholesterol was predominantly detected in all tissues examined. Dietary phytosterols and desmosterol, a putative biosynthetic intermediate from phytosterols to cholesterol, were also detected, indicating that imperfect intestinal conversion from phytosterols to cholesterol influences the sterol composition in larval tissues.

Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons

NASA Astrophysics Data System (ADS)

Wang, Feng; Wang, Riyuan; Wang, Yuancheng; Zhao, Ping; Xia, Qingyou

2015-11-01

With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a “safe harbor” locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

Indigenous Indonesian Wild Silkworm Cocoon of Attacus atlas as Biocompatible Film Biomaterial

NASA Astrophysics Data System (ADS)

Nindhia, T. G. T.; Knejzlik, Z.; Ruml, T.; Surata, I. W.; Nindhia, T. S.

2017-05-01

The biocompatible film made from wild silkworm cocoon of Attacus atlas is intorduced in this research for anticipation of demand on biocompatible film for regenerative medicine. The wild silkworm cocoon was indigenous Indonesia and was taken from it original location in Indonesia. Protocol for degumming method was obtained in this research by using treatment with NaOH solution at 0.1 M for 1 hour. The film was prepared by grinding the wet degummed fiber until pulp like state was obtained. The mixture was dropped in sequence on the hot ceramic plate with temperature around 60°C. The film thickness can be controlled precisely by using this technique. The film is soaked in alcohor for 1 day for stability testing and the result is found stable. The film is introduced in to COS-1 Cell suspension with previously washed in PBS solution and put in a chamber for biocompatibility testing. The cell are found able to grow and attach in first day observation and dramatically increase after 3 days observation. This is an indicate that the film that is produced from wild silkworm cocon of Attacus atlas has excellent biocompatibility.

Purification and Functional Characterization of a Protein: Bombyx mori Human Growth Hormone Like Protein in Silkworm Pupa

PubMed Central

Lv, Zhengbing; Nie, Zuoming; Chen, Jian; Chen, Hao; Yu, Wei; Gai, Qijing; Zhang, Yaozhou

2014-01-01

Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people’s interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs. PMID:25469649

Purification and functional characterization of a protein: Bombyx mori human growth hormone like protein in silkworm pupa.

PubMed

Chen, Jianqing; Shu, Tejun; Lv, Zhengbing; Nie, Zuoming; Chen, Jian; Chen, Hao; Yu, Wei; Gai, Qijing; Zhang, Yaozhou

2014-01-01

Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people's interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.

Identification of a Serratia marcescens virulence factor that promotes hemolymph bleeding in the silkworm, Bombyx mori.

PubMed

Ishii, Kenichi; Adachi, Tatsuo; Hara, Takashi; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-03-01

Injection of culture supernatant of Serratia marcescens, a Gram-negative bacterium pathogenic to a wide range of host animals including insects and mammals, into the hemolymph of silkworm (Bombyx mori) larvae led to continuous flow of the hemolymph (blood of insects) from the injection site. The amount of hemolymph lost within 60 min reached 15-20% of the total larval weight. Using a bioassay with live silkworms, we purified Serralysin, a metalloprotease that requires divalent cations for its activity, as the factor responsible for the promotion of hemolymph bleeding from the culture supernatant of S. marcescens. Recombinant protein also induced hemolymph bleeding in silkworms. Moreover, the culture supernatant of an S. marcescens disruption mutant of the ser gene showed attenuated ability to promote hemolymph bleeding. In addition, this bleeding-promoting activity of the S. marcescens culture supernatant was attenuated by disruption of the wecA gene, which is involved in the biosynthesis of the lipopolysaccharide O-antigen. These findings suggest that Serralysin metalloprotease contributes to the pathogenesis of S. marcescens by inhibiting wound healing, which leads to a massive loss of hemolymph from silkworm larvae. Copyright © 2014 Elsevier Inc. All rights reserved.

Photosensitivity in the circadian hatching rhythm of the carotenoid-depleted silkworm, Bombyx mori.

PubMed

Sakamoto, K; Shimizu, I

1994-01-01

Silkworms (Bombyx mori) were reared on a carotenoid-deprived artificial diet, and the carotenoid-depleted eggs of the next generation were incubated so that we could observe the effect of the depletion on the circadian rhythm of hatching. The phototactic response curves of newly hatched larvae showed that the visual photosensitivity in ocelli of larvae from the carotenoid-depleted eggs was at least 4 log units lower than that of a carotenoid-rich control group. However, the phase-shift experiment revealed that carotenoid depletion did not reduce the photosensitivity in the hatching rhythm. When the hatching rhythm was generated by exposure to a single light pulse in constant darkness, the first peak in the rhythm of the carotenoid-depleted silkworms occurred significantly earlier than that of the carotenoid-rich group, but the following second peaks of both groups were found at the same time. These results suggest that for the silkworm, carotenoid is not involved in photoreception for the hatching rhythm, but is involved in the timing of hatching.

Effect of Venom from the Jellyfish Nemopilema nomurai on the Silkworm Bombyx mori L.

PubMed

Yu, Huahua; Li, Rongfeng; Chen, Xiaolin; Yue, Yang; Xing, Ronge; Liu, Song; Li, Pengcheng

2015-09-24

The silkworm Bombyx mori L. (B. mori) has a significant impact on the economy by producing more than 80% of the globally produced raw silk. The exposure of silkworm to pesticides may cause adverse effects on B. mori, such as a reduction in the production and quality of silk. This study aims to assay the effect of venom from the jellyfish Nemopilema nomurai on growth, cuticle and acetylcholinesterase (AChE) activity of the silkworm B. mori by the leaf dipping method. The experimental results revealed that the four samples caused neither antifeeding nor a lethal effect on B. mori. The sample SFV inhibited B. mori growth after 6 days of treatment in a dose-dependent manner. The samples SFV, DSFV and Fr-1 inhibited the precipitation and synthesis of chitin in the cuticle after 12 and 14 days of treatment. In the case of the four samples, the AChE was significantly improved after 14 days of treatment.

The advances and perspectives of recombinant protein production in the silk gland of silkworm Bombyx mori.

PubMed

Xu, Hanfu

2014-10-01

The silk gland of silkworm Bombyx mori, is one of the most important organs that has been fully studied and utilized so far. It contributes finest silk fibers to humankind. The silk gland has excellent ability of synthesizing silk proteins and is a kind tool to produce some useful recombinant proteins, which can be widely used in the biological, biotechnical and pharmaceutical application fields. It's a very active area to express recombinant proteins using the silk gland as a bioreactor, and great progress has been achieved recently. This review recapitulates the progress of producing recombinant proteins and silk-based biomaterials in the silk gland of silkworm in addition to the construction of expression systems. Current challenges and future trends in the production of valuable recombinant proteins using transgenic silkworms are also discussed.

Evaluation of the innate immune-stimulating activity of amazake using a silkworm muscle contraction assay.

PubMed

Maruki-Uchida, Hiroko; Sai, Masahiko; Sekimizu, Kazuhisa

2017-11-22

We evaluated the innate immune-stimulating activity of amazake using a silkworm muscle contraction assay. Sake cake, a raw material used to make amazake, had high innate immunity-stimulating activity, whereas rice malt, another raw material used to make amazake, did not, even after fermentation. These results suggest that the silkworm muscle contraction assay is a useful tool to screen foods with high innate immune-stimulating activity and that amazake made from sake cake has immunomodulatory potential.

Isolation and characterization of lipase-producing bacteria in the intestine of the silkworm, Bombyx mori, reared on different forage.

PubMed

Feng, Wei; Wang, Xiao-Qiang; Zhou, Wei; Liu, Guang-Ying; Wan, Yong-Ji

2011-01-01

The silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), an oligophagous insect that mainly feeds on mulberry leaves, is susceptible to entomopathogen infection when reared with tricuspid cudrania leaves. A total of 56 dominant bacterial strains, classified into 12 phylotypes based on bacteriological properties and analysis of 16S rRNA genes, were isolated from the intestine of the fourth and fifth instar silkworm larvae. Ten and seven phylotypes exist in the intestine of the silkworm larvae reared with mulberry leaves and tricuspid cudrania leaves, respectively. Four of them are common in the intestine of the two treatment groups. By screening their lipolytic ability on a Rhodamine B agar plate, nine lipase-producing bacterial strains were obtained and classified into six genera, including Bacillus, Brevibacterium, Corynebacterium, Staphylococcus, Klebsiella, and Stenotrophomonas. Except for Stenotrophomonas, which is common in both, the other genera only exist in the intestine of the silkworm larvae fed with mulberry leaves. In addition, by culture and fermentation in vitro, the maximum cell density and lipase activity of lipase-producing bacteria were examined at about 48 hours. The results indicate that diet has a significant impact on the gut bacterial community, especially lipase-producing bacteria. We suggest that the difference of lipase-producing bacterial diversity might be related to disease resistance of the silkworm.

A Review of the Implications of Heterozygosity and Inbreeding on Germplasm Biodiversity and Its Conservation in the Silkworm, Bombyx mori

PubMed Central

Jingade, A.H.; Vijayan, K.; Somasundaram, P.; Srivasababu, G.K.; Kamble, C.K.

2011-01-01

Silkworm genebanks assume paramount importance as the reservoirs of biodiversity and source of alleles that can be easily retrieved for genetic enhancement of popular breeds. More than 4000 Bombyx mori L (Lepidoptera: Bombycidae) strains are currently available and these strains are maintained through continuous sibling mating. This repeated sibling mating makes the populations of each strain more homozygous, but leads to loss of unique and valuable genes through the process of inbreeding depression. Hence, it is essential to maintain a minimal degree of heterozygosity within the population of each silkworm strain, especially in the traditional geographic strains, to avoid such loss. As a result, accurate estimation of genetic diversity is becoming more important in silkworm genetic resources conservation. Application of molecular markers help estimate genetic diversity much more accurately than that of morphological traits. Since a minimal amount of heterozygosity in each silkworm strain is essential for better conservation by avoiding inbreeding depression, this article overviews both theoretical and practical importance of heterozygosity together with impacts of inbreeding depression and the merits and demerits of neutral molecular markers for measurements of both heterozygosity and inbreeding depression in the silkworm Bombyx mori. PMID:21521139

A review of the implications of heterozygosity and inbreeding on germplasm biodiversity and its conservation in the silkworm, Bombyx mori.

PubMed

Jingade, A H; Vijayan, K; Somasundaram, P; Srivasababu, G K; Kamble, C K

2011-01-01

Abstract Silkworm genebanks assume paramount importance as the reservoirs of biodiversity and source of alleles that can be easily retrieved for genetic enhancement of popular breeds. More than 4000 Bombyx mori L (Lepidoptera: Bombycidae) strains are currently available and these strains are maintained through continuous sibling mating. This repeated sibling mating makes the populations of each strain more homozygous, but leads to loss of unique and valuable genes through the process of inbreeding depression. Hence, it is essential to maintain a minimal degree of heterozygosity within the population of each silkworm strain, especially in the traditional geographic strains, to avoid such loss. As a result, accurate estimation of genetic diversity is becoming more important in silkworm genetic resources conservation. Application of molecular markers help estimate genetic diversity much more accurately than that of morphological traits. Since a minimal amount of heterozygosity in each silkworm strain is essential for better conservation by avoiding inbreeding depression, this article overviews both theoretical and practical importance of heterozygosity together with impacts of inbreeding depression and the merits and demerits of neutral molecular markers for measurements of both heterozygosity and inbreeding depression in the silkworm Bombyx mori.

2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori

PubMed Central

Wang, Yuancheng; Wang, Feng; Wang, Riyuan; Zhao, Ping; Xia, Qingyou

2015-01-01

Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas. PMID:26537835

Molecular architecture of silk fibroin of Indian golden silkmoth, Antheraea assama.

PubMed

Gupta, Adarsh K; Mita, Kazuei; Arunkumar, Kallare P; Nagaraju, Javaregowda

2015-08-03

The golden silk spun by Indian golden silkmoth Antheraea assama, is regarded for its shimmering golden luster, tenacity and value as biomaterial. This report describes the gene coding for golden silk H-fibroin (AaFhc), its expression, full-length sequence and structurally important motifs discerning the underlying genetic and biochemical factors responsible for its much sought-after properties. The coding region, with biased isocodons, encodes highly repetitious crystalline core, flanked by a pair of 5' and 3' non-repetitious ends. AaFhc mRNA expression is strictly territorial, confined to the posterior silk gland, encoding a protein of size 230 kDa, which makes homodimers making the elementary structural units of the fibrous core of the golden silk. Characteristic polyalanine repeats that make tight β-sheet crystals alternate with non-polyalanine repeats that make less orderly antiparallel β-sheets, β-turns and partial α-helices. Phylogenetic analysis of the conserved N-terminal amorphous motif and the comparative analysis of the crystalline region with other saturniid H-fibroins reveal that AaFhc has longer, numerous and relatively uniform repeat motifs with lower serine content that assume tighter β-crystals and denser packing, which are speculated to be responsible for its acclaimed properties of higher tensile strength and higher refractive index responsible for golden luster.

Transcriptional Profiling of Midgut Immunity Response and Degeneration in the Wandering Silkworm, Bombyx mori

PubMed Central

Xiao, Guohua; Yang, Bing; Zhang, Jie; Li, Xuquan; Guan, Jingmin; Shao, Qimiao; Beerntsen, Brenda T.; Zhang, Peng; Wang, Chengshu; Ling, Erjun

2012-01-01

Background Lepidoptera insects have a novel development process comprising several metamorphic stages during their life cycle compared with vertebrate animals. Unlike most Lepidoptera insects that live on nectar during the adult stage, the Bombyx mori silkworm adults do not eat anything and die after egg-laying. In addition, the midguts of Lepidoptera insects produce antimicrobial proteins during the wandering stage when the larval tissues undergo numerous changes. The exact mechanisms responsible for these phenomena remain unclear. Principal Findings We used the silkworm as a model and performed genome-wide transcriptional profiling of the midgut between the feeding stage and the wandering stage. Many genes concerned with metabolism, digestion, and ion and small molecule transportation were down-regulated during the wandering stage, indicating that the wandering stage midgut loses its normal functions. Microarray profiling, qRT-PCR and western blot proved the production of antimicrobial proteins (peptides) in the midgut during the wandering stage. Different genes of the immune deficiency (Imd) pathway were up-regulated during the wandering stage. However, some key genes belonging to the Toll pathway showed no change in their transcription levels. Unlike butterfly (Pachliopta aristolochiae), the midgut of silkworm moth has a layer of cells, indicating that the development of midgut since the wandering stage is not usual. Cell division in the midgut was observed only for a short time during the wandering stage. However, there was extensive cell apoptosis before pupation. The imbalance of cell division and apoptosis probably drives the continuous degeneration of the midgut in the silkworm since the wandering stage. Conclusions This study provided an insight into the mechanism of the degeneration of the silkworm midgut and the production of innate immunity-related proteins during the wandering stage. The imbalance of cell division and apoptosis induces irreversible

The nicotinic acetylcholine receptor gene family of the silkworm, Bombyx mori

PubMed Central

Shao, Ya-Ming; Dong, Ke; Zhang, Chuan-Xi

2007-01-01

Background Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic cholinergic transmission in the insect central nervous system. The insect nAChR is the molecular target of a class of insecticides, neonicotinoids. Like mammalian nAChRs, insect nAChRs are considered to be made up of five subunits, coded by homologous genes belonging to the same family. The nAChR subunit genes of Drosophila melanogaster, Apis mellifera and Anopheles gambiae have been cloned previously based on their genome sequences. The silkworm Bombyx mori is a model insect of Lepidoptera, among which are many agricultural pests. Identification and characterization of B. mori nAChR genes could provide valuable basic information for this important family of receptor genes and for the study of the molecular mechanisms of neonicotinoid action and resistance. Results We searched the genome sequence database of B. mori with the fruit fly and honeybee nAChRs by tBlastn and cloned all putative silkworm nAChR cDNAs by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. B. mori appears to have the largest known insect nAChR gene family to date, including nine α-type subunits and three β-type subunits. The silkworm possesses three genes having low identity with others, including one α and two β subunits, α9, β2 and β3. Like the fruit fly and honeybee counterparts, silkworm nAChR gene α6 has RNA-editing sites, and α4, α6 and α8 undergo alternative splicing. In particular, alternative exon 7 of Bmα8 may have arisen from a recent duplication event. Truncated transcripts were found for Bmα4 and Bmα5. Conclusion B. mori possesses a largest known insect nAChR gene family characterized to date, including nine α-type subunits and three β-type subunits. RNA-editing, alternative splicing and truncated transcripts were found in several subunit genes, which might enhance the diversity of the gene family. PMID:17868469

Silvernanotherapy on the viral borne disease of silkworm Bombyx mori L.

NASA Astrophysics Data System (ADS)

Govindaraju, K.; Tamilselvan, S.; Kiruthiga, V.; Singaravelu, G.

2011-12-01

Antiviral assays of chemically and biologically synthesized silver nanoparticles were made against BmNPV ( Bombyx mori Nuclear Polyhedrosis Virus). Reduction of silver ions by sodium citrate and Spirulina platensis led to the formation of spherical silver nanoparticles of 40-60 and 7-16 nm size. Single cell protein ( Spirulina platensis)-synthesized silver nanoparticles showed the strongest antiviral activity. Immunological studies made on the silkworm Bombyx mori disclosed that a significant increase in the total hemocyte count and differential hemocyte count due to S. platensis-synthesized silver nanoparticles supplementation. Improvement in the defense mechanism was noticed from the strengthened peritrophic membrane of the digestive tract and the increased total protein. Overall, the results presented illustrate that single cell protein-synthesized silver nanoparticles supplementation is effective in controlling viral-borne diseases of the silkworm.

Differentially expressed genes in the silk gland of silkworm (Bombyx mori) treated with TiO2 NPs.

PubMed

Xue, Bin; Li, Fanchi; Hu, Jingsheng; Tian, Jianghai; Li, Jinxin; Cheng, Xiaoyu; Hu, Jiahuan; Li, Bing

2017-05-05

Silk gland is a silkworm organ where silk proteins are synthesized and secreted. Dietary supplement of TiO 2 nanoparticles (NPs) promotes silk protein synthesis in silkworms. In this study, digital gene expression (DGE) tag was used to analyze the gene expression profile of the posterior silk gland of silkworms that were fed with TiO 2 NPs. In total, 5,702,823 and 6,150,719 clean tags, 55,096 and 74,715 distinct tags were detected in TiO 2 NPs treated and control groups, respectively. Compared with the control, TiO 2 NPs treated silkworms showed 306 differentially expressed genes, including 137 upregulated genes and 169 downregulated genes. Of these differentially expressed genes, 106 genes were related to silk protein synthesis, among which 97 genes were upregulated and 9 genes were downregulated. Pathway mapping using the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that 20 pathways were significantly enriched in TiO 2 NPs treated silkworms, and the metabolic pathway-related genes were the most significantly enriched. The DGE results were verified by qRT-PCR analysis of eight differentially expressed genes. The DGE and qRT-PCR results were consistent for all three upregulated genes and three of the five downregulated genes, but the expression trends of the remaining two genes were different between qRT-PCR and DGE analysis. This study enhances our understanding of the mechanism of TiO 2 NPs promoted silk protein synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanical properties of cocoons constructed consecutively by a single silkworm caterpillar, Bombyx mori

NASA Astrophysics Data System (ADS)

Huang, S. Q.; Zhao, H. P.; Feng, X. Q.; Cui, W.; Lin, Z.; Xu, M. Q.

2008-04-01

Most animals have the ability to adapt, to some extends and in different ways, the variation or disturbance of environment. In our experiments, we forced a silkworm caterpillar to spin two, three or four thin cocoons by taking it out from the cocoon being constructed. The mechanical properties of these cocoons were studied by static tensile tests and dynamic mechanical thermal analysis. Though external disturbances may cause the decrease in the total weight of silk spun by the silkworm, a gradual enhancement was interestingly found in the mechanical properties of these thin cocoons. Scanning electron microscopy observations of the fractured specimens of the cocoons showed that there exist several different energy dissipation mechanisms occurred simultaneously at macro-, meso-, and micro-scales, yielding a superior capacity of cocoons to adsorb the energy of possible attacks from the outside and to protect efficiently its pupa against damage. Through evolution of millions of years, therefore, the silkworm Bombyx mori seems to have gained the ability to adapt external disturbances and to redesign a new cocoon with optimized protective function when its first cocoon has been damaged for some reasons.

Animal welfare and use of silkworm as a model animal.

PubMed

Sekimizu, N; Paudel, A; Hamamoto, H

2012-08-01

Sacrificing model animals is required for developing effective drugs before being used in human beings. In Japan today, at least 4,210,000 mice and other mammals are sacrificed to a total of 6,140,000 per year for the purpose of medical studies. All the animals treated in Japan, including test animals, are managed under control of "Act on Welfare and Management of Animals". Under the principle of this Act, no person shall kill, injure, or inflict cruelty on animals without due cause. "Animal" addressed in the Act can be defined as a "vertebrate animal". If we can make use of invertebrate animals in testing instead of vertebrate ones, that would be a remarkable solution for the issue of animal welfare. Furthermore, there are numerous advantages of using invertebrate animal models: less space and small equipment are enough for taking care of a large number of animals and thus are cost-effective, they can be easily handled, and many biological processes and genes are conserved between mammals and invertebrates. Today, many invertebrates have been used as animal models, but silkworms have many beneficial traits compared to mammals as well as other insects. In a Genome Pharmaceutical Institute's study, we were able to achieve a lot making use of silkworms as model animals. We would like to suggest that pharmaceutical companies and institutes consider the use of the silkworm as a model animal which is efficacious both for financial value by cost cutting and ethical aspects in animals' welfare.

A comparative analysis of serpin genes in the silkworm genome

PubMed Central

Zou, Zhen; Picheng, Zhao; Weng, Hua; Mita, Kazuei; Jiang, Haobo

2009-01-01

Serine protease inhibitors (serpins) are a superfamily of proteins, most of which control protease-mediated processes by inhibiting their cognate enzymes. Sequencing of the silkworm genome provides an opportunity to investigate serpin structure, function, and evolution at the genome level. There are thirty-four serpin genes in Bombyx mori. Six are highly similar to their Manduca sexta orthologs that regulate innate immunity. Three alternative exons in serpin1 gene and four in serpin28 encode a variable region including the reactive site loop. Splicing of serpin2 pre-mRNA yields variations in serpin2A, 2A′ and 2B. Sequence similarity and intron positions reveal the evolutionary pathway of seven serpin genes in group C. RT-PCR indicates an increase in the mRNA levels of serpin1, 3, 5, 6, 9, 12, 13, 25, 27, 32 and 34 in fat body and hemocytes of larvae injected with bacteria. These results suggest that the silkworm serpins play regulatory roles in defense responses. PMID:19150649

[Effect of fluoride on gut microflora of silkworm (Bombyx mori)].

PubMed

Li, Guannan; Xia, Xuejuan; Sendegeya, Parfait; Zhao, Huanhuan; Long, Yaohang; Zhu, Yong

2015-07-04

We examined the effect of fluoride on gut microflora of silkworm. After DNA extraction and PCR amplification, clone libraries of 16S rRNA gene fragment were constructed. Amplified ribosomal DNA restriction analysis (ARDRA) was performed by digestion of the 16S rRNA gene, and each unique restriction fragment polymorphism pattern was designated as an operational taxonomic unit (OTU). A total of 14 OTUs were identified from intestinal samples of both T6 and 734. Phylogenetic trees of bacterial 16S rRNA nucleotide sequences were constructed and analyzed. Furthermore, the dominant bacteria were studied by the nested polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DDGE) technology. After fluorosis, the flora of Enterococcus and Bacillus reduced. However, the flora of Staphylococcus increased. Fluoride can destroy the balance of microflora in the gut of silkworm by changing the bacteria diversity and proportion, which has bigger effect to 734 than T6.

Production of an active feline interferon in the cocoon of transgenic silkworms using the fibroin H-chain expression system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurihara, H.; Sezutsu, H.; Tamura, T.

2007-04-20

We constructed the fibroin H-chain expression system to produce recombinant proteins in the cocoon of transgenic silkworms. Feline interferon (FeIFN) was used for production and to assess the quality of the product. Two types of FeIFN fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the FeIFN/H-chain fusion gene was regulated by the fibroin H-chain promoter domain. The transgenic silkworms introduced these constructs with the piggyBac transposon-derived vector, which produced the normal sized cocoons containing each FeIFN/H-chain fusion protein. Although themore » native-protein produced by transgenic silkworms have almost no antiviral activity, the proteins after the treatment with PreScission protease to eliminate fibroin H-chain derived N- and C-terminal sequences from the products, had very high antiviral activity. This H-chain expression system, using transgenic silkworms, could be an alternative method to produce an active recombinant protein and silk-based biomaterials.« less

BmCyclin B and BmCyclin B3 are required for cell cycle progression in the silkworm, Bombyx mori.

PubMed

Pan, Minhui; Hong, Kaili; Chen, Xiangyun; Pan, Chun; Chen, Xuemei; Kuang, Xiuxiu; Lu, Cheng

2013-04-01

Cyclin B is an important regulator of the cell cycle G2 to M phase transition. The silkworm genomic database shows that there are two Cyclin B genes in the silkworm (Bombyx mori), BmCyclin B and BmCyclin B3. Using silkworm EST data, the cyclin B3 (EU074796) gene was cloned. Its complete cDNA was 1665 bp with an ORF of 1536 bp derived from seven exons and six introns. The BmCyclin B3 gene encodes 511 amino acids, and the predicted molecular weight is 57.8 kD with an isoelectric point of 9.18. The protein contains one protein damage box and two cyclin boxes. RNA interference-mediated reduction of BmCyclin B and BmCyclin B3 expression induced cell cycle arrest in G2 or M phase in BmN-SWU1 cells, thus inhibiting cell proliferation. These results suggest that BmCyclin B and BmCyclin B3 are necessary for completing the cell cycle in silkworm cells.

The genomic underpinnings of apoptosis in the silkworm, Bombyx mori

PubMed Central

2010-01-01

Background Apoptosis is regulated in an orderly fashion by a series of genes, and has a crucial role in important physiological processes such as growth development, immunological response and so on. Recently, substantial studies have been undertaken on apoptosis in model animals including humans, fruit flies, and the nematode. However, the lack of genomic data for silkworms limits their usefulness in apoptosis studies, despite the advantages of silkworm as a representative of Lepidoptera and an effective model system. Herein we have identified apoptosis-related genes in the silkworm Bombyx mori and compared them to those from insects, mammals, and nematodes. Results From the newly assembled genome databases, a genome-wide analysis of apoptosis-related genes in Bombyx mori was performed using both nucleotide and protein Blast searches. Fifty-two apoptosis-related candidate genes were identified, including five caspase family members, two tumor necrosis factor (TNF) superfamily members, one Bcl-2 family member, four baculovirus IAP (inhibitor of apoptosis) repeat (BIR) domain family members and 1 RHG (Reaper, Hid, Grim, and Sickle; Drosophila cell death activators) family member. Moreover, we identified a new caspase family member, BmCaspase-New, two splice variants of BmDronc, and Bm3585, a mammalian TNF superfamily member homolog. Twenty-three of these apoptosis-related genes were cloned and sequenced using cDNA templates isolated from BmE-SWU1 cells. Sequence analyses revealed that these genes could have key roles in apoptosis. Conclusions Bombyx mori possesses potential apoptosis-related genes. We hypothesized that the classic intrinsic and extrinsic apoptotic pathways potentially are active in Bombyx mori. These results lay the foundation for further apoptosis-related study in Bombyx mori. PMID:21040523

Bombykol receptors in the silkworm moth and the fruit fly

PubMed Central

Syed, Zainulabeuddin; Kopp, Artyom; Kimbrell, Deborah A.; Leal, Walter S.

2010-01-01

Male moths are endowed with odorant receptors (ORs) to detect species-specific sex pheromones with remarkable sensitivity and selectivity. We serendipitously discovered that an endogenous OR in the fruit fly, Drosophila melanogaster, is highly sensitive to the sex pheromone of the silkworm moth, bombykol. Intriguingly, the fruit fly detectors are more sensitive than the receptors of the silkworm moth, although its ecological significance is unknown. By expression in the “empty neuron” system, we identified the fruit fly bombykol-sensitive OR as DmelOR7a (= DmOR7a). The profiles of this receptor in response to bombykol in the native sensilla (ab4) or expressed in the empty neuron system (ab3 sensilla) are indistinguishable. Both WT and transgenic flies responded with high sensitivity, in a dose-dependent manner, and with rapid signal termination. In contrast, the same empty neuron expressing the moth bombykol receptor, BmorOR1, demonstrated low sensitivity and slow signal inactivation. When expressed in the trichoid sensilla T1 of the fruit fly, the neuron housing BmorOR1 responded with sensitivity comparable to that of the native trichoid sensilla in the silkworm moth. By challenging the native bombykol receptor in the fruit fly with high doses of another odorant to which the receptor responds with the highest sensitivity, we demonstrate that slow signal termination is induced by overdose of a stimulus. As opposed to the empty neuron system in the basiconic sensilla, the structural, biochemical, and/or biophysical features of the sensilla make the T1 trichoid system of the fly a better surrogate for the moth receptor. PMID:20439725

Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system.

PubMed

Imai, Saki; Kusakabe, Takahiro; Xu, Jian; Li, Zhiqing; Shirai, Shintaro; Mon, Hiroaki; Morokuma, Daisuke; Lee, Jae Man

2015-11-01

Baculovirus expression vector system (BEVS) is widely used for production of recombinant eukaryotic proteins in insect larvae or cultured cells. BEVS has advantages over bacterial expression system in producing post-translationally modified secreted proteins. However, for some unknown reason, it is very difficult for insects to secrete sufficiently for certain proteins of interest. To understand the reasons why insect cells fail to secrete some kinds of recombinant proteins, we here employed three mammalian proteins as targets, EPO, HGF, and Wnt3A, with different secretion levels in BEVS and investigated their mRNA transcriptions from the viral genome, subcellular localizations, and interactions with silkworm ER chaperones. Moreover, we observed that no significantly influence on the secretion amounts of all three proteins when depleting or overexpressing most endogenous ER chaperone genes in cultured silkworm cells. However, among all detected ER chaperones, the depletion of BiP severely decreased the recombinant protein secretion in BEVS, indicating the possible central role of Bip in silkworm secretion pathway.

Efficacy of silkworm (Bombyx mori L.) chrysalis oil as a lipid source in adult Wistar rats.

PubMed

Mentang, Feny; Maita, Masashi; Ushio, Hideki; Ohshima, Toshiaki

2011-08-01

The effects of silkworm chrysalis oil, rich in n-3 α-linolenic acid (ALA), on lipid metabolism in Wistar rats were investigated. The rats were fed diets containing 7% soybean oil (control), silkworm chrysalis oil (SWO), or fish oil (FO) for 8weeks. Plasma triglyceride and glucose levels were significantly lower in the SWO group after 8weeks compared to the control and FO groups. The total cholesterol and blood urea nitrogen levels were higher in the control group than in the SWO and FO groups at 8weeks post-consumption. However, aspartate amino transferase and alanine amino transferase levels were not significantly different among all groups. A higher arachidonic acid (AA) content was detected in the control group, while lower AA levels were observed with the increase in EPA and DHA in the SWO and FO groups. These results suggest that n-3 α-linolenic acid-rich silkworm chrysalis oil can improve hyperlipidaemia and hyperglycaemia. Copyright © 2011 Elsevier Ltd. All rights reserved.

Characterization and expression patterns of let-7 microRNA in the silkworm (Bombyx mori).

PubMed

Liu, Shiping; Xia, Qingyou; Zhao, Ping; Cheng, Tingcai; Hong, Kaili; Xiang, Zhonghuai

2007-07-25

lin-4 and let-7, the two founding members of heterochronic microRNA genes, are firstly confirmed in Caenorhabditis elegans to control the proper timing of developmental programs in a heterochronic pathway. let-7 has been thought to trigger the onset of adulthood across animal phyla. Ecdysone and Broad-Complex are required for the temporal expression of let-7 in Drosophila melanogaster. For a better understanding of the conservation and functions of let-7, we seek to explore how it is expressed in the silkworm (Bombyx mori). One member of let-7 family has been identified in silkworm computationally and experimentally. All known members of this family share the same nucleotides at ten positions within the mature sequences. Sequence logo and phylogenetic tree show that they are not only conserved but diversify to some extent among some species. The bmo-let-7 was very lowly expressed in ova harvested from newborn unmated female adult and in individuals from the first molt to the early third instar, highly expressed after the third molt, and the most abundant expression was observed after mounting, particularly after pupation. The expression levels were higher at the end of each instar and at the beginning of each molt than at other periods, coinciding with the pulse of ecdysone and BR-C as a whole. Using cultured ovary cell line, BmN-SWU1, we examined the effect of altered ecdysone levels on bmo-let-7 expression. The expression was also detected in various tissues of day 3 of the fifth instar and of from day 7 of the fifth to pupa, suggesting a wide distributing pattern with various signal intensities. bmo-let-7 is stage- and tissue-specifically expressed in the silkworm. Although no signals were detected during embryonic development and first larval instar stages, the expression of bmo-let-7 was observed from the first molt, suggesting that it might also function at early larval stage of the silkworm. The detailed expression profiles in the whole life cycle and

Design and optimization of an experimental bioregenerative life support system with higher plants and silkworms

NASA Astrophysics Data System (ADS)

Hu, Enzhu; Bartsev, Sergey I.; Zhao, Ming; Liu, Professor Hong

The conceptual scheme of an experimental bioregenerative life support system (BLSS) for planetary exploration was designed, which consisted of four elements - human metabolism, higher plants, silkworms and waste treatment. 15 kinds of higher plants, such as wheat, rice, soybean, lettuce, mulberry, et al., were selected as regenerative component of BLSS providing the crew with air, water, and vegetable food. Silkworms, which producing animal nutrition for crews, were fed by mulberry-leaves during the first three instars, and lettuce leaves last two instars. The inedible biomass of higher plants, human wastes and silkworm feces were composted into soil like substrate, which can be reused by higher plants cultivation. Salt, sugar and some household material such as soap, shampoo would be provided from outside. To support the steady state of BLSS the same amount and elementary composition of dehydrated wastes were removed periodically. The balance of matter flows between BLSS components was described by the system of algebraic equations. The mass flows between the components were optimized by EXCEL spreadsheets and using Solver. The numerical method used in this study was Newton's method.

Potential of inherent RGD containing silk fibroin-poly (Є-caprolactone) nanofibrous matrix for bone tissue engineering.

PubMed

Bhattacharjee, Promita; Kundu, Banani; Naskar, Deboki; Kim, Hae-Won; Bhattacharya, Debasis; Maiti, T K; Kundu, S C

2016-02-01

The current study deals with the fabrication and characterization of blended nanofibrous scaffolds of tropical tasar silk fibroin of Antheraea mylitta and poly (Є-caprolactone) to act as an ideal scaffold for bone regeneration. The use of poly (Є-caprolactone) in osteogenesis is well-recognized. At the same time, the osteoconductive nature of the non-mulberry tasar fibroin is also established due to its internal integrin binding peptide RGD (Arg-Gly-Asp) sequences, which enhance cellular interaction and proliferation. Considering that the materials have the required and favorable properties, the blends are formed using an equal volume ratio of fibroin (2 and 4 wt%) and poly (Є-caprolactone) solution (10 wt%) to fabricate nanofibers. The nanofibers possess an average diameter of 152 ± 18 nm (2 % fibroin/PCL) and 175 ± 15 nm (4% fibroin/PCL). The results of Fourier transform infrared spectroscopy substantiates the preservation of the secondary structure of the fibroin in the blends indicating the structural stability of the neo-matrix. With an increase in the fibroin percentage, the hydrophobicity and thermal stability of the matrices as measured from melting temperature Tm (using DSC) decrease, while the mechanical strength is improved. The blended nanofibrous scaffolds are biodegradable, and support the viability and proliferation of human osteoblast-like cells as observed through scanning electron and confocal microscopes. Alkaline phosphatase assay indicates the cell proliferation and the generation of the neo-bone matrix. Taken together, these findings illustrate that the silk-poly (Є-caprolactone) blended nanofibrous scaffolds have an excellent prospect as scaffolding material in bone tissue engineering.

Genomic analysis of carboxyl/cholinesterase genes in the silkworm Bombyx mori

PubMed Central

2010-01-01

Background Carboxyl/cholinesterases (CCEs) have pivotal roles in dietary detoxification, pheromone or hormone degradation and neurodevelopment. The recent completion of genome projects in various insect species has led to the identification of multiple CCEs with unknown functions. Here, we analyzed the phylogeny, expression and genomic distribution of 69 putative CCEs in the silkworm, Bombyx mori (Lepidoptera: Bombycidae). Results A phylogenetic tree of CCEs in B. mori and other lepidopteran species was constructed. The expression pattern of each B. mori CCE was also investigated by a search of an expressed sequence tag (EST) database, and the relationship between phylogeny and expression was analyzed. A large number of B. mori CCEs were identified from a midgut EST library. CCEs expressed in the midgut formed a cluster in the phylogenetic tree that included not only B. mori genes but also those of other lepidopteran species. The silkworm, and possibly also other lepidopteran species, has a large number of CCEs, and this might be a consequence of the large cluster of midgut CCEs. Investigation of intron-exon organization in B. mori CCEs revealed that their positions and splicing site phases were strongly conserved. Several B. mori CCEs, including juvenile hormone esterase, not only showed clustering in the phylogenetic tree but were also closely located on silkworm chromosomes. We investigated the phylogeny and microsynteny of neuroligins in detail, among many CCEs. Interestingly, we found the evolution of this gene appeared not to be conserved between B. mori and other insect orders. Conclusions We analyzed 69 putative CCEs from B. mori. Comparison of these CCEs with other lepidopteran CCEs indicated that they had conserved expression and function in this insect order. The analyses showed that CCEs were unevenly distributed across the genome of B. mori and suggested that neuroligins may have a distinct evolutionary history from other insect order. It is possible

Bombyx mori histone methyltransferase BmAsh2 is essential for silkworm piRNA-mediated sex determination.

PubMed

Li, Zhiqian; You, Lang; Yan, Dong; James, Anthony A; Huang, Yongping; Tan, Anjiang

2018-02-01

Sex determination is a hierarchically-regulated process with high diversity in different organisms including insects. The W chromosome-derived Fem piRNA has been identified as the primary sex determination factor in the lepidopteran insect, Bombyx mori, revealing a distinctive piRNA-mediated sex determination pathway. However, the comprehensive mechanism of silkworm sex determination is still poorly understood. We show here that the silkworm PIWI protein BmSiwi, but not BmAgo3, is essential for silkworm sex determination. CRISPR/Cas9-mediated depletion of BmSiwi results in developmental arrest in oogenesis and partial female sexual reversal, while BmAgo3 depletion only affects oogenesis. We identify three histone methyltransferases (HMTs) that are significantly down-regulated in BmSiwi mutant moths. Disruption one of these, BmAsh2, causes dysregulation of piRNAs and transposable elements (TEs), supporting a role for it in the piRNA signaling pathway. More importantly, we find that BmAsh2 mutagenesis results in oogenesis arrest and partial female-to-male sexual reversal as well as dysregulation of the sex determination genes, Bmdsx and BmMasc. Mutagenesis of other two HMTs, BmSETD2 and BmEggless, does not affect piRNA-mediated sex determination. Histological analysis and immunoprecipitation results support a functional interaction between the BmAsh2 and BmSiwi proteins. Our data provide the first evidence that the HMT, BmAsh2, plays key roles in silkworm piRNA-mediated sex determination.

Silkworm Pupa Protein Hydrolysate Induces Mitochondria-Dependent Apoptosis and S Phase Cell Cycle Arrest in Human Gastric Cancer SGC-7901 Cells.

PubMed

Li, Xiaotong; Xie, Hongqing; Chen, Yajie; Lang, Mingzi; Chen, Yuyin; Shi, Liangen

2018-03-28

Silkworm pupae ( Bombyx mori ) are a high-protein nutrition source consumed in China since more than 2 thousand years ago. Recent studies revealed that silkworm pupae have therapeutic benefits to treat many diseases. However, the ability of the compounds of silkworm pupae to inhibit tumourigenesis remains to be elucidated. Here, we separated the protein of silkworm pupae and performed alcalase hydrolysis. Silkworm pupa protein hydrolysate (SPPH) can specifically inhibit the proliferation and provoke abnormal morphologic features of human gastric cancer cells SGC-7901 in a dose- and time-dependent manner. Moreover, flow cytometry indicated that SPPH can induce apoptosis and arrest the cell-cycle in S phase. Furthermore, SPPH was shown to provoke accumulation of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential. Western blotting analysis indicated that SPPH inhibited Bcl-2 expression and promoted Bax expression, and subsequently induced apoptosis-inducing factor and cytochrome C release, which led to the activation of initiator caspase-9 and executioner caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), eventually caused cell apoptosis. Moreover, SPPH-induced S-phase arrest was mediated by up-regulating the expression of E2F1 and down-regulating those of cyclin E, CDK2 and cyclin A2. Transcriptome sequencing and gene set enrichment analysis (GSEA) also revealed that SPPH treatment could affect gene expression and pathway regulation related to tumourigenesis, apoptosis and cell cycle. In summary, our results suggest that SPPH could specifically suppress cell growth of SGC-7901 through an intrinsic apoptotic pathway, ROS accumulation and cell cycle arrest, and silkworm pupae have a potential to become a source of anticancer agents in the future.

The homeodomain transcription factors antennapedia and POU-M2 regulate the transcription of the steroidogenic enzyme gene Phantom in the silkworm.

PubMed

Meng, Meng; Cheng, Dao-Jun; Peng, Jian; Qian, Wen-Liang; Li, Jia-Rui; Dai, Dan-Dan; Zhang, Tian-Lei; Xia, Qing-You

2015-10-02

The steroid hormone ecdysone, which controls insect molting and metamorphosis, is synthesized in the prothoracic gland (PG), and several steroidogenic enzymes that are expressed specifically in the PG are involved in ecdysteroidogenesis. In this study, we identified new regulators that are involved in the transcriptional control of the silkworm steroidogenic enzyme genes. In silico analysis predicted several potential cis-regulatory elements (CREs) for the homeodomain transcription factors Antennapedia (Antp) and POU-M2 in the proximal promoters of steroidogenic enzyme genes. Antp and POU-M2 are expressed dynamically in the PG during larval development, and their overexpression in silkworm embryo-derived (BmE) cells induced the expression of steroidogenic enzyme genes. Importantly, luciferase reporter analyses, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays revealed that Antp and POU-M2 promote the transcription of the silkworm steroidogenic enzyme gene Phantom (Phm) by binding directly to specific motifs within overlapping CREs in the Phm promoter. Mutations of these CREs in the Phm promoter suppressed the transcriptional activities of both Antp and POU-M2 in BmE cells and decreased the activities of mutated Phm promoters in the silkworm PG. In addition, pulldown and co-immunoprecipitation assays demonstrated that Antp can interact with POU-M2. Moreover, RNA interference-mediated down-regulation of either Antp or POU-M2 during silkworm wandering not only decreased the ecdysone titer but also led to the failure of metamorphosis. In summary, our results suggest that Antp and POU-M2 coordinate the transcription of the silkworm Phm gene directly, indicating new roles for homeodomain proteins in regulating insect ecdysteroidogenesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

TIL-type protease inhibitors may be used as targeted resistance factors to enhance silkworm defenses against invasive fungi.

PubMed

Li, Youshan; Zhao, Ping; Liu, Huawei; Guo, Xiaomeng; He, Huawei; Zhu, Rui; Xiang, Zhonghuai; Xia, Qingyou

2015-02-01

Entomopathogenic fungi penetrate the insect cuticle using their abundant hydrolases. These hydrolases, which include cuticle-degrading proteases and chitinases, are important virulence factors. Our recent findings suggest that many serine protease inhibitors, especially TIL-type protease inhibitors, are involved in insect resistance to pathogenic microorganisms. To clarify the molecular mechanism underlying this resistance to entomopathogenic fungi and identify novel genes to improve the silkworm antifungal capacity, we conducted an in-depth study of serine protease inhibitors. Here, we cloned and expressed a novel silkworm TIL-type protease inhibitor, BmSPI39. In activity assays, BmSPI39 potently inhibited the virulence protease CDEP-1 of Beauveria bassiana, suggesting that it might suppress the fungal penetration of the silkworm integument by inhibiting the cuticle-degrading proteases secreted by the fungus. Phenol oxidase activation studies showed that melanization is involved in the insect immune response to fungal invasion, and that fungus-induced excessive melanization is suppressed by BmSPI39 by inhibiting the fungal cuticle-degrading proteases. To better understand the mechanism involved in the inhibition of fungal virulence by protease inhibitors, their effects on the germination of B. bassiana conidia was examined. BmSPI38 and BmSPI39 significantly inhibited the germination of B. bassiana conidia. Survival assays showed that BmSPI38 and BmSPI39 markedly improved the survival rates of silkworms, and can therefore be used as targeted resistance proteins in the silkworm. These results provided new insight into the molecular mechanisms whereby insect protease inhibitors confer resistance against entomopathogenic fungi, suggesting their potential application in medicinal or agricultural fields. Copyright © 2014 Elsevier Ltd. All rights reserved.

Molecular cloning, characterization and expression analysis of ATG1 in the silkworm, Bombyx mori.

PubMed

Casati, Barbara; Terova, Genciana; Cattaneo, Anna Giulia; Rimoldi, Simona; Franzetti, Eleonora; de Eguileor, Magda; Tettamanti, Gianluca

2012-12-15

Atg1 is a Serine/Threonine protein kinase that plays a pivotal role in autophagy. A complete coding sequence of ATG1 is not available for the silkworm, Bombyx mori which is a good model for studying the autophagic process. In the present study we isolated two full-length cDNAs of 2175 (transcript variant A) and 2271 (transcript variant B) bases representing ATG1 in the silkworm. Phylogenetic analysis indicated that BmATG1 was closely related to orthologs of other insects. The encoded BmAtg1 proteins shared extensive homology with orthologs from yeast to mammals, showing high conservation at the N-terminal region where the catalytic domain and ATP- and Mg-binding sites are located. A de novo prediction of the three-dimensional structure for each protein is presented. We used real-time RT-PCR to quantify dynamic changes in mRNA copy number of BmATG1 in the midgut and fat body of fifth instar larvae undergoing starvation, as well as in other tissues of silkworm at the end of last larval instar. Our qPCR results revealed that BmATG1 expression levels at the end of larval life were comparable in the midgut, fat body and Malpighian tubules, while these were higher in the gonads; moreover, the mRNA copy number of ATG1 was very different among the anterior, middle and posterior silk glands. Real-time PCR analysis also showed that starvation significantly influenced BmATG1 mRNA copy number in the fat body of silkworm, inducing an upregulation 24h after food withdrawal, with only a slight effect in the midgut. Low expression levels of BmATG1 were observed in both tissues of control animals up to the second day of spinning phase. Copyright © 2012 Elsevier B.V. All rights reserved.

Bombyx mori cyclin-dependent kinase inhibitor is involved in regulation of the silkworm cell cycle.

PubMed

Tang, X-F; Zhou, X-L; Zhang, Q; Chen, P; Lu, C; Pan, M-H

2018-06-01

Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of the cell cycle. They can bind to cyclin-dependent kinase (CDK)-cyclin complexes and inhibit CDK activities. We identified a single homologous gene of the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family, BmCKI, in the silkworm, Bombyx mori. The gene transcribes two splice variants: a 654-bp-long BmCKI-L (the longer splice variant) encoding a protein with 217 amino acids and a 579-bp-long BmCKI-S (the shorter splice variant) encoding a protein with 192 amino acids. BmCKI-L and BmCKI-S contain the Cip/Kip family conserved cyclin-binding domain and the CDK-binding domain. They are localized in the nucleus and have an unconventional bipartite nuclear localization signal at amino acid residues 181-210. Overexpression of BmCKI-L or BmCKI-S affected cell cycle progression; the cell cycle was arrested in the first gap phase of cell cycle (G1). RNA interference of BmCKI-L or BmCKI-S led to cells accumulating in the second gap phase and the mitotic phase of cell cycle (G2/M). Both BmCKI-L and BmCKI-S are involved in cell cycle regulation and probably have similar effects. The transgenic silkworm with BmCKI-L overexpression (BmCKI-L-OE), exhibited embryonic lethal, larva developmental retardation and lethal phenotypes. These results suggest that BmCKI-L might regulate the growth and development of silkworm. These findings clarify the function of CKIs and increase our understanding of cell cycle regulation in the silkworm. © 2018 The Royal Entomological Society.

Induced Hyperproteinemia and Its Effects on the Remodeling of Fat Bodies in Silkworm, Bombyx mori

PubMed Central

Chen, Xue-Dong; Wang, Yong-Feng; Wang, Yu-Long; Li, Qiu-Ying; Ma, Huan-Yu; Wang, Lu; Sima, Yang-Hu; Xu, Shi-Qing

2018-01-01

Hyperproteinemia, which is characterized by an abnormally elevated plasma protein concentration (PPC), is a high-mortality, metabolic complication associated with severe liver and kidney disease. It is difficult to clinically distinguish the difference between the impacts of primary diseases and hyperproteinemia on tissues and organs, and there are no available animal models of hyperproteinemia. Here, we constructed an animal model of hyperproteinemia with a controllable PPC and no primary disease effects in the silkworm Bombyx mori that has attracted interest owing to its potential use in the pathological analysis of model animals. Silkworm have an open circulatory system in which each organ is directly immersed in hemolymph. The fat body (FB) of a silkworm, as a major organ for nutrient storage and energy metabolism, can effectively reflect hyperproteinemia-induced metabolic abnormalities in damaged visceral tissues. A pathogenesis study showed that hyperproteinemia attenuated cell autophagy and apoptosis by attenuating an endocrine hormone, thereby preventing FB remodeling during metamorphosis. Meanwhile, hyperproteinemia increased oxidative stress in the FB and resulted in a dysfunction of amino acid conversion. Supplementation with exogenous 20-hydroxyecdysone effectively mitigated the hyperproteinemia-mediated inhibition of FB remodeling. PMID:29651251

An efficient strategy for producing a stable, replaceable, highly efficient transgene expression system in silkworm, Bombyx mori

PubMed Central

Long, Dingpei; Lu, Weijian; Zhang, Yuli; Bi, Lihui; Xiang, Zhonghuai; Zhao, Aichun

2015-01-01

We developed an efficient strategy that combines a method for the post-integration elimination of all transposon sequences, a site-specific recombination system, and an optimized fibroin H-chain expression system to produce a stable, replaceable, highly efficient transgene expression system in the silkworm (Bombyx mori) that overcomes the disadvantages of random insertion and post-integration instability of transposons. Here, we generated four different transgenic silkworm strains, and of one the transgenic strains, designated TS1-RgG2, with up to 16% (w/w) of the target protein in the cocoons, was selected. The subsequent elimination of all the transposon sequences from TS1-RgG2 was completed by the heat-shock-induced expression of the transposase in vivo. The resulting transgenic silkworm strain was designated TS3-g2 and contained only the attP-flanked optimized fibroin H-chain expression cassette in its genome. A phiC31/att-system-based recombinase-mediated cassette exchange (RMCE) method could be used to integrate other genes of interest into the same genome locus between the attP sites in TS3-g2. Controlling for position effects with phiC31-mediated RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs. PMID:25739894

Comprehensive profiling of lysine acetylation suggests the widespread function is regulated by protein acetylation in the silkworm, Bombyx mori.

PubMed

Nie, Zuoming; Zhu, Honglin; Zhou, Yong; Wu, Chengcheng; Liu, Yue; Sheng, Qing; Lv, Zhengbing; Zhang, Wenping; Yu, Wei; Jiang, Caiying; Xie, Longfei; Zhang, Yaozhou; Yao, Juming

2015-09-01

Lysine acetylation in proteins is a dynamic and reversible PTM and plays an important role in diverse cellular processes. In this study, using lysine-acetylation (Kac) peptide enrichment coupled with nano HPLC/MS/MS, we initially identified the acetylome in the silkworms. Overall, a total of 342 acetylated proteins with 667 Kac sites were identified in silkworm. Sequence motifs analysis around Kac sites revealed an enrichment of Y, F, and H in the +1 position, and F was also enriched in the +2 and -2 positions, indicating the presences of preferred amino acids around Kac sites in the silkworm. Functional analysis showed the acetylated proteins were primarily involved in some specific biological processes. Furthermore, lots of nutrient-storage proteins, such as apolipophorin, vitellogenin, storage proteins, and 30 K proteins, were highly acetylated, indicating lysine acetylation may represent a common regulatory mechanism of nutrient utilization in the silkworm. Interestingly, Ser2 proteins, the coating proteins of larval silk, were found to contain many Kac sites, suggesting lysine acetylation may be involved in the regulation of larval silk synthesis. This study is the first to identify the acetylome in a lepidoptera insect, and expands greatly the catalog of lysine acetylation substrates and sites in insects. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role of cholesterol 10-methyl group and effect of "extra" 14-methyl group on silkworm growth and development.

PubMed

Mamiya, M; Takahashi, K; Eguchi, S; Morisaki, M

1989-07-01

In order to establish the functional importance of the 10-methyl group of cholesterol and the planarity of the steroid ring, silkworms (Bombyx mori) were reared on an artificial diet containing 19-norcholesterol (1), 14 alpha-methylcholesterol (3) or 19,19-difluorocholesterol (2). The former two sterols (1 and 3) only partially satisfied the silkworm sterol requirement; growth and development were seriously retarded. The fluorinated sterol (2) was much more deleterious and was totally inadequate in meeting the sterol requirement.

Effects of BmCPV Infection on Silkworm Bombyx mori Intestinal Bacteria

PubMed Central

Zhang, Hao; Kumar, Dhiraj; Liu, Bo; Gong, Yongchang; Zhu, Min; Zhu, Liyuan; Liang, Zi; Kuang, Sulan; Chen, Fei; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2016-01-01

The gut microbiota has a crucial role in the growth, development and environmental adaptation in the host insect. The objective of our work was to investigate the microbiota of the healthy silkworm Bombyx mori gut and changes after the infection of B. mori cypovirus (BmCPV). Intestinal contents of the infected and healthy larvae of B. mori of fifth instar were collected at 24, 72 and 144 h post infection with BmCPV. The gut bacteria were analyzed by pyrosequencing of the 16S rRNA gene. 147(135) and 113(103) genera were found in the gut content of the healthy control female (male) larvae and BmCPV-infected female (male) larvae, respectively. In general, the microbial communities in the gut content of healthy larvae were dominated by Enterococcus, Delftia, Pelomonas, Ralstonia and Staphylococcus, however the abundance change of each genus was depended on the developmental stage and gender. Microbial diversity reached minimum at 144 h of fifth instar larvae. The abundance of Enterococcus in the females was substantially lower and the abundance of Delftia, Aurantimonas and Staphylococcus was substantially higher compared to the males. Bacterial diversity in the intestinal contents decreased after post infection with BmCPV, whereas the abundance of both Enterococcus and Staphylococcus which belongs to Gram-positive were increased. Therefore, our findings suggested that observed changes in relative abundance was related to the immune response of silkworm to BmCPV infection. Relevance analysis of plenty of the predominant genera showed the abundance of the Enterococcus genus was in negative correlation with the abundance of the most predominant genera. These results provided insight into the relationship between the gut microbiota and development of the BmCPV-infected silkworm. PMID:26745627

Effects of BmCPV Infection on Silkworm Bombyx mori Intestinal Bacteria.

PubMed

Sun, Zhenli; Lu, Yahong; Zhang, Hao; Kumar, Dhiraj; Liu, Bo; Gong, Yongchang; Zhu, Min; Zhu, Liyuan; Liang, Zi; Kuang, Sulan; Chen, Fei; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2016-01-01

The gut microbiota has a crucial role in the growth, development and environmental adaptation in the host insect. The objective of our work was to investigate the microbiota of the healthy silkworm Bombyx mori gut and changes after the infection of B. mori cypovirus (BmCPV). Intestinal contents of the infected and healthy larvae of B. mori of fifth instar were collected at 24, 72 and 144 h post infection with BmCPV. The gut bacteria were analyzed by pyrosequencing of the 16S rRNA gene. 147(135) and 113(103) genera were found in the gut content of the healthy control female (male) larvae and BmCPV-infected female (male) larvae, respectively. In general, the microbial communities in the gut content of healthy larvae were dominated by Enterococcus, Delftia, Pelomonas, Ralstonia and Staphylococcus, however the abundance change of each genus was depended on the developmental stage and gender. Microbial diversity reached minimum at 144 h of fifth instar larvae. The abundance of Enterococcus in the females was substantially lower and the abundance of Delftia, Aurantimonas and Staphylococcus was substantially higher compared to the males. Bacterial diversity in the intestinal contents decreased after post infection with BmCPV, whereas the abundance of both Enterococcus and Staphylococcus which belongs to Gram-positive were increased. Therefore, our findings suggested that observed changes in relative abundance was related to the immune response of silkworm to BmCPV infection. Relevance analysis of plenty of the predominant genera showed the abundance of the Enterococcus genus was in negative correlation with the abundance of the most predominant genera. These results provided insight into the relationship between the gut microbiota and development of the BmCPV-infected silkworm.

Production of recombinant Bombyx mori nucleopolyhedrovirus in silkworm by intrahaemocoelic injection with invasive diaminopimelate auxotrophic Escherichia coli containing BmNPV-Bacmid.

PubMed

Sun, Jingchen; Yao, Lunguang; Yao, Ning; Xu, Hua; Jin, Pengfei; Kan, Yunchao

2010-12-01

The present study elaborates a cost-effective and transfectant-free method for generating recombinant Bombyx mori (silkworm) nucleopolyhedrovirus in silkworm larvae and pupae by injecting invasive Escherichia coli carrying BmBacmid [BmNPV (B. mori nucleopolyhedrovirus)-Bacmid] into larval haemocoel. Up to 109 PFU (plaque-forming units)/ml of infective recombinant baculovirus was generated in the silkworm by intrahaemocoelic injection with 106 DAP (diaminopimelic acid) auxotrophic and BmBacmid containing E. coli cells expressing both invasin and listeriolysin. Thus 1 ml of overnight culture of E. coli is sufficient to inject more than 2000 larvae, while DAP costing up to $1 is enough to inject about 4000 larvae. Recombinant proteins can be controlled to be expressed mainly in pupae by adjusting the injection dose, too. In this new method, many original manipulations have been eliminated, including BmBacmid preparation and the subsequent complex transfection procedures. Hence it is a time- and cost-saving means for large-scale injection of B. mori for recombinant baculovirus production in comparison with the traditional transfection methods, which may play an important role in the industrial development of the BmNPV-silkworm bioreactor.

Identification and Characterization of Novel Chitin-Binding Proteins from the Larval Cuticle of Silkworm, Bombyx mori.

PubMed

Dong, Zhaoming; Zhang, Weiwei; Zhang, Yan; Zhang, Xiaolu; Zhao, Ping; Xia, Qingyou

2016-05-06

Cuticle is mainly made of chitin filaments embedded in a matrix of cuticular proteins (CPs). Cuticular chitins have minor differences, whereas CPs are widely variable with respect to their sequences and structures. To understand the molecular basis underlying the mechanical properties of cuticle, it is necessary to know which CPs interact with chitin and how they are assembled into the cuticle structure. In the present study, a chitin-binding assay was performed followed by liquid chromatography-tandem mass spectrometry to identify the extracted proteins from the larval cuticle of silkworm, Bombyx mori. There were 463 proteins identified from the silkworm larval cuticle, 200 of which were recovered in the chitin-binding fraction. A total of 103 proteins were annotated as CPs, which were classified into 11 CP families based on their conserved motifs, including CPR, CPAP, CPT, CPF and CPFL, CPCFC, chitin_bind 3, BmCPH2 homologues, BmCPH9 homologues, BmCPG1 homologues, BmCPG20 homologues, and BmCPG21 homologues. A total of five CP families were newly identified in the chitin-binding fraction, thereby providing new information and insight into the composition, structure, and function of the silkworm larval cuticle.

Aspartate Decarboxylase is Required for a Normal Pupa Pigmentation Pattern in the Silkworm, Bombyx mori.

PubMed

Dai, Fangyin; Qiao, Liang; Cao, Cun; Liu, Xiaofan; Tong, Xiaoling; He, Songzhen; Hu, Hai; Zhang, Li; Wu, Songyuan; Tan, Duan; Xiang, Zhonghuai; Lu, Cheng

2015-06-16

The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of β-alanine and N-β-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of β-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera.

Aspartate Decarboxylase is Required for a Normal Pupa Pigmentation Pattern in the Silkworm, Bombyx mori

PubMed Central

Dai, Fangyin; Qiao, Liang; Cao, Cun; Liu, Xiaofan; Tong, Xiaoling; He, Songzhen; Hu, Hai; Zhang, Li; Wu, Songyuan; Tan, Duan; Xiang, Zhonghuai; Lu, Cheng

2015-01-01

The pigmentation pattern of Lepidoptera varies greatly in different development stages. To date, the effects of key genes in the melanin metabolism pathway on larval and adult body color are distinct, yet the effects on pupal pigmentation remains unclear. In the silkworm, Bombyx mori, the black pupa (bp) mutant is only specifically melanized at the pupal stage. Using positional cloning, we found that a mutation in the Aspartate decarboxylase gene (BmADC) is causative in the bp mutant. In the bp mutant, a SINE-like transposon with a length of 493 bp was detected ~2.2 kb upstream of the transcriptional start site of BmADC. This insertion causes a sharp reduction in BmADC transcript levels in bp mutants, leading to deficiency of β-alanine and N-β-alanyl dopamine (NBAD), but accumulation of dopamine. Following injection of β-alanine into bp mutants, the color pattern was reverted that of the wild-type silkworms. Additionally, melanic pupae resulting from knock-down of BmADC in the wild-type strain were obtained. These findings show that BmADC plays a crucial role in melanin metabolism and in the pigmentation pattern of the silkworm pupal stage. Finally, this study contributes to a better understanding of pupa pigmentation patterns in Lepidoptera. PMID:26077025

Combined toxicity of chlorantraniliprole, lambda-cyhalothrin, and imidacloprid to the silkworm Bombyx mori (Lepidoptera: Bombycidae).

PubMed

Liu, Yanmei; Zhang, Hui; He, Fengmei; Li, Xuesheng; Tan, Huihua; Zeng, Dongqiang

2018-05-29

Insecticides with different modes of action may act in combination, in ways such as drifting, spray equipment residual, or utilizing concurrently in mulberry orchards or nearby agricultural fields. Silkworms may suffer from a diverse impact on the survival. In this study, the toxicity of chlorantraniliprole, lambda-cyhalothrin, and imidacloprid and their combinations to the second instar of silkworms (Bombyx mori (L.)(Lepidoptera: Bombycidae)) were evaluated after 48 and 72 h treatment by the leaf-dipping method and the combination index (CI)-isobologram equation. After 48 h treatment, results indicated that (1) the increasing order of toxicity was imidacloprid < chlorantraniliprole < lambda-cyhalothrin, and that (2) synergism was predominated in most combinations excepted for the lambda-cyhalothrin + imidacloprid combination which displayed an additive effect at f a value 0.5. Then, after 72 h treatment, results exhibited that (1) the increasing order of toxicity was imidacloprid < lambda-cyhalothrin < chlorantraniliprole, and that (2) only the chlorantraniliprole + imidacloprid mixture yielded antagonism at f a value 0.5; the other combinations performed an additive effect at least. Consequently, combined toxicity of mixtures may pose a worse effect on silkworm than single toxicity of insecticides. Therefore, we suggest that insecticide mixtures should be added into ecotoxicological risk assessment.

A discovery of novel microRNAs in the silkworm (Bombyx mori) genome.

PubMed

Yu, Xiaomin; Zhou, Qing; Cai, Yimei; Luo, Qibin; Lin, Hongbin; Hu, Songnian; Yu, Jun

2009-12-01

MicroRNAs (miRNAs) are pivotal regulators involved in various physiological and pathological processes via their post-transcriptional regulation of gene expressions. We sequenced 14 libraries of small RNAs constructed from samples spanning the life cycle of silkworms, and discovered 50 novel miRNAs previously not known in animals and verified 43 of them using stem-loop RT-PCR. Our genome-wide analyses of 27 species-specific miRNAs suggest they arise from transposable elements, protein-coding genes duplication/transposition and random foldback sequences; which is consistent with the idea that novel animal miRNAs may evolve from incomplete self-complementary transcripts and become fixed in the process of co-adaptation with their targets. Computational prediction suggests that the silkworm-specific miRNAs may have a preference of regulating genes that are related to life-cycle-associated traits, and these genes can serve as potential targets for subsequent studies of the modulating networks in the development of Bombyx mori.

Comparative transcriptome profiling of a thermal resistant vs. sensitive silkworm strain in response to high temperature under stressful humidity condition

PubMed Central

Xiao, Jinshu; Wang, La; Liu, Taihang; Wu, Yunfei; Dong, Feifan; Jiang, Yaming; Pan, Minhui; Zhang, Youhong; Lu, Cheng

2017-01-01

Thermotolerance is important particularly for poikilotherms such as insects. Understanding the mechanisms by which insects respond to high temperatures can provide insights into their adaptation to the environment. Therefore, in this study, we performed a transcriptome analysis of two silkworm strains with significantly different resistance to heat as well as humidity; the thermo-resistant strain 7532 and the thermos-sensitive strain Knobbed. We identified in total 4,944 differentially expressed genes (DEGs) using RNA-Seq. Among these, 4,390 were annotated and 554 were novel. Gene Ontology (GO) analysis of 747 DEGs identified between RT_48h (Resistant strain with high-temperature Treatment for 48 hours) and ST_48h (Sensitive strain with high-temperature Treatment for 48 hours) showed significant enrichment of 12 GO terms including metabolic process, extracellular region and serine-type peptidase activity. Moreover, we discovered 12 DEGs that may contribute to the heat-humidity stress response in the silkworm. Our data clearly showed that 48h post-exposure may be a critical time point for silkworm to respond to high temperature and humidity. These results provide insights into the genes and biological processes involved in high temperature and humidity tolerance in the silkworm, and advance our understanding of thermal tolerance in insects. PMID:28542312

Comparative transcriptome profiling of a thermal resistant vs. sensitive silkworm strain in response to high temperature under stressful humidity condition.

PubMed

Xiao, Wenfu; Chen, Peng; Xiao, Jinshu; Wang, La; Liu, Taihang; Wu, Yunfei; Dong, Feifan; Jiang, Yaming; Pan, Minhui; Zhang, Youhong; Lu, Cheng

2017-01-01

Thermotolerance is important particularly for poikilotherms such as insects. Understanding the mechanisms by which insects respond to high temperatures can provide insights into their adaptation to the environment. Therefore, in this study, we performed a transcriptome analysis of two silkworm strains with significantly different resistance to heat as well as humidity; the thermo-resistant strain 7532 and the thermos-sensitive strain Knobbed. We identified in total 4,944 differentially expressed genes (DEGs) using RNA-Seq. Among these, 4,390 were annotated and 554 were novel. Gene Ontology (GO) analysis of 747 DEGs identified between RT_48h (Resistant strain with high-temperature Treatment for 48 hours) and ST_48h (Sensitive strain with high-temperature Treatment for 48 hours) showed significant enrichment of 12 GO terms including metabolic process, extracellular region and serine-type peptidase activity. Moreover, we discovered 12 DEGs that may contribute to the heat-humidity stress response in the silkworm. Our data clearly showed that 48h post-exposure may be a critical time point for silkworm to respond to high temperature and humidity. These results provide insights into the genes and biological processes involved in high temperature and humidity tolerance in the silkworm, and advance our understanding of thermal tolerance in insects.

Reducing blood glucose levels in TIDM mice with an orally administered extract of sericin from hIGF-I-transgenic silkworm cocoons.

PubMed

Song, Zuowei; Zhang, Mengyao; Xue, Renyu; Cao, Guangli; Gong, Chengliang

2014-05-01

In previous studies, we reported that the blood glucose levels of mice with type I diabetes mellitus (TIDM) was reduced with orally administered silk gland powder from silkworms transgenic for human insulin-like growth factor-I (hIGF-I). However, potential safety hazards could not be eliminated because the transgenic silk gland powder contained heterologous DNA, including the green fluorescent protein (gfp) and neomycin resistance (neo) genes. These shortcomings might be overcome if the recombinant hIGF-I were secreted into the sericin layer of the cocoon. In this study, silkworm eggs were transfected with a novel piggyBac transposon vector, pigA3GFP-serHS-hIGF-I-neo, containing the neo, gfp, and hIGF-I genes controlled by the sericin-1 (ser-1) promoter with the signal peptide DNA sequence of the fibrin heavy chain (Fib-H) and a helper plasmid containing the piggyBac transposase sequence under the control of the Bombyx mori actin 3 (A3) promoter, using sperm-mediated gene transfer to generate the transformed silkworms. The hIGF-I content estimated by enzyme-linked immunosorbent assay was approximately 162.7 ng/g. To estimate the biological activity of the expressed hIGF-I, streptozotocin-induced TIDM mice were orally administered sericin from the transgenic silkworm. The blood glucose levels of the mice were significantly reduced, suggesting that the extract from the transgenic hIGF-I silkworm cocoons can be used as an orally administered drug. Copyright © 2014 Elsevier Ltd. All rights reserved.

Impacts on silkworm larvae midgut proteomics by transgenic Trichoderma strain and analysis of glutathione S-transferase sigma 2 gene essential for anti-stress response of silkworm larvae.

PubMed

Li, Yingying; Dou, Kai; Gao, Shigang; Sun, Jianan; Wang, Meng; Fu, Kehe; Yu, Chuanjin; Wu, Qiong; Li, Yaqian; Chen, Jie

2015-08-03

Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72 h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain. Copyright © 2015 Elsevier B.V. All rights reserved.

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial.

PubMed

Otsuki, Ryosuke; Yamamoto, Masafumi; Matsumoto, Erika; Iwamoto, Shin-Ichi; Sezutsu, Hideki; Suzui, Masumi; Takaki, Keiko; Wakabayashi, Keiji; Mori, Hajime; Kotani, Eiji

2017-06-27

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm ( Bombyx mori ) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly ( Pieris rapae ) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori -derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential.

Effects of added CeCl3 on resistance of fifth-instar larvae of silkworm to Bombyx mori nucleopolyhedrovirus infection.

PubMed

Li, Bing; Xie, Yi; Cheng, Zhe; Cheng, Jie; Hu, Rengping; Cui, Yaling; Gong, Xiaolan; Shen, Weide; Hong, Fashui

2012-06-01

One of the most important agents causing lethal disease in the silkworm is the Bombyx mori nucleopolyhedrovirus (BmNPV), while low-dose rare earths are demonstrated to increase immune capacity in animals. However, very little is known about the effects of added CeCl(3) on decreasing BmNPV infection of silkworm. The present study investigated the effects of added CeCl(3) to an artificial diet on resistance of fifth-instar larvae of silkworm to BmNPV infection. Our findings indicated that added CeCl(3) significantly decreased inhibition of growth and mortality of fifth-instar larvae caused by BmNPV infection. Furthermore, the added CeCl(3) obviously decreased lipid peroxidation level and accumulation of reactive oxygen species such as O(2)(-), H(2)O(2), (·)OH, and NO and increased activities of the antioxidant enzymes including superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, ascorbate, and glutathione contents in the BmNPV-infected fifth-instar larvae. In addition, the added CeCl(3) could significantly promote acetylcholine esterase activity and attenuate the activity of inducible nitric oxide synthase in the BmNPV-infected fifth-instar larvae. These findings suggested that added CeCl(3) may relieve oxidative damage and neurotoxicity of silkworm caused by BmNPV infection via increasing antioxidant capacity and acetylcholine esterase activity.

Nanofibrous nonmulberry silk/PVA scaffold for osteoinduction and osseointegration.

PubMed

Bhattacharjee, Promita; Kundu, Banani; Naskar, Deboki; Maiti, Tapas K; Bhattacharya, Debasis; Kundu, Subhas C

2015-05-01

Poly-vinyl alcohol and nonmulberry tasar silk fibroin of Antheraea mylitta are blended to fabricate nanofibrous scaffolds for bone regeneration. Nanofibrous matrices are prepared by electrospinning the equal volume ratio blends of silk fibroin (2 and 4 wt%) with poly-vinyl alcohol solution (10 wt%) and designated as 2SF/PVA and 4SF/PVA, respectively with average nanofiber diameters of 177 ± 13 nm (2SF/PVA) and 193 ± 17 nm (4SF/PVA). Fourier transform infrared spectroscopy confirms retention of the secondary structure of fibroin in blends indicating the structural stability of neo-matrix. Both thermal stability and contact angle of the blends decrease with increasing fibroin percentage. Conversely, fibroin imparts mechanical stability to the blends; greater tensile strength is observed with increasing fibroin concentration. Blended scaffolds are biodegradable and support well the neo-bone matrix synthesis by human osteoblast like cells. The findings indicate the potentiality of nanofibrous scaffolds of nonmulberry fibroin as bone scaffolding material. © 2014 Wiley Periodicals, Inc.

Conceptual design of a bioregenerative life support system containing crops and silkworms

NASA Astrophysics Data System (ADS)

Hu, Enzhu; Bartsev, Sergey I.; Liu, Hong

2010-04-01

This article summarizes a conceptual design of a bioregenerative life support system for permanent lunar base or planetary exploration. The system consists of seven compartments - higher plants cultivation, animal rearing, human habitation, water recovery, waste treatment, atmosphere management, and storages. Fifteen kinds of crops, such as wheat, rice, soybean, lettuce, and mulberry, were selected as main life support contributors to provide the crew with air, water, and vegetable food. Silkworms fed by crop leaves were designated to produce partial animal nutrition for the crew. Various physical-chemical and biological methods were combined to reclaim wastewater and solid waste. Condensate collected from atmosphere was recycled into potable water through granular activated carbon adsorption, iodine sterilization, and trace element supplementation. All grey water was also purified though multifiltration and ultraviolet sterilization. Plant residue, human excrement, silkworm feces, etc. were decomposed into inorganic substances which were finally absorbed by higher plants. Some meat, ingredients, as well as nitrogen fertilizer were prestored and resupplied periodically. Meanwhile, the same amount and chemical composition of organic waste was dumped to maintain the steady state of the system. A nutritional balanced diet was developed by means of the linear programming method. It could provide 2721 kcal of energy, 375.5 g of carbohydrate, 99.47 g of protein, and 91.19 g of fat per capita per day. Silkworm powder covered 12.54% of total animal protein intakes. The balance of material flows between compartments was described by the system of stoichiometric equations. Basic life support requirements for crews including oxygen, food, potable and hygiene water summed up to 29.68 kg per capita per day. The coefficient of system material closure reached 99.40%.

Development of advanced antimicrobial and sterilized plasma polypropylene grafted muga (Antheraea assama) silk as suture biomaterial.

PubMed

Gogoi, Dolly; Choudhury, Arup Jyoti; Chutia, Joyanti; Pal, Arup Ratan; Khan, Mojibur; Choudhury, Manash; Pathak, Pallabi; Das, Gouranga; Patil, Dinkar S

2014-04-01

Surface modification of silk fibroin (SF) materials using environmentally friendly and non-hazardous process to tailor them for specific application as biomaterials has drawn a great deal of interest in the field of biomedical research. To further explore this area of research, in this report, polypropylene (PP) grafted muga (Antheraea assama) SF (PP-AASF) suture is developed using plasma treatment and plasma graft polymerization process. For this purpose, AASF is first sterilized in argon (Ar) plasma treatment followed by grafting PP onto its surface. AASF is a non-mulberry variety having superior qualities to mulberry SF and is still unexplored in the context of suture biomaterial. AASF, Ar plasma treated AASF (AASFAr) and PP-AASF are subjected to various characterization techniques for better comparison and the results are attempted to correlate with their observed properties. Excellent mechanical strength, hydrophobicity, antibacterial behavior, and remarkable wound healing activity of PP-AASF over AASF and AASFAr make it a promising candidate for application as sterilized suture biomaterial. Copyright © 2013 Wiley Periodicals, Inc.

GC/MS-based metabolomic studies reveal key roles of glycine in regulating silk synthesis in silkworm, Bombyx mori.

PubMed

Chen, Quanmei; Liu, Xinyu; Zhao, Ping; Sun, Yanhui; Zhao, Xinjie; Xiong, Ying; Xu, Guowang; Xia, Qingyou

2015-02-01

Metabolic profiling of silkworm, especially the factors that affect silk synthesis at the metabolic level, is little known. Herein, metabolomic method based on gas chromatography-mass spectrometry was applied to identify key metabolic changes in silk synthesis deficient silkworms. Forty-six differential metabolites were identified in Nd group with the defect of silk synthesis. Significant changes in the levels of glycine and uric acid (up-regulation), carbohydrates and free fatty acids (down-regulation) were observed. The further metabolomics of silk synthesis deficient silkworms by decreasing silk proteins synthesis using knocking out fibroin heavy chain gene or extirpating silk glands operation showed that the changes of the metabolites were almost consistent with those of the Nd group. Furthermore, the increased silk yields by supplying more glycine or its related metabolite confirmed that glycine is a key metabolite to regulate silk synthesis. These findings provide important insights into the regulation between metabolic profiling and silk synthesis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Microwave-assisted extraction of silkworm pupal oil and evaluation of its fatty acid composition, physicochemical properties and antioxidant activities.

PubMed

Hu, Bin; Li, Cheng; Zhang, Zhiqing; Zhao, Qing; Zhu, Yadong; Su, Zhao; Chen, Yizi

2017-09-15

Microwave-assisted extraction (MAE) of oil from silkworm pupae was firstly performed in the present research. The response surface methodology was applied to optimize the parameters for MAE. The yield of oil by MAE was 30.16% under optimal conditions of a mixed solvent consisting of ethanol and n-hexane (1:1, v/v), microwave power (360W), liquid to solid ratio (7.5/1mL/g), microwave time (29min). Moreover, oil extracted by MAE was quantitatively (yield) and qualitatively (fatty acid profile) similar to those obtained using Soxhlet extraction (SE), but oil extracted by MAE exhibited favourable physicochemical properties and oxidation stability. Additionally, oil extracted by MAE had a higher content of total phenolic, and it showed stronger antioxidant activities. Scanning electron microscopy revealed that microwave technique efficiently promoted the release of oil by breaking down the cell structure of silkworm pupae. Therefore, MAE can be an effective method for the silkworm pupal oil extraction. Copyright © 2017 Elsevier Ltd. All rights reserved.

Osiris9a is a major component of silk fiber in lepidopteran insects.

PubMed

Liu, Chun; Hu, Wenbo; Cheng, Tingcai; Peng, Zhangchuan; Mita, Kazuei; Xia, Qingyou

2017-10-01

In a previous high-throughput proteomics study, it was found that the silkworm cocoon contains hundreds of complex proteins, many of which have unknown functions, in addition to fibroins, sericins, and some protease inhibitors. Osiris was one of the proteins with no known function. In this study, we identified the Osiris gene family members and constructed a phylogenetic tree based on the sequences from different species. Our results indicate that the Osiris9 gene subfamily contains six members; it is specifically expressed in lepidopteran insects and has evolved by gene duplication. An Osiris gene family member from Bombyx mori was designated as BmOsiris9a (BmOsi9a) on the basis of its homology to Drosophila melanogaster Osiris9. The expression pattern of BmOsi9a showed that it was highly expressed only in the middle silk gland of silkworm larvae, similar to Sericin1 (Ser1). BmOsi9a was visualized as two bands in western blot analysis, implying that it probably undergoes post-translational modifications. Immunohistochemistry analysis revealed that BmOsi9a was synthesized and secreted into the lumen of the middle silk gland, and was localized in the sericin layer in the silk fiber. BmOsi9a was found in the silk fibers of not only three Bombycidae species, viz. B. mori, B. mandarina, and B. huttoni, but also in the fibers collected from Saturniidae species, including Antheraea assama, Antheraea mylitta, and Samia cynthia. Although the exact biological function of Osi9a in the silk fibers is unknown, our results are important because they demonstrate that Osi9a is a common structural component of silk fiber and is expressed widely among the silk-producing Bombycidae and Saturniidae insects. Our results should help in understanding the role of Osi9a in silk fibers. Copyright © 2017 Elsevier Ltd. All rights reserved.

Experimental RNomics and genomic comparative analysis reveal a large group of species-specific small non-message RNAs in the silkworm Bombyx mori

PubMed Central

Li, Dandan; Wang, Yanhong; Zhang, Kun; Jiao, Zhujin; Zhu, Xiaopeng; Skogerboe, Geir; Guo, Xiangqian; Chinnusamy, Viswanathan; Bi, Lijun; Huang, Yongping; Dong, Shuanglin; Chen, Runsheng; Kan, Yunchao

2011-01-01

Accumulating evidences show that small non-protein coding RNAs (ncRNAs) play important roles in development, stress response and other cellular processes. The silkworm is an important model for studies on insect genetics and control of lepidopterous pests. Here, we have performed the first systematic identification and analysis of intermediate size ncRNAs (50–500 nt) in the silkworm. We identified 189 novel ncRNAs, including 141 snoRNAs, six snRNAs, three tRNAs, one SRP and 38 unclassified ncRNAs. Forty ncRNAs showed significantly altered expression during silkworm development or across specific stage transitions. Genomic comparisons revealed that 123 of these ncRNAs are potentially silkworm-specific. Analysis of the genomic organization of the ncRNA loci showed that 32.62% of the novel snoRNA loci are intergenic, and that all the intronic snoRNAs follow the pattern of one-snoRNA-per-intron. Target site analysis predicted a total of 95 2′-O-methylation and pseudouridylation modification sites of rRNAs, snRNAs and tRNAs. Together, these findings provide new clues for future functional study of ncRNA during insect development and evolution. PMID:21227919

Antheraea pernyi (Lepidoptera: Saturniidae) and Its Importance in Sericulture, Food Consumption, and Traditional Chinese Medicine.

PubMed

Li, Wenli; Zhang, Zhengyao; Lin, Lan; Terenius, Olle

2017-08-01

Sericulture was developed in China in ancient times. Antheraea pernyi Guérin-Méneville was domesticated at least 2,000 yr ago, and Chinese farmers developed artificial rearing of A. pernyi before the 17th century. Today, >60,000 tons of cocoons are produced in China each year, which accounts for 90% of the world production. Despite the widespread utilization of A. pernyi in China and a long history of domestic research, the knowledge of A. pernyi outside China is limited. Therefore, we have in this paper summarized the production, usage, and breeding of A. pernyi. The foremost usage of A. pernyi is as silk producers; however, about 55-70% is used for other purposes. In this paper, we give examples of how the different developmental stages are used as a food source for human consumption and in traditional Chinese medicine, both directly in different preparations and also as a nutrient source for rearing medicinal fungi. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Thromboelastometric and platelet responses to silk biomaterials.

PubMed

Kundu, Banani; Schlimp, Christoph J; Nürnberger, Sylvia; Redl, Heinz; Kundu, S C

2014-05-13

Silkworm's silk is natural biopolymer with unique properties including mechanical robustness, all aqueous base processing and ease in fabrication into different multifunctional templates. Additionally, the nonmulberry silks have cell adhesion promoting tri-peptide (RGD) sequences, which make it an immensely potential platform for regenerative medicine. The compatibility of nonmulberry silk with human blood is still elusive; thereby, restricts its further application as implants. The present study, therefore, evaluate the haematocompatibility of silk biomaterials in terms of platelet interaction after exposure to nonmulberry silk of Antheraea mylitta using thromboelastometry (ROTEM). The mulberry silk of Bombyx mori and clinically used Uni-Graft W biomaterial serve as references. Shortened clotting time, clot formation times as well as enhanced clot strength indicate the platelet mediated activation of blood coagulation cascade by tested biomaterials; which is comparable to controls.

Bioengineered silkworms with butterfly cytotoxin-modified silk glands produce sericin cocoons with a utility for a new biomaterial

PubMed Central

Otsuki, Ryosuke; Yamamoto, Masafumi; Matsumoto, Erika; Iwamoto, Shin-ichi; Sezutsu, Hideki; Suzui, Masumi; Takaki, Keiko; Wakabayashi, Keiji; Mori, Hajime; Kotani, Eiji

2017-01-01

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm (Bombyx mori) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly (Pieris rapae) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori-derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential. PMID:28607081

BmRobo2/3 is required for axon guidance in the silkworm Bombyx mori.

PubMed

Li, Xiao-Tong; Yu, Qi; Zhou, Qi-Sheng; Zhao, Xiao; Liu, Zhao-Yang; Cui, Wei-Zheng; Liu, Qing-Xin

2016-02-15

Axon guidance is critical for proper wiring of the nervous system. During the neural development, the axon guidance molecules play a key role and direct axons to choose the correct way to reach the target. Robo, as the receptor of axon guidance molecule Slit, is evolutionarily conserved from planarians to humans. However, the function of Robo in the silkworm, Bombyx mori, remained unknown. In this study, we cloned robo2/3 from B. mori (Bmrobo2/3), a homologue of robo2/3 in Tribolium castaneum. Moreover, BmRobo2/3 was localized in the neuropil, and RNAi-mediated knockdown of Bmrobo2/3 resulted in the longitudinal connectives forming closer to the midline. These data demonstrate that BmRobo2/3 is required for axon guidance in the silkworm. Copyright © 2015 Elsevier B.V. All rights reserved.

Biological and molecular characterization of silkworm strains from the Brazilian germplasm bank of Bombyx mori.

PubMed

Pereira, N C; Munhoz, R E F; Bignotto, T S; Bespalhuk, R; Garay, L B; Saez, C R N; Fassina, V A; Nembri, A; Fernandez, M A

2013-06-28

Brazil has only one public genetic pool of Bombyx mori strains, which was established in 2005 at Universidade Estadual de Maringá, Maringá, Paraná State. This genetic bank has been maintained, and the strains have been characterized using genetic and morphological tools. The quantitative and qualitative traits, directly or indirectly related to productivity, were evaluated in 14 silkworm strains. In addition to biological and productivity analyses, DNA markers related to susceptibility to the B. mori nucleopolyhedrovirus (BmNPV) were analyzed. BmNPV is a major cause of production loss and is a serious problem for Paraná sericulture. The silkworm strains from diverse geographic origins were found to have different characteristics, including body weight, larval stage duration, cocoon weight, and other biological traits. In terms of productivity, the raw silk percentages were almost uniform, with an overall average of 16.28%. Overall, the Chinese strain C37 gave the best performance in many of the quantitative traits, and it surpassed the other strains in productivity traits. Therefore, it can be used as one of the strains that compose the elite germplasm for silkworm breeding programs. Additionally, genetic molecular markers were efficient in discriminating between B. mori strains that had been identified based on their geographical origin. We found that all Japanese strains produced a 400-bp molecular marker that has been associated with susceptibility to BmNPV.

Genome-Wide Transcriptional Response of Silkworm (Bombyx mori) to Infection by the Microsporidian Nosema bombycis

PubMed Central

Pan, Guoqing; Li, Zhihong; Han, Bing; Xu, Jinshan; Lan, Xiqian; Chen, Jie; Yang, Donglin; Chen, Quanmei; Sang, Qi; Ji, Xiaocun; Li, Tian; Long, Mengxian; Zhou, Zeyang

2013-01-01

Microsporidia have attracted much attention because they infect a variety of species ranging from protists to mammals, including immunocompromised patients with AIDS or cancer. Aside from the study on Nosema ceranae, few works have focused on elucidating the mechanism in host response to microsporidia infection. Nosema bombycis is a pathogen of silkworm pébrine that causes great economic losses to the silkworm industry. Detailed understanding of the host (Bombyx mori) response to infection by N. bombycis is helpful for prevention of this disease. A genome-wide survey of the gene expression profile at 2, 4, 6 and 8 days post-infection by N. bombycis was performed and results showed that 64, 244, 1,328, 1,887 genes were induced, respectively. Up to 124 genes, which are involved in basal metabolism pathways, were modulated. Notably, B. mori genes that play a role in juvenile hormone synthesis and metabolism pathways were induced, suggesting that the host may accumulate JH as a response to infection. Interestingly, N. bombycis can inhibit the silkworm serine protease cascade melanization pathway in hemolymph, which may be due to the secretion of serpins in the microsporidia. N. bombycis also induced up-regulation of several cellular immune factors, in which CTL11 has been suggested to be involved in both spore recognition and immune signal transduction. Microarray and real-time PCR analysis indicated the activation of silkworm Toll and JAK/STAT pathways. The notable up-regulation of antimicrobial peptides, including gloverins, lebocins and moricins, strongly indicated that antimicrobial peptide defense mechanisms were triggered to resist the invasive microsporidia. An analysis of N. bombycis-specific response factors suggested their important roles in anti-microsporidia defense. Overall, this study primarily provides insight into the potential molecular mechanisms for the host-parasite interaction between B. mori and N. bombycis and may provide a foundation for

Dietary sterol preference in the silkworm, Bombyx mori.

PubMed

Nagata, Shinji; Omori, Yukie; Nagasawa, Hiromichi

2006-12-01

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that beta-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: beta-sitosterol > ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouth part might be different from the sterol recognition mechanism present in sterol metabolism.

Systematic cloning and analysis of autophagy-related genes from the silkworm Bombyx mori

PubMed Central

Zhang, Xuan; Hu, Zhan-Ying; Li, Wei-Fang; Li, Qing-Rong; Deng, Xiao-Juan; Yang, Wan-Ying; Cao, Yang; Zhou, Cong-Zhao

2009-01-01

Background Through the whole life of eukaryotes, autophagy plays an important role in various biological events including development, differentiation and determination of lifespan. A full set of genes and their encoded proteins of this evolutionarily conserved pathway have been identified in many eukaryotic organisms from yeast to mammals. However, this pathway in the insect model organism, the silkworm Bombyx mori, remains poorly investigated. Results Based on the autophagy pathway in several model organisms and a series of bioinformatic analyses, we have found more than 20 autophagy-related genes from the current database of the silkworm Bombyx mori. These genes could be further classified into the signal transduction pathway and two ubiquitin-like pathways. Using the mRNA extracted from the silkgland, we cloned the full length cDNA fragments of some key genes via reverse transcription PCR and 3' rapid amplification of cDNA ends (RACE). In addition, we found that the transcription levels of two indicator genes BmATG8 and BmATG12 in the silkgland tend to be increased from 1st to 8th day of the fifth instar larvae. Conclusion Bioinformatics in combination with RT-PCR enable us to remodel a preliminary pathway of autophagy in the silkworm. Amplification and cloning of most autophagy-related genes from the silkgland indicated autophagy is indeed an activated process. Furthermore, the time-course transcriptional profiles of BmATG8 and BmATG12 revealed that both genes are up-regulated along the maturation of the silkgland during the fifth instar. These findings suggest that the autophagy should play an important role in Bombyx mori silkgland. PMID:19470186

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry.

PubMed

Hou, Yong; Zhang, Yan; Gong, Jing; Tian, Sha; Li, Jianwei; Dong, Zhaoming; Guo, Chao; Peng, Li; Zhao, Ping; Xia, Qingyou

2016-05-01

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC-MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity-related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant-binding, and chemosensory proteins, showed stage-specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The crucial role of cyclic GMP in the eclosion hormone mediated signal transduction in the silkworm metamorphoses.

PubMed

Shibanaka, Y; Hayashi, H; Okada, N; Fujita, N

1991-10-31

The signal transduction of the peptide, eclosion hormone, in the silkworm Bombyx mori appears to be mediated via the second messenger cyclic GMP throughout their life cycle. Injection of 8-bromo-cGMP induced the ecdysis behavior in pharate adults with similar latency to eclosion hormone-induced ecdysis; the moulting occurred 50-70 min after the injection. The potency of 8Br-cGMP was 10(2) fold higher than that of cGMP and the efficacy was increased by the co-injection of the phosphodiesterase inhibitor IBMX. On the other hand, in the silkworm pupal ecdysis the eclosion hormone and also 8Br-cGMP induced the moulting behavior in a dose-dependent manner. The adult development of the ability to respond to 8Br-cGMP took place concomitantly with the response to the eclosion hormone. Both the developmental time courses were shifted by a shift of light and dark cycles. Accordingly, the sensitivities to the peptide and cyclic nucleotide developed correspondently under the light and dark circadian rhythm. Thus throughout the silkworm life cycle, eclosion hormone is effective to trigger the ecdysis behavior and cGMP plays a crucial role as the second messenger in the eclosion hormone-mediated signal transduction.

Insect food for astronauts: gas exchange in silkworms fed on mulberry and lettuce and the nutritional value of these insects for human consumption during deep space flights.

PubMed

Tong, L; Yu, X; Liu, H

2011-10-01

In this study, silkworm moth (Bombyx mori L.) larvae were regarded as an animal protein source for astronauts in the bioregenerative life support system during long-term deep space exploration in the future. They were fed with mulberry and stem lettuce leaves during the first three instars and the last two instars, respectively. In addition, this kind of environmental approach, which utilised inedible biomass of plants to produce animal protein of high quality, can likewise be applied terrestrially to provide food for people living in extreme environments and/or impoverished agro-ecosystems, such as in polar regions, isolated military bases, ships, submarines, etc. Respiration characteristics of the larvae during development under two main physiological conditions, namely eating and not-eating of leaves, were studied. Nutrient compositions of silkworm powder (SP), ground and freeze-dried silkworms on the 3rd day of the 5th instar larvae, including protein, fat, vitamins, minerals and fatty acids, were measured using international standard methods. Silkworms' respiration rates, measured when larvae were eating mulberry leaves, were higher than those of similar larvae that hadn't eaten such leaves. There was a significant difference between silkworms fed on mulberry leaves and those fed on stem lettuce in the 4th and 5th instars (P<0.01). Amounts of CO2 exhaled by the silkworms under the two physiological regimes differed from each other (P<0.01). There was also a significant difference between the amount of O2 inhaled when the insects were under the two physiological statuses (P<0.01). Moreover, silkworms' respiration quotient under the eating regime was larger than when under the not-eating regime. The SP was found to be rich in protein and amino acids in total; 12 essential vitamins, nine minerals and twelve fatty acids were detected. Moreover, 359 kcal could be generated per 100 gram of SP (dry weight).

Hormonal regulation and developmental role of Krüppel homolog 1, a repressor of metamorphosis, in the silkworm Bombyx mori.

PubMed

Kayukawa, Takumi; Murata, Mika; Kobayashi, Isao; Muramatsu, Daisuke; Okada, Chieko; Uchino, Keiro; Sezutsu, Hideki; Kiuchi, Makoto; Tamura, Toshiki; Hiruma, Kiyoshi; Ishikawa, Yukio; Shinoda, Tetsuro

2014-04-01

Juvenile hormone (JH) has an ability to repress the precocious metamorphosis of insects during their larval development. Krüppel homolog 1 (Kr-h1) is an early JH-inducible gene that mediates this action of JH; however, the fine hormonal regulation of Kr-h1 and the molecular mechanism underlying its antimetamorphic effect are little understood. In this study, we attempted to elucidate the hormonal regulation and developmental role of Kr-h1. We found that the expression of Kr-h1 in the epidermis of penultimate-instar larvae of the silkworm Bombyx mori was induced by JH secreted by the corpora allata (CA), whereas the CA were not involved in the transient induction of Kr-h1 at the prepupal stage. Tissue culture experiments suggested that the transient peak of Kr-h1 at the prepupal stage is likely to be induced cooperatively by JH derived from gland(s) other than the CA and the prepupal surge of ecdysteroid, although involvement of unknown factor(s) could not be ruled out. To elucidate the developmental role of Kr-h1, we generated transgenic silkworms overexpressing Kr-h1. The transgenic silkworms grew normally until the spinning stage, but their development was arrested at the prepupal stage. The transgenic silkworms from which the CA were removed in the penultimate instar did not undergo precocious pupation or larval-larval molt but fell into prepupal arrest. This result demonstrated that Kr-h1 is indeed involved in the repression of metamorphosis but that Kr-h1 alone is incapable of implementing normal larval molt. Moreover, the expression profiles and hormonal responses of early ecdysone-inducible genes (E74, E75, and Broad) in transgenic silkworms suggested that Kr-h1 is not involved in the JH-dependent modulation of these genes, which is associated with the control of metamorphosis. Copyright © 2014 Elsevier Inc. All rights reserved.

The complete mitogenome sequence of the Japanese oak silkmoth, Antheraea yamamai (Lepidoptera: Saturniidae).

PubMed

Kim, Seong Ryeol; Kim, Man Il; Hong, Mee Yeon; Kim, Kee Young; Kang, Pil Don; Hwang, Jae Sam; Han, Yeon Soo; Jin, Byung Rae; Kim, Iksoo

2009-09-01

The 15,338-bp long complete mitochondrial genome (mitogenome) of the Japanese oak silkmoth, Antheraea yamamai (Lepidoptera: Saturniidae) was determined. This genome has a gene arrangement identical to those of all other sequenced lepidopteran insects, but differs from the most common type, as the result of the movement of tRNA(Met) to a position 5'-upstream of tRNA(Ile). No typical start codon of the A. yamamai COI gene is available. Instead, a tetranucleotide, TTAG, which is found at the beginning context of all sequenced lepidopteran insects was tentatively designated as the start codon for A. yamamai COI gene. Three of the 13 protein-coding genes (PCGs) harbor the incomplete termination codon, T or TA. All tRNAs formed stable stem-and-loop structures, with the exception of tRNA(Ser)(AGN), the DHU arm of which formed a simple loop as has been observed in many other metazoan mt tRNA(Ser)(AGN). The 334-bp long A + T-rich region is noteworthy in that it harbors tRNA-like structures, as has also been seen in the A + T-rich regions of other insect mitogenomes. Phylogenetic analyses of the available species of Bombycoidea, Pyraloidea, and Tortricidea bolstered the current morphology-based hypothesis that Bombycoidea and Pyraloidea are monophyletic (Obtectomera). As has been previously suggested, Bombycidae (Bombyx mori and B. mandarina) and Saturniidae (A. yamamai and Caligula boisduvalii) formed a reciprocal monophyletic group.

Characterization of feeding-delaying factors from the silkworm Bombyx mori.

PubMed

Nagata, Shinji; Morooka, Nobukatsu; Matsumoto, Sumihiro; Nagasawa, Hiromichi

2009-04-01

In several phytophagous insects, feeding behavior occurs regularly. Recently, we demonstrated that feeding behavior in larvae of the silkworm Bombyx mori had a regular frequency. To address the control of the feeding cycle in B. mori, we aimed to characterize factors influencing feeding initiation and termination. Injection of extracts of the midgut, foregut, and fat body into starved Bombyx larvae delayed the initiation of feeding. This result indicates the presence in these tissues of factors capable of decreasing the likelihood of feeding initiation.

The effect of calorie restriction on growth and development in silkworm, Bombyx mori.

PubMed

Li, Yijia; Chen, Keping; Yao, Qin; Li, Jun; Wang, Yong; Liu, Haijun; Zhang, Chiyu; Huang, Guoping

2009-07-01

Caloric restriction (CR) is known to extend the life span in different species from yeast to mammals. In this report, a simple organism silkworm (Bombyx mori) was used to study the effect of moderate CR on the growth and development processes of insects. Here we show that an extension of life span upon moderate CR was observed in the silkworm. The total protein level in the 5th instar larvae hemolymph appeared to decline significantly under CR. SDS-PAGE analysis showed that the influence of CR was sex-dependent. The CR effects on female animals were much more significant than on the males. The MALDI-TOF MS study identified 16 proteins that expressed differentially among six groups of the male or female larvae fed at different time frequencies. Four of them, storage protein 1 (SP1), arylphorin (SP2), imaginal disk growth factor (IDGF), and 30-kDa lipoprotein, showed significant differences. It was demonstrated that these four proteins were up-regulated when the larvae were over-fed and down-regulated when the larvae were less-fed. (c) 2009 Wiley Periodicals, Inc.

Influence of clove oil and eugenol on muscle contraction of silkworm (Bombyx mori).

PubMed

Kheawfu, Kantaporn; Pikulkaew, Surachai; Hamamoto, Hiroshi; Sekimizu, Kazuhisa; Okonogi, Siriporn

2017-05-30

Clove oil is used in fish anesthesia and expected to have a mechanism via glutamic receptor. The present study explores the activities of clove oil and its major compound, eugenol, in comparison with L-glutamic acid on glutamic receptor of silkworm muscle and fish anesthesia. It was found that clove oil and eugenol had similar effects to L-glutamic acid on inhibition of silkworm muscle contraction after treated with D-glutamic acid and kainic acid. Anesthetic activity of the test samples was investigated in goldfish. The results demonstrated that L-glutamic acid at 20 and 40 mM could induce the fish to stage 3 of anesthesia that the fish exhibited total loss of equilibrium and muscle tone, whereas clove oil and eugenol at 60 ppm could induce the fish to stage 4 of anesthesia that the reflex activity of the fish was lost. These results suggest that clove oil and eugenol have similar functional activities and mechanism to L-glutamic acid on muscle contraction and fish anesthesia.

Can the silkworm (Bombyx mori) be used as a human disease model?

PubMed

Tabunoki, Hiroko; Bono, Hidemasa; Ito, Katsuhiko; Yokoyama, Takeshi

2016-02-01

Bombyx mori (silkworm) is the most famous lepidopteran in Japan. B. mori has long been used in the silk industry and also as a model insect for agricultural research. In recent years, B. mori has attracted interest in its potential for use in pathological analysis of model animals. For example, the human macular carotenoid transporter was discovered using information of B. mori carotenoid transporter derived from yellow-cocoon strain. The B. mori carotenoid transport system is useful in human studies. To develop a human disease model, we characterized the human homologs of B. mori, and by constructing KAIKO functional annotation pipeline, and to analyze gene expression profile of a unique B. mori mutant strain using microarray analysis. As a result, we identified a novel molecular network involved in Parkinson's disease. Here we describe the potential use of a spontaneous mutant silkworm strain as a human disease model. We also summarize recent progress in the application of genomic information for annotation of human homologs in B. mori. The B. mori mutant will provide a clue to pathological mechanisms, and the findings will be helpful for the development of therapies and for medical drug discovery.

Molecular characterization of a peritrophic membrane protein from the silkworm, Bombyx mori.

PubMed

Hu, Xiaolong; Chen, Lin; Yang, Rui; Xiang, Xingwei; Wu, Xiaofeng

2013-02-01

The peritrophic membrane lines the gut of most insects at one or more stages of their life cycles. It facilitates the digestive processes in the guts and protects from invasion by pathogens or food particles. In the current study, a novel PM protein, designated as BmMtch, was identified from the silkworm, Bombyx mori. The open reading frame of BmMtch is 888 bp in length, encoding 295 amino acid residues consisting of two domains (Mito_carr domains) and three transmembrane regions. They are localized on the 11th chromosome as single copy with one exon only. Quantitative real time PCR analysis (qRT-PCR) revealed that BmMtch was mainly expressed in larval fat bodies, Malpighian tubules, testis and ovaries, and could be detected through all stages of the life cycle of silkworm. Immuno-fluorescence analysis indicated that BmMtch was localized within the goblet cell of larval midgut. Western blotting analysis showed that BmMtch were detected in total proteins of PM and larval midgut. The characteristics of BmMtch indicated that BmMtch represents a novel member of insect PM proteins, without chitin-binding domains.

Comparative analysis of the integument transcriptomes of the black dilute mutant and the wild-type silkworm Bombyx mori

PubMed Central

Wu, Songyuan; Tong, Xiaoling; Peng, Chenxing; Xiong, Gao; Lu, Kunpeng; hu, Hai; Tan, Duan; Li, Chunlin; Han, Minjin; Lu, Cheng; Dai, Fangyin

2016-01-01

The insect cuticle is a critical protective shell that is composed predominantly of chitin and various cuticular proteins and pigments. Indeed, insects often change their surface pigment patterns in response to selective pressures, such as threats from predators, sexual selection and environmental changes. However, the molecular mechanisms underlying the construction of the epidermis and its pigmentation patterns are not fully understood. Among Lepidoptera, the silkworm is a favorable model for color pattern research. The black dilute (bd) mutant of silkworm is the result of a spontaneous mutation; the larval body color is notably melanized. We performed integument transcriptome sequencing of the wild-type strain Dazao and the mutant strains +/bd and bd/bd. In these experiments, during an early stage of the fourth molt, a stage at which approximately 51% of genes were expressed genome wide (RPKM ≥1) in each strain. A total of 254 novel transcripts were characterized using Cuffcompare and BLAST analyses. Comparison of the transcriptome data revealed 28 differentially expressed genes (DEGs) that may contribute to bd larval melanism, including 15 cuticular protein genes that were remarkably highly expressed in the bd/bd mutant. We suggest that these significantly up-regulated cuticular proteins may promote melanism in silkworm larvae. PMID:27193628

Silkworm cocoons inspire models for random fiber and particulate composites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Fujia; Porter, David; Vollrath, Fritz

The bioengineering design principles evolved in silkworm cocoons make them ideal natural prototypes and models for structural composites. Cocoons depend for their stiffness and strength on the connectivity of bonding between their constituent materials of silk fibers and sericin binder. Strain-activated mechanisms for loss of bonding connectivity in cocoons can be translated directly into a surprisingly simple yet universal set of physically realistic as well as predictive quantitative structure-property relations for a wide range of technologically important fiber and particulate composite materials.

Silkworm cocoons inspire models for random fiber and particulate composites

NASA Astrophysics Data System (ADS)

Chen, Fujia; Porter, David; Vollrath, Fritz

2010-10-01

The bioengineering design principles evolved in silkworm cocoons make them ideal natural prototypes and models for structural composites. Cocoons depend for their stiffness and strength on the connectivity of bonding between their constituent materials of silk fibers and sericin binder. Strain-activated mechanisms for loss of bonding connectivity in cocoons can be translated directly into a surprisingly simple yet universal set of physically realistic as well as predictive quantitative structure-property relations for a wide range of technologically important fiber and particulate composite materials.

Two Geminin homologs regulate DNA replication in silkworm, Bombyx mori.

PubMed

Tang, Xiao-Fang; Chen, Xiang-Yun; Zhang, Chun-Dong; Li, Yao-Feng; Liu, Tai-Hang; Zhou, Xiao-Lin; Wang, La; Zhang, Qian; Chen, Peng; Lu, Cheng; Pan, Min-Hui

2017-05-03

DNA replication is rigorously controlled in cells to ensure that the genome duplicates exactly once per cell cycle. Geminin is a small nucleoprotein, which prevents DNA rereplication by directly binding to and inhibiting the DNA replication licensing factor, Cdt1. In this study, we have identified 2 Geminin genes, BmGeminin1 and BmGeminn2, in silkworm, Bombyx mori. These genes contain the Geminin conserved coiled-coil domain and are periodically localized in the nucleus during the S-G2 phase but are degraded at anaphase in mitosis. Both BmGeminin1 and BmGeminin2 are able to homodimerize and interact with BmCdt1 in cells. In addition, BmGeminin1 and BmGeminin2 can interact with each other. Overexpression of BmGeminin1 affects cell cycle progression: cell cycle is arrested in S phase, and RNA interference of BmGeminin1 leads to rereplication. In contrast, overexpression or knockdown of BmGeminin2 with RNAi did not significantly affect cell cycle, while more rereplication occurred when BmGeminin1 and BmGeminin2 together were knocked down in cells than when only BmGeminin1 was knocked down. These data suggest that both BmGeminin1 and BmGeminin2 are involved in the regulation of DNA replication. These findings provide insight into the function of Geminin and contribute to our understanding of the regulation mechanism of cell cycle in silkworm.

Two Geminin homologs regulate DNA replication in silkworm, Bombyx mori

PubMed Central

Tang, Xiao-Fang; Chen, Xiang-Yun; Zhang, Chun-Dong; Li, Yao-Feng; Liu, Tai-Hang; Zhou, Xiao-Lin; Wang, La; Zhang, Qian; Chen, Peng; Lu, Cheng; Pan, Min-Hui

2017-01-01

ABSTRACT DNA replication is rigorously controlled in cells to ensure that the genome duplicates exactly once per cell cycle. Geminin is a small nucleoprotein, which prevents DNA rereplication by directly binding to and inhibiting the DNA replication licensing factor, Cdt1. In this study, we have identified 2 Geminin genes, BmGeminin1 and BmGeminn2, in silkworm, Bombyx mori. These genes contain the Geminin conserved coiled-coil domain and are periodically localized in the nucleus during the S-G2 phase but are degraded at anaphase in mitosis. Both BmGeminin1 and BmGeminin2 are able to homodimerize and interact with BmCdt1 in cells. In addition, BmGeminin1 and BmGeminin2 can interact with each other. Overexpression of BmGeminin1 affects cell cycle progression: cell cycle is arrested in S phase, and RNA interference of BmGeminin1 leads to rereplication. In contrast, overexpression or knockdown of BmGeminin2 with RNAi did not significantly affect cell cycle, while more rereplication occurred when BmGeminin1 and BmGeminin2 together were knocked down in cells than when only BmGeminin1 was knocked down. These data suggest that both BmGeminin1 and BmGeminin2 are involved in the regulation of DNA replication. These findings provide insight into the function of Geminin and contribute to our understanding of the regulation mechanism of cell cycle in silkworm. PMID:28379781

Immune function of a Rab-related protein by modulating the JAK-STAT signaling pathway in the silkworm, Bombyx mori.

PubMed

Chen, Chen; Eldein, Salah; Zhou, Xiaosan; Sun, Yu; Gao, Jin; Sun, Yuxuan; Liu, Chaoliang; Wang, Lei

2018-01-01

The Rab-family GTPases mainly regulate intracellular vesicle transport, and play important roles in the innate immune response in invertebrates. However, the function and signal transduction of Rab proteins in immune reactions remain unclear in silkworms. In this study, we analyzed a Rab-related protein of silkworm Bombyx mori (BmRABRP) by raising antibodies against its bacterially expressed recombinant form. Tissue distribution analysis showed that BmRABRP mRNA and protein were high expressed in the Malpighian tubule and fat body, respectively. However, among the different stages, only the fourth instar larvae and pupae showed significant BmRABRP levels. After challenge with four pathogenic microorganisms (Escherichia coli, BmNPV, Beauveria bassiana, Micrococcus luteus), the expression of BmRABRP mRNA in the fat body was significantly upregulated. In contrast, the BmRABRP protein was significantly upregulated after infection with BmNPV, while it was downregulated by E. coli, B. bassiana, and M. luteus. A specific dsRNA was used to explore the immune function and relationship between BmRABRP and the JAK-STAT signaling pathway. After BmRABRP gene interference, significant reduction in the number of nodules and increased mortality suggested that BmRABRP plays an important role in silkworm's response to bacterial challenge. In addition, four key genes (BmHOP, BmSTAT, BmSOCS2, and BmSOCS6) of the JAK-STAT signaling pathway showed significantly altered expressions after BmRABRP silencing. BmHOP and BmSOCS6 expressions were significantly decreased, while BmSTAT and BmSOCS2 were significantly upregulated. Our results suggested that BmRABRP is involved in the innate immune response against pathogenic microorganisms through the JAK-STAT signaling pathway in silkworm. © 2017 Wiley Periodicals, Inc.

MicroRNA profile of silk gland reveals different silk yields of three silkworm strains.

PubMed

Qin, Sheng; Danso, Blessing; Zhang, Jing; Li, Juan; Liu, Na; Sun, Xia; Hou, Chengxiang; Luo, Heng; Chen, Keping; Zhang, Guozheng; Li, Muwang

2018-05-05

Silk proteins are synthesized and secreted by the silk gland. The differential gene expression in it leads to different silk yield among various silkworm strains. As crucial factors, microRNAs (miRNAs) regulate protein synthesis at post-transcriptional level in silk gland. MiRNAs expression level in the silk gland of three silkworm strains (Jingsong, Lan10 and Dazao) was analyzed and 33 differentially expressed miRNAs (DEMs) were discovered between JingSong (JS) and Lan10 (L10), 60 DEMs between JS and Dazao, 54 DEMs between L10 and Dazao respectively. The DEMs target genes were predicted combing with two different methods and their functions were annotated according to gene ontology. Our previous studies showed that a batch of genes related to silk yield were identified in JS and L10 strains by comparative transcriptome and quantitative trait loci (QTL) method. Thirteen DEMs whose target genes are related to protein biosynthesis processes were screened by combining with these researches. Twelve DEMs potentially regulate nineteen genes which exist in our QTL results. Six common DEMs potentially regulate the genes in both of previous results. Finally, five DEMs were selected to verify their expression levels between JS and L10 by qRT-PCR, which showed similar difference as the results of small RNA-sequencing. MiRNAs in the silk gland may directly affect silk protein biosynthesis in different silkworm strains. In current work, we identified a batch of DEMs which potentially regulate the genes related to silk yield. Further functionally study of these miRNAs will contribute to improve varieties and boost the silk yield. Our research provides a basis for studying these miRNAs and their functions in silk production. Copyright © 2018 Elsevier B.V. All rights reserved.

Artificial “ping-pong” cascade of PIWI-interacting RNA in silkworm cells

PubMed Central

Shoji, Keisuke; Suzuki, Yutaka; Sugano, Sumio; Shimada, Toru; Katsuma, Susumu

2017-01-01

PIWI-interacting RNAs (piRNAs) play essential roles in the defense system against selfish elements in animal germline cells by cooperating with PIWI proteins. A subset of piRNAs is predicted to be generated via the “ping-pong” cascade, which is mainly controlled by two different PIWI proteins. Here we established a cell-based artificial piRNA production system using a silkworm ovarian cultured cell line that is believed to possess a complete piRNA pathway. In addition, we took advantage of a unique silkworm sex-determining one-to-one ping-pong piRNA pair, which enabled us to precisely monitor the behavior of individual artificial piRNAs. With this novel strategy, we successfully generated artificial piRNAs against endogenous protein-coding genes via the expected back-and-forth traveling mechanism. Furthermore, we detected “primary” piRNAs from the upstream region of the artificial “ping-pong” site in the endogenous gene. This artificial piRNA production system experimentally confirms the existence of the “ping-pong” cascade of piRNAs. Also, this system will enable us to identify the factors involved in both, or each, of the “ping” and “pong” cascades and the sequence features that are required for efficient piRNA production. PMID:27777367

Cathepsin O is involved in the innate immune response and metamorphosis of Antheraea pernyi.

PubMed

Sun, Yu-Xuan; Zhu, Bao-Jian; Tang, Lin; Sun, Yu; Chen, Chen; Nadeem Abbas, Muhammad; Wang, Lei; Qian, Cen; Wei, Guo-Qing; Liu, Chao-Liang

2017-11-01

Cathepsins are key members of mammalian papain-like cysteine proteases that play an important role in the immune response. In this study, a fragment of cDNA encoding cathepsin O proteinase (ApCathepsin O) was cloned from Antheraea pernyi. It contains an open reading frame of 1170bp and encodes a protein with 390 amino acid residues, including a conserved I29 inhibitor domain and a peptidase C1A (clan CA of cysteine proteases, papain family C1 subfamily) domain. Comparison with other previously reported cathepsin O proteins showed identity ranging from 45% to 79%. Quantitative real-time PCR (qRT-PCR) and Western blot analysis revealed that ApCathepsin O was highly expressed in the fat body; furthermore, the high expression during the pupal stage indicated that it might be involved during metamorphosis. After exposure to four different heat-killed pathogens (Escherichia coli, Beauveria bassiana, Micrococcus luteus, and A. pernyi nucleopolyhedrovirus), the expression levels of ApCathepsin O mRNA significantly increased and showed variable expression patterns. This indicates that ApCathepsin O is potentially involved in the innate immune system of A. pernyi. Interestingly, ApCathepsin O expression was upregulated after 20-hydroxyecdysone (20E) injection, which suggested that it might be regulated by 20E. In conclusion, ApCathepsin O is a protease that may play an important role in the innate immune response and metamorphosis of A. pernyi. Copyright © 2017. Published by Elsevier Inc.

Antheraea pernyi silk fibroin for targeted gene delivery of VEGF165-Ang-1 with PEI.

PubMed

Ma, Caili; Lv, Linlin; Liu, Yu; Yu, Yanni; You, Renchuan; Yang, Jicheng; Li, Mingzhong

2014-06-01

Vascularization is a crucial challenge in tissue engineering. One solution for this problem is to implant scaffolds that contain functional genes that promote vascularization by providing angiogenic growth factors via a gene delivery carrier. Poly(ethylenimine) (PEI) is a gene delivery carrier with high transfection efficiency but with cytotoxicity. To solve this problem, we utilized Antheraea pernyi silk fibroin (ASF), which has favorable cytocompatibility and biodegradability, RGD sequences and a negative charge, in conjunction with PEI, as the delivery vector for vascular endothelial growth factor (VEGF) 165-angiopoietin-1 (Ang-1) dual gene simultaneous expression plasmid, creating an ASF/PEI/pDNA complex. The results suggested that the zeta potential of the ASF/PEI/pDNA complex was significantly lower than that of the PEI/pDNA complex. Decreased nitrogen and increased oxygen on the surface of the complex demonstrated that the ASF had successfully combined with the surface of the PEI/pDNA. Furthermore, the complexes resisted digestion by nucleic acid enzymes and degradation by serum. L929 cells were cultured and transfected in vitro and improved cytotoxicity was found when the cells were transfected with ASF/PEI/pDNA compared with PEI/pDNA. In addition, the transfection efficiency and VEGF secretion increased. In general, this study provides a novel method for decreasing the cytotoxicity of PEI gene delivery vectors and increasing transfection efficiency of angiogenesis-related genes.

Silkworm dropping extract ameliorate trimellitic anhydride-induced allergic contact dermatitis by regulating Th1/Th2 immune response.

PubMed

Choi, Dae Woon; Kwon, Da-Ae; Jung, Sung Keun; See, Hye-Jeong; Jung, Sun Young; Shon, Dong-Hwa; Shin, Hee Soon

2018-05-26

Allergic contact dermatitis (ACD) is an inflammatory skin disease caused by hapten-specific immune response. Silkworm droppings are known to exert beneficial effects during the treatment of inflammatory diseases. Here, we studied whether topical treatment and oral administration of silkworm dropping extract (SDE) ameliorate trimellitic anhydride (TMA)-induced ACD. In ACD mice model, SDE treatment significantly suppressed the increase in both ear thickness and serum IgE levels. Furthermore, IL-1β and TNF-α levels were reduced by SDE. In allergic responses, SDE treatment significantly attenuated the production of the Th2-associated cytokine IL-4 in both ear tissue and draining lymph nodes. However, it increased the production of the Th1-mediated cytokine IL-12. Thus, these results showed that SDE attenuated TMA-induced ACD symptoms through regulation of Th1/Th2 immune response. Taken together, we suggest that SDE treatment might be a potential agent in the prevention or therapy of Th2-mediated inflammatory skin diseases such as ACD and atopic dermatitis. ACD: allergic contact dermatitis; AD: atopic dermatitis; APC: antigen presenting cells; CCL: chemokine (C-C motif) ligand; CCR: C-C chemokine receptor; Dex: dexamethasone; ELISA: enzyme-linked immunosorbent assay; IFN: interferon; Ig: immunoglobulin; IL: interleukin; OVA: ovalbumin; PS: prednisolone; SDE: silkworm dropping extract; Th: T helper; TMA: trimellitic anhydride; TNF: tumor necrosis factor.

Quantitative proteomic and transcriptomic analyses of molecular mechanisms associated with low silk production in silkworm Bombyx mori.

PubMed

Wang, Shao-Hua; You, Zheng-Ying; Ye, Lu-Peng; Che, Jiaqian; Qian, Qiujie; Nanjo, Yohei; Komatsu, Setsuko; Zhong, Bo-Xiong

2014-02-07

To investigate the molecular mechanisms underlying the low fibroin production of the ZB silkworm strain, we used both SDS-PAGE-based and gel-free-based proteomic techniques and transcriptomic sequencing technique. Combining the data from two different proteomic techniques was preferable in the characterization of the differences between the ZB silkworm strain and the original Lan10 silkworm strain. The correlation analysis showed that the individual protein and transcript were not corresponded well, however, the differentially changed proteins and transcripts showed similar regulated direction in function at the pathway level. In the ZB strain, numerous ribosomal proteins and transcripts were down-regulated, along with the transcripts of translational related elongation factors and genes of important components of fibroin. The proteasome pathway was significantly enhanced in the ZB strain, indicating that protein degradation began on the third day of fifth instar when fibroin would have been produced in the Lan10 strain normally and plentifully. From proteome and transcriptome levels of the ZB strain, the energy-metabolism-related pathways, oxidative phosphorylation, glycolysis/gluconeogenesis, and citrate cycle were enhanced, suggesting that the energy metabolism was vigorous in the ZB strain, while the silk production was low. This may due to the inefficient energy employment in fibroin synthesis in the ZB strain. These results suggest that the reason for the decreasing of the silk production might be related to the decreased ability of fibroin synthesis, the degradation of proteins, and the inefficiency of the energy exploiting.

Shotgun proteomics approach to characterizing the embryonic proteome of the silkworm, Bombyx mori, at labrum appearance stage.

PubMed

Li, J-Y; Chen, X; Hosseini Moghaddam, S H; Chen, M; Wei, H; Zhong, B-X

2009-10-01

The shotgun approach has gained considerable acknowledgement in recent years as a dominant strategy in proteomics. We observed a dramatic increase of specific protein spots in two-dimensional electrophoresis (2-DE) gels of the silkworm (Bombyx mori) embryo at labrum appearance, a characteristic stage during embryonic development of silkworm which is involved with temperature increase by silkworm raiser. We employed shotgun liquid chromatography tandem mass spectrometry (LC-MS/MS) technology to analyse the proteome of B. mori embryos at this stage. A total of 2168 proteins were identified with an in-house database. Approximately 47% of them had isoelectric point (pI) values distributed theoretically in the range pI 5-7 and approximately 60% of them had molecular weights of 15-45 kDa. Furthermore, 111 proteins had an pI greater than 10 and were difficult to separate by 2-DE. Many important functional proteins related to embryonic development, stress response, DNA transcription/translation, cell growth, proliferation and differentiation, organogenesis and reproduction were identified. Among them proteins related to nervous system development were noticeable. All known heat shock proteins (HSPs) were detected in this developmental stage of B. mori embryo. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed energetic metabolism at this stage. These results were expected to provide more information for proteomic monitoring of the insect embryo and better understanding of the spatiotemporal expression of genes during embryonic developmental processes.

Structure and physical properties of silkworm cocoons

PubMed Central

Chen, Fujia; Porter, David; Vollrath, Fritz

2012-01-01

Silkworm cocoons have evolved a wide range of different structures and combinations of physical and chemical properties in order to cope with different threats and environmental conditions. We present our observations and measurements on 25 diverse types of cocoons in a first attempt to correlate physical properties with the structure and morphology of the cocoons. These two architectural parameters appear to be far more important than the material properties of the silk fibres themselves. We consider tensile and compressive mechanical properties and gas permeation of the cocoon walls, and in each case identify mechanisms or models that relate these properties to cocoon structure, usually based upon non-woven fibre composites. These properties are of relevance also for synthetic non-woven composite materials and our studies will help formulate bio-inspired design principles for new materials. PMID:22552916

Spermidine enhances the silk production by mulberry silkworm.

PubMed

Lattala, Gayatri Manogna; Kandukuru, Kasturaiah; Gangupantula, Shamitha; Mamillapalli, Anitha

2014-01-01

Polyamines are ubiquitous low molecular weight polycationic aliphatic amines involved in diverse cellular processes. Spermidine (Spd), a polyamine, has been proved to be crucial for cell survival in various organisms. Our study reports the effect of Spd on the growth of Bombyx mori. Silkworms showed improved silk gland weight and economic parameters in the fifth instar larval stage when treated with different concentrations of Spd, in the range of 25-75 µM. The worms treated with Spd produced 31% more silk when compared with the control worms. Altogether, this study establishes that Spd-treated leaves can be fed into the larvae for better silk production. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

Stress induction of Bm1 RNA in silkworm larvae: SINEs, an unusual class of stress genes

PubMed Central

Kimura, Richard H.; Choudary, Prabhakara V.; Stone, Koni K.; Schmid, Carl W.

2001-01-01

This study surveys the induction of RNA polymerase III (Pol III)–directed expression of short interspersed element (SINE) transcripts by various stresses in an animal model, silkworm larvae. Sublethal heat shock and exposure to several toxic compounds increase the level of Bm1 RNA, the silkworm SINE transcript, while also transiently increasing expression of a well-characterized stress-induced transcript, Hsp70 messenger RNA (mRNA). In certain cases, the Bm1 RNA response coincides with that of Hsp70 mRNA, but more often Bm1 RNA responds later in recovery. Baculovirus infection and exposure to certain toxic compounds increase Bm1 RNA but not Hsp70 mRNA, showing that SINE induction is not necessarily coupled to transcription of this particular heat shock gene. SINEs behave as an additional class of stress-inducible genes in living animals but are unusual as stress genes because of their high copy number, genomic dispersion, and Pol III–directed transcription. PMID:11599568

A novel immune-related gene HDD1 of silkworm Bombyx mori is involved in bacterial response.

PubMed

Zhang, Kui; Pan, Guangzhao; Zhao, Yuzu; Hao, Xiangwei; Li, Chongyang; Shen, Li; Zhang, Rui; Su, Jingjing; Cui, Hongjuan

2017-08-01

Insects have evolved an effective immune system to respond to various challenges. In this study, a novel immune-related gene, called BmHDD1, was first charactered in silkworm, Bombyx mori. BmHDD1 contained an ORF of 837bp and encoding a deduced protein of 278 amino acids. BmHDD1 was specifically expressed in hemocytes, and highly expressed at the molting and metamorphosis stages under normal physiological conditions. Our results suggested that BmHDD1 was mainly generated by hemocytes and secreted into hemolymph. Our results also showed that the expression level of BmHDD1 was significantly increased after 20E injection, which indicated that BmHDD1 might be regulated by ecdysone. More importantly, BmHDD1 was dramatically induced after injected with different types of PAMPs or bacteria, either in hemocytes or fat body. Those results suggested that BmHDD1 plays a role in developing and immunity system in silkworm, Bombyx mori. Copyright © 2017. Published by Elsevier Ltd.

De novo transcriptome assembly and analysis of differential gene expression following peptidoglycan (PGN) challenge in Antheraea pernyi.

PubMed

Liu, Yu; Xin, Zhao-Zhe; Zhang, Dai-Zhen; Zhu, Xiao-Yu; Wang, Ying; Chen, Li; Tang, Bo-Ping; Zhou, Chun-Lin; Chai, Xin-Yue; Tian, Ji-Wu; Liu, Qiu-Ning

2018-06-01

Antheraea pernyi is not only an important economic insect, it is increasingly employed as a model organism due to a variety of advantages, including ease of rearing and experimental manipulation compared with other Lepidoptera. Peptidoglycan (PGN) is a major component of the bacterial cell wall, and interactions between PGN and A. pernyi cause a series of physiological changes in the insect. In the present study, we constructed cDNA libraries from a A. pernyi PGN-infected group and a control group stimulated with phosphate-buffered saline (PBS). The transcriptome was de novo assembled using the Trinity platform, and 1698 differentially expressed genes (DEGs) were identified, comprising 894 up-regulated and 804 down-regulated genes. To further investigate immune-related DEGs, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed. GO analysis identified major immune-related GO terms and KEGG enrichment indicated gene responses to three pathways related to the insect immune system. Several homologous genes related to the immune response of the A. pernyi fat body post-PGN infection were identified and categorised. Taken together, the results provide insight into the complex molecular mechanisms of the responses to bacterial infection at the transcriptional level. Copyright © 2018 Elsevier B.V. All rights reserved.

Bombyx mori P-element Somatic Inhibitor (BmPSI) Is a Key Auxiliary Factor for Silkworm Male Sex Determination

PubMed Central

Chen, Shuqing; Zeng, Baosheng; James, Anthony A.; Tan, Anjiang; Huang, Yongping

2017-01-01

Manipulation of sex determination pathways in insects provides the basis for a wide spectrum of strategies to benefit agriculture and public health. Furthermore, insects display a remarkable diversity in the genetic pathways that lead to sex differentiation. The silkworm, Bombyx mori, has been cultivated by humans as a beneficial insect for over two millennia, and more recently as a model system for studying lepidopteran genetics and development. Previous studies have identified the B. mori Fem piRNA as the primary female determining factor and BmMasc as its downstream target, while the genetic scenario for male sex determination was still unclear. In the current study, we exploite the transgenic CRISPR/Cas9 system to generate a comprehensive set of knockout mutations in genes BmSxl, Bmtra2, BmImp, BmImpM, BmPSI and BmMasc, to investigate their roles in silkworm sex determination. Absence of Bmtra2 results in the complete depletion of Bmdsx transcripts, which is the conserved downstream factor in the sex determination pathway, and induces embryonic lethality. Loss of BmImp or BmImpM function does not affect the sexual differentiation. Mutations in BmPSI and BmMasc genes affect the splicing of Bmdsx and the female reproductive apparatus appeared in the male external genital. Intriguingly, we identify that BmPSI regulates expression of BmMasc, BmImpM and Bmdsx, supporting the conclusion that it acts as a key auxiliary factor in silkworm male sex determination. PMID:28103247

Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

PubMed Central

Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

2015-01-01

The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future. PMID:26198671

Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

NASA Astrophysics Data System (ADS)

Nie, Hongyi; Cheng, Tingcai; Huang, Xiaofeng; Zhou, Mengting; Zhang, Yinxia; Dai, Fangyin; Mita, Kazuei; Xia, Qingyou; Liu, Chun

2015-07-01

The thermoprotective mechanisms of insects remain largely unknown. We reported the Bombyx mori contractile (cot) behavioral mutant with thermo-sensitive seizures phenotype. At elevated temperatures, the cot mutant exhibit seizures associated with strong contractions, rolling, vomiting, and a temporary lack of movement. We narrowed a region containing cot to ~268 kb by positional cloning and identified the mutant gene as Bmsei which encoded a potassium channel protein. Bmsei was present in both the cell membrane and cytoplasm in wild-type ganglia but faint in cot. Furthermore, Bmsei was markedly decreased upon high temperature treatment in cot mutant. With the RNAi method and injecting potassium channel blockers, the wild type silkworm was induced the cot phenotype. These results demonstrated that Bmsei was responsible for the cot mutant phenotype and played an important role in thermoprotection in silkworm. Meanwhile, comparative proteomic approach was used to investigate the proteomic differences. The results showed that the protein of Hsp-1 and Tn1 were significantly decreased and increased on protein level in cot mutant after thermo-stimulus, respectively. Our data provide insights into the mechanism of thermoprotection in insect. As cot phenotype closely resembles human epilepsy, cot might be a potential model for the mechanism of epilepsy in future.

Fabrication and Characterization of Conductive Conjugated Polymer-Coated Antheraea mylitta Silk Fibroin Fibers for Biomedical Applications.

PubMed

Gh, Darshan; Kong, Dexu; Gautrot, Julien; Vootla, Shyam Kumar

2017-07-01

Conductive polymers are interesting materials for a number of biological and medical applications requiring electrical stimulation of cells or tissues. Highly conductive polymers (polypyrrole and polyaniline)/Antheraea mylitta silk fibroin coated fibers are fabricated successfully by in situ polymerization without any modification of the native silk fibroin. Coated fibers characterized by scanning electron microscopy confirm the silk fiber surface is covered by conductive polymers. Thermogravimetric analysis reveals preserved thermal stability of silk fiber after coating process. X-ray diffraction of degummed fiber diffraction peaks at around 2θ = 20.4 and 16.5 confirms the preservation of the β-sheet structure typical of degummed silk II fibers. This phenomenon implies that both polypyrrole and polyaniline chains form interactions with peptide linkages in degummed fiber macromolecules, without significantly disrupting protein assembly. Fourier transform infrared spectroscopy of coated fibers indicates hydrogen bonding and electrostatic interactions exist between silk fibroin macromolecules and conductive polymers. Resulting fibers display good conductive properties compared to corresponding conjugated polymers. In vitro analysis (live/dead assay) of the behavior of human immortalized keratinocytes (HaCaTs) on coated fibers demonstrates improved cell-adhesive properties and viability after polymers coating. Hence, polypyrrole- and polyaniline-coated A. mylitta silk fibers are suitable for application in cell culture and for tissue engineering, where electrical conduction properties are required. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Silkworm larvae plasma (SLP) assay for detection of bacteria: False positives secondary to inflammation in vivo.

PubMed

Ma, Michelle; Rice, Tyler A; Percopo, Caroline M; Rosenberg, Helene F

2017-01-01

The silkworm larvae plasma (SLP) assay has been developed as a means to detect bacterial peptidoglycan as a surrogate for live bacteria. Here, we present results that indicate that generation of melanin by this assay is not fully reliable as a surrogate marker for bacterial count. Published by Elsevier B.V.

MicroRNA expression profiling during the life cycle of the silkworm (Bombyx mori)

PubMed Central

Liu, Shiping; Zhang, Liang; Li, Qibin; Zhao, Ping; Duan, Jun; Cheng, Daojun; Xiang, Zhonghuai; Xia, Qingyou

2009-01-01

Background MicroRNAs (miRNAs) are expressed by a wide range of eukaryotic organisms, and function in diverse biological processes. Numerous miRNAs have been identified in Bombyx mori, but the temporal expression profiles of miRNAs corresponding to each stage transition over the entire life cycle of the silkworm remain to be established. To obtain a comprehensive overview of the correlation between miRNA expression and stage transitions, we performed a whole-life test and subsequent stage-by-stage examinations on nearly one hundred miRNAs in the silkworm. Results Our results show that miRNAs display a wide variety of expression profiles over the whole life of the silkworm, including continuous expression from embryo to adult (miR-184), up-regulation over the entire life cycle (let-7 and miR-100), down-regulation over the entire life cycle (miR-124), expression associated with embryogenesis (miR-29 and miR-92), up-regulation from early 3rd instar to pupa (miR-275), and complementary pulses in expression between miR-34b and miR-275. Stage-by-stage examinations revealed further expression patterns, such as emergence at specific time-points during embryogenesis and up-regulation of miRNA groups in late embryos (miR-1 and bantam), expression associated with stage transition between instar and molt larval stages (miR-34b), expression associated with silk gland growth and spinning activity (miR-274), continuous high expression from the spinning larval to pupal and adult stages (miR-252 and miR-31a), a coordinate expression trough in day 3 pupae of both sexes (miR-10b and miR-281), up-regulation in pupal metamorphosis of both sexes (miR-29b), and down-regulation in pupal metamorphosis of both sexes (miR-275). Conclusion We present the full-scale expression profiles of miRNAs throughout the life cycle of Bombyx mori. The whole-life expression profile was further investigated via stage-by-stage analysis. Our data provide an important resource for more detailed functional

MicroRNA expression profiling during the life cycle of the silkworm (Bombyx mori).

PubMed

Liu, Shiping; Zhang, Liang; Li, Qibin; Zhao, Ping; Duan, Jun; Cheng, Daojun; Xiang, Zhonghuai; Xia, Qingyou

2009-09-28

MicroRNAs (miRNAs) are expressed by a wide range of eukaryotic organisms, and function in diverse biological processes. Numerous miRNAs have been identified in Bombyx mori, but the temporal expression profiles of miRNAs corresponding to each stage transition over the entire life cycle of the silkworm remain to be established. To obtain a comprehensive overview of the correlation between miRNA expression and stage transitions, we performed a whole-life test and subsequent stage-by-stage examinations on nearly one hundred miRNAs in the silkworm. Our results show that miRNAs display a wide variety of expression profiles over the whole life of the silkworm, including continuous expression from embryo to adult (miR-184), up-regulation over the entire life cycle (let-7 and miR-100), down-regulation over the entire life cycle (miR-124), expression associated with embryogenesis (miR-29 and miR-92), up-regulation from early 3rd instar to pupa (miR-275), and complementary pulses in expression between miR-34b and miR-275. Stage-by-stage examinations revealed further expression patterns, such as emergence at specific time-points during embryogenesis and up-regulation of miRNA groups in late embryos (miR-1 and bantam), expression associated with stage transition between instar and molt larval stages (miR-34b), expression associated with silk gland growth and spinning activity (miR-274), continuous high expression from the spinning larval to pupal and adult stages (miR-252 and miR-31a), a coordinate expression trough in day 3 pupae of both sexes (miR-10b and miR-281), up-regulation in pupal metamorphosis of both sexes (miR-29b), and down-regulation in pupal metamorphosis of both sexes (miR-275). We present the full-scale expression profiles of miRNAs throughout the life cycle of Bombyx mori. The whole-life expression profile was further investigated via stage-by-stage analysis. Our data provide an important resource for more detailed functional analysis of miRNAs in this animal.

Diversity in copy number and structure of a silkworm morphogenetic gene as a result of domestication.

PubMed

Sakudoh, Takashi; Nakashima, Takeharu; Kuroki, Yoko; Fujiyama, Asao; Kohara, Yuji; Honda, Naoko; Fujimoto, Hirofumi; Shimada, Toru; Nakagaki, Masao; Banno, Yutaka; Tsuchida, Kozo

2011-03-01

The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time. © 2011 by the Genetics Society of America

Diversity in Copy Number and Structure of a Silkworm Morphogenetic Gene as a Result of Domestication

PubMed Central

Sakudoh, Takashi; Nakashima, Takeharu; Kuroki, Yoko; Fujiyama, Asao; Kohara, Yuji; Honda, Naoko; Fujimoto, Hirofumi; Shimada, Toru; Nakagaki, Masao; Banno, Yutaka; Tsuchida, Kozo

2011-01-01

The carotenoid-binding protein (CBP) of the domesticated silkworm, Bombyx mori, a major determinant of cocoon color, is likely to have been substantially influenced by domestication of this species. We analyzed the structure of the CBP gene in multiple strains of B. mori, in multiple individuals of the wild silkworm, B. mandarina (the putative wild ancestor of B. mori), and in a number of other lepidopterans. We found the CBP gene copy number in genomic DNA to vary widely among B. mori strains, ranging from 1 to 20. The copies of CBP are of several types, based on the presence of a retrotransposon or partial deletion of the coding sequence. In contrast to B. mori, B. mandarina was found to possess a single copy of CBP without the retrotransposon insertion, regardless of habitat. Several other lepidopterans were found to contain sequences homologous to CBP, revealing that this gene is evolutionarily conserved in the lepidopteran lineage. Thus, domestication can generate significant diversity of gene copy number and structure over a relatively short evolutionary time. PMID:21242537

Absorption of mulberry root urease to the hemolymph of the silkworm, Bombyx mori.

PubMed

Kurahashi, Hitoshi; Atiwetin, Panida; Nagaoka, Sumiharu; Miyata, Seiji; Kitajima, Sakihito; Sugimura, Yukio

2005-09-01

Mulberry leaves are the sole diet of the silkworm, Bombyx mori. The host urease is incorporated into the larval hemolymph and involved in nitrogen metabolism in the insect. To investigate the selective absorption of the host urease to the larvae, crude urease was prepared from mulberry leaves and roots. Root urease was identical to leaf urease on the basis of electrophoretic analyses: (1) the urease activity appeared in the same migration position in a native gel; (2) There was no difference in molecular mass of the subunit. The root urease was orally injected to the fifth instar larvae of the silkworm. Just before spinning, the larvae absorbed intact urease from the midgut lumen to the hemolymph without the loss of activity. The capacity to absorb urease occurred only at the specific stage. Localization of host urease in midgut tissue was observed using confocal laser scanning microscopy and transmission electron microscopy. Based on spatial distribution of immunofluorescent signals and immunogold particles, host urease specifically attached to the surfaces of microvilli existing in the apical side of columnar cells and appeared in the cytoplasm of the cells for transport to the hemolymph. The incorporation efficiency of root urease into the hemolymph was significantly higher than for ureases from jack bean seeds and Bacillus pasteurii. The urease that was transported to the hemolymph was electrophoretically altered, compared with the host urease extracted.

Resistance to BmNPV via Overexpression of an Exogenous Gene Controlled by an Inducible Promoter and Enhancer in Transgenic Silkworm, Bombyx mori

PubMed Central

Jiang, Liang; Cheng, Tingcai; Zhao, Ping; Yang, Qiong; Wang, Genhong; Jin, Shengkai; Lin, Ping; Xiao, Yang; Xia, Qingyou

2012-01-01

The hycu-ep32 gene of Hyphantria cunea NPV can inhibit Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in co-infected cells, but it is not known whether the overexpression of the hycu-ep32 gene has an antiviral effect in the silkworm, Bombyx mori. Thus, we constructed four transgenic vectors, which were under the control of the 39 K promoter of BmNPV (39 KP), Bombyx mori A4 promoter (A4P), hr3 enhancer of BmNPV combined with 39 KP, and hr3 combined with A4P. Transgenic lines were created via embryo microinjection using practical diapause silkworm. qPCR revealed that the expression level of hycu-ep32 could be induced effectively after BmNPV infection in transgenic lines where hycu-ep32 was controlled by hr3 combined with 39 KP (i.e., HEKG). After oral inoculation of BmNPV with 3 × 105 occlusion bodies per third instar, the mortality with HEKG-B was approximately 30% lower compared with the non-transgenic line. The economic characteristics of the transgenic lines remained unchanged. These results suggest that overexpression of an exogenous antiviral gene controlled by an inducible promoter and enhancer is a feasible method for breeding silkworms with a high antiviral capacity. PMID:22870254

Effects of Ag Nanoparticles on Growth and Fat Body Proteins in Silkworms (Bombyx mori).

PubMed

Meng, Xu; Abdlli, Nouara; Wang, Niannian; Lü, Peng; Nie, Zhichao; Dong, Xin; Lu, Shuang; Chen, Keping

2017-12-01

Ag nanoparticles (AgNPs), a widely used non-antibiotic, antibacterial material, have shown toxic and other potentially harmful effects in mammals. However, the deleterious effects of AgNPs on insects are still unknown. Here, we studied the effects of AgNPs on the model invertebrate organism Bombyx mori. After feeding silkworm larvae different concentrations of AgNPs, we evaluated the changes of B. mori body weights, survival rates, and proteomic differences. The results showed that low concentrations (<400 mg/L) of AgNPs promoted the growth and cocoon weights of B. mori. Although high concentrations (≥800 mg/L) of AgNPs also improved B. mori growth, they resulted in silkworm death. An analysis of fat body proteomic differences revealed 13 significant differences in fat body protein spots, nine of which exhibited significantly downregulated expression, while four showed significantly upregulated expression. Reverse transcription-polymerase chain reaction results showed that at an AgNP concentration of 1600 mg/L, the expression levels of seven proteins were similar to the transcription levels of their corresponding genes. Our results suggest that AgNPs lowered the resistance to oxidative stress, affected cell apoptosis, and induced cell necrosis by regulating related protein metabolism and metabolic pathways in B. mori.

Molecular and Physiological Characterization of Two Novel Multirepeat β-Thymosins from Silkworm, Bombyx mori

PubMed Central

Lü, Peng; Yang, Yanhua; Yao, Qin; Xia, Hengchuan; Chen, Keping

2015-01-01

β-thymosin plays important roles in the development of the lymphatic system and the central nervous system in vertebrates. However, its role and function in invertebrates remain much less explored. Here, we firstly isolated a gene encoding β-thymosin in silkworm (Bombyx mori L.). Interestingly, this gene encodes two polypeptides, named as BmTHY1 and BmTHY2, via two different modes of RNA splicing. The recombinant proteins fused with an N-term GST tag were over-expressed in Escherichia coli (E. coli) and further purified to near homogenity to prepare mouse antibodies. The Western blot analysis showed that these proteins were expressed in various tissues and organs, as well as in different developmental stages. Amazingly, the expression of BmTHY2 was hugely increased during the pupae stage, indicating a specialized role in this period. The expression of these proteins was gradually decreased in BmN cells infected by BmNPV, suggesting they may play different roles in the virus infection. In addition, both BmTHY1 and BmTHY2 can interact with 14-3-3 of silkworm and Ubiquitin of BmNPV as shown by GST pull down and Co-IP assays, consistent with their roles in the regulation of the development of nervous system. PMID:26474303

Understanding the nano- and macromechanical behaviour, the failure and fatigue mechanisms of advanced and natural polymer fibres by Raman/IR microspectrometry

NASA Astrophysics Data System (ADS)

Colomban, Philippe

2013-03-01

The coupled mechanical and Raman/infrared (IR) analysis of the (nano)structure and texture of synthetic and natural polymer fibres (polyamides (PA66), polyethylene terephthalate (PET), polypropylene (PP), poly(paraphenylene benzobisoxazole) (PBO), keratin/hair, Bombyx mori, Gonometa rufobrunea/postica Antheraea/Tussah silkworms and Nephila Madagascarensis spider silks) is applied so as to differentiate between crystalline and amorphous macromolecules. Bonding is very similar in the two cases but a broader distribution of conformations is observed for the amorphous macromolecules. These conclusions are then used to discuss the modifications induced by the application of a tensile or compressive stress, including the effects of fatigue. Detailed attention is paid to water and the inter-chain coupling for which the importance of hydrogen bonding is reconsidered. The significant role of the ‘amorphous’ bonds/domains in the process of fracture/fatigue is shown. Invited talk at the 6th International Workshop on Advanced Materials Science and Nanotechnology IWAMSN2012, 30 October-2 November 2012, Ha Long, Vietnam.

Properties of a recombinant bovine tissue factor expressed by Silkworm pupae and its performance as an Owren-type prothrombin time reagent for warfarin monitoring.

PubMed

Okuda, Masahiro; Taniguchi, Tomokuni; Takamiya, Osamu

2012-09-01

Tissue factor (TF), or thromboplastin, is a glycoprotein that triggers the extrinsic coagulation pathway. In blood coagulation testing, TF has been used as a natural source for determining Quick prothrombin time (PT) or the Owren PT (OBT). Currently, natural sources are being replaced with recombinant proteins because of their uniform characteristics and the possibility of stable mass production of PT reagents. Because bovine spongiform encephalopathy (BSE)-infected cows are widespread in Japan, we prepared a recombinant bovine TF (rbTF) with a baculovirus expression system using silkworms. To overcome the limitations of natural TF, especially in bovine brain, we expressed a full-length rbTF protein in Silkworm pupae with a baculovirus expression system. Baculovirus inactivation and the presence of DNA fragments in the rbTF fraction were confirmed using Reed-Muench and polymerase chain reaction methods after inactivation with a detergent. The rbTF fraction prepared by an immobilized anti-Silkworm pupae fluid protein Sepharose 4B column was identified as a visible band on western blots with a polyclonal antibody against human TF with cross-reactivity with TFs. The inhibition of the polyclonal antibody against human TF by the clotting assay for PT was identified, and amidolytic biological activity through activated factor VII on S-2288 substrate was observed. In conclusion, the rbTF expressed by the baculovirus system using Silkworm pupae was uniformly specific for bovine TF. The OBT reagent incorporated by this rbTF was similar to those of commercial reagents. It also showed a suitable International Sensitivity Index and reproducibility precision, thereby allowing for diagnostic use. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cloning and Expression Analysis of the Bombyx mori α-amylase Gene (Amy) from the Indigenous Thai Silkworm Strain, Nanglai

PubMed Central

Ngernyuang, Nipaporn; Kobayashi, Isao; Promboon, Amornrat; Ratanapo, Sunanta; Tamura, Toshiki; Ngernsiri, Lertluk

2011-01-01

α-Amylase is a common enzyme for hydrolyzing starch. In the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), α-amylase is found in both digestive fluid and hemolymph. Here, the complete genomic sequence of the Amy gene encoding α-amylase from a local Thai silkworm, the Nanglai strain, was obtained. This gene was 7981 bp long with 9 exons. The full length Amy cDNA sequence was 1749 bp containing a 1503 bp open reading frame. The ORF encoded 500 amino acid residues. The deduced protein showed 81–54% identity to other insect α-amylases and more than 50% identity to mammalian enzymes. Southern blot analysis revealed that in the Nanglai strain Amy is a single-copy gene. RT- PCR showed that Amy was transcribed only in the foregut. Transgenic B. mori also showed that the Amy promoter activates expression of the transgene only in the foregut. PMID:21529256

Transcriptome analysis of the epidermis of the purple quail-like (q-lp) mutant of silkworm, Bombyx mori.

PubMed

Wang, Pingyang; Qiu, Zhiyong; Xia, Dingguo; Tang, Shunming; Shen, Xingjia; Zhao, Qiaoling

2017-01-01

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.

Mutation of a vitelline membrane protein, BmEP80, is responsible for the silkworm "Ming" lethal egg mutant.

PubMed

Chen, Anli; Gao, Peng; Zhao, Qiaoling; Tang, Shunming; Shen, Xingjia; Zhang, Guozheng; Qiu, Zhiyong; Xia, Dingguo; Huang, Yongping; Xu, Yunmin; He, Ningjia

2013-02-25

The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive "Ming" lethal egg mutants (l-e(m)) lose water and become concaved around 1h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e(m) mutant to 360 kb on chromosome 10 using 2287 F(2) individuals. Using expression analysis and RNA interference, the best l-e(m) candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~1.9 kb region from the 3' untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a >100 kb DNA fragment in the l-e(m) mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e(m) mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e(m) mutant. These results indicate that BmEP80 is responsible for the l-e(m) mutation. Copyright © 2012 Elsevier B.V. All rights reserved.

Silkworm Thermal Biology: A Review of Heat Shock Response, Heat Shock Proteins and Heat Acclimation in the Domesticated Silkworm, Bombyx mori

PubMed Central

Manjunatha, H. B.; Rajesh, R. K.; Aparna, H. S.

2010-01-01

Heat shock proteins (HSPs) are known to play ecological and evolutionary roles in this postgenomic era. Recent research suggests that HSPs are implicated in cardiovascular biology and disease development, proliferation and regulation of cancer cells, cell death via apoptosis, and several other key cellular functions. These activities have generated great interest amongst cell and molecular biologists, and these biologists are keen to unravel other hitherto unknown potential functions of this group of proteins. Consequently, the biological significance of HSPs has led to cloning and characterization of genes encoding HSPs in many organisms including the silkworm, Bombyx mori L. (Lepidoptera: Bombycidae). However, most of the past investigations in B. mori were confined to expression of HSPs in tissues and cell lines, whereas information on their specific functional roles in biological, physiological, and molecular processes is scarce. Naturally occurring or domesticated polyvoltines (known to be the tropical race) are more resistant to high temperatures and diseases than bi- or univoltines (temperate races). The mechanism of ecological or evolutionary modification of HSPs during the course of domestication of B. mori - particularly in relation to thermotolerance in geographically distinct races/strains - is still unclear. In addition, the heat shock response, thermal acclimation, and hardening have not been studied extensively in B. mori compared to other organisms. Towards this, recent investigations on differential expression of HSPs at various stages of development, considering the concept of the whole organism, open ample scope to evaluate their biological and commercial importance in B. mori which has not been addressed in any of the representative organisms studied so far. Comparatively, heat shock response among different silkworm races/strains of poly-, bi-, and univoltines varies significantly and thermotolerance increases as the larval development proceeds

Changes in glutathione redox cycle during diapause determination and termination in the bivoltine silkworm, Bombyx mori.

PubMed

Zhao, Lin-Chuan; Hou, Yi-Sheng; Sima, Yang-Hu

2014-02-01

To explore whether glutathione regulates diapause determination and termination in the bivoltine silkworm Bombyx mori, we monitored the changes in glutathione redox cycle in the ovary of both diapause- and nondiapause-egg producers, as well as those in diapause eggs incubated at different temperatures. The activity of thioredoxin reductase (TrxR) was detected in ovaries but not in eggs, while neither ovaries nor eggs showed activity of glutathione peroxidase. A lower reduced glutathione/oxidized glutathione (GSH/GSSG) ratio was observed in the ovary of diapause-egg producers, due to weaker reduction of oxidized glutathione (GSSG) to the reduced glutathione (GSH) catalyzed by glutathione reductase (GR) and TrxR. This indicates an oxidative shift in the glutathione redox cycle during diapause determination. Compared with the 25°C-treated diapause eggs, the 5°C-treated diapause eggs showed lower GSH/GSSG ratio, a result of stronger oxidation of GSH catalyzed by thioredoxin peroxidase and weaker reduction of GSSG catalyzed by GR. Our study demonstrated the important regulatory role of glutathione in diapause determination and termination of the bivoltine silkworm. © 2013 Institute of Zoology, Chinese Academy of Sciences.

Middle region of FancM interacts with Mhf and Rmi1 in silkworms, a species lacking the Fanconi anaemia (FA) core complex.

PubMed

Sugahara, R; Mon, H; Lee, J M; Kusakabe, T

2014-04-01

The Fanconi anaemia (FA) pathway is responsible for interstrand crosslink (ICL) repair. Among the FA core complex components, FANCM is believed to act as a damage sensor for the ICL-blocked replication fork and also as a molecular platform for FA core complex assembly and interaction with Bloom's syndrome (BS) complex that is thought to play an important role in the processing of DNA structures such as stalled replication forks. In the present study, we found that in silkworms, Bombyx mori, a species lacking the major FA core complex components (FANCA, B, C, E, F, and G), FancM is required for FancD2 monoubiquitination and cell proliferation in the presence of mitomycin C (MMC). Silkworm FancM (BmFancM) was phosphorylated in the middle regions, and the modification was associated with its subcellular localization. In addition, BmFancM interacted with Mhf1, a histone-fold protein, and Rmi1, a subunit of the BS complex, in the different regions. The interaction region containing at least these two protein-binding domains played an essential role in FancM-dependent resistance to MMC. Our results suggest that BmFancM also acts as a platform for recruitment of both the FA protein and the BS protein, although the silkworm genome seems to lose FAAP24, a FancM-binding partner protein in mammals. © 2013 The Royal Entomological Society.

Effects of transient high temperature treatment on the intestinal flora of the silkworm Bombyx mori.

PubMed

Sun, Zhenli; Kumar, Dhiraj; Cao, Guangli; Zhu, Liyuan; Liu, Bo; Zhu, Min; Liang, Zi; Kuang, Sulan; Chen, Fei; Feng, Yongjie; Hu, Xiaolong; Xue, Renyu; Gong, Chengliang

2017-06-13

The silkworm Bombyx mori is a poikilotherm and is therefore sensitive to various climatic conditions. The influence of temperature on the intestinal flora and the relationship between the intestinal flora and gene expression in the silkworm remain unknown. In the present study, changes of the intestinal flora at 48, 96 and 144 h following transient high temperature treatment (THTT) of 37 °C for 8 h were investigated. According to principal component analysis, the abundances of Enterococcus and Staphylococcus showed a negative correlation with other dominant genera. After THTT, the gene expression levels of spatzle-1 and dicer-2 were increased and decreased, respectively, which suggested that the Toll and RNAi pathways were activated and suppressed, respectively. The species-gene expression matrix confirmed that the spatzle-1 and dicer-2 gene expression levels were negatively and positively correlated, respectively, with the abundance of Enterococcus and Staphylococcus in the control. The abundance of Variovorax post-THTT was positively correlated with the spatzle-1 gene expression level, whereas the community richness of Enterococcus was negatively correlated with the spatzle-1 gene expression level and positively correlated with the dicer-2. The results of the present investigation provide new evidence for understanding the relationships among THTT, intestinal flora and host gene expression.

The effect of hemoperfusion on patients with toxic encephalopathy induced by silkworm chrysalis ingestion.

PubMed

Hu, Haixia; Wang, Xu; Lv, Jiaqi; Sun, Jing; Xing, Jihong; Liu, Xiaoliang

2016-08-01

This study aims to determine therapeutic effect of hemoperfusion on patients with acute toxic encephalopathy induced by silkworm chrysalis ingestion. Three patients who developed toxic encephalopathy after chrysalis ingestion were analysed. Two patients lost their consciousness, while two patients had typical extrapyramidal tremor symptoms. Further neurological examination revealed various degrees of muscle strength impairment in these patients. All of them received treatments of omeprazole (40 mg/day), furosemide (one dose of 20 mg), vitamin C (2.0 g/day), calcium gluconate (2.0 g/day) and rehydration with glucose and sodium chloride (1500 ml/day). In addition, they received hemoperfusion treatment for 1.5 h. All patients recovered well after hemoperfusion. Two patients with loss of consciousness significantly recovered at 45 min and 65 min after hemoperfusion, respectively. All tremor symptoms were completely resolved in these patients at 30 min, 50 min, and 70 min following treatment, respectively. After the hemoperfusion treatment, encephalopathy symptoms of two patients had completely disappeared. All patients were followed up for one month and did not report any abnormalities. Our study indicates that hemoperfusion could be a useful and efficient treatment strategy for patients with acute encephalopathy after silkworm chrysalis ingestion. Larger clinical trials with longer follow-up are warranted to confirm the clinical benefit of hemoperfusion. © The Author(s) 2015.

Quantification of silkworm coactivator of MBF1 mRNA by SYBR Green I real-time RT-PCR reveals tissue- and stage-specific transcription levels.

PubMed

Li, Guang-li; Roy, Bhaskar; Li, Xing-hua; Yue, Wan-fu; Wu, Xiao-feng; Liu, Jian-mei; Zhang, Chuan-xi; Miao, Yun-gen

2009-05-01

Transcriptional coactivators play a crucial role in gene transcription and expression. Multiprotein bridging factor 1 (MBF1) is a transcriptional coactivator necessary for transcriptional activation caused by DNA-binding activators, such as FTZ-F1 and GCN4. Until now, very few studies have been reported in the silkworm. We selected the Bombyx mori because it is a model insect and acts as an economic animal for silk industry. In this study, we conducted the quantitative analysis of MBF1 mRNA in silkworm B. mori L. with actin (A3) as internal standard by means of SYBR Green I real-time RT-PCR method. The total RNA was extracted from the silk gland, epidermis, fat body, and midguts of the fifth instar B. mori larvae. The mRNA was reverse transcripted, and the cDNA fragments of MBF1 mRNA and actin gene were amplified by RT-PCR using specific primers. MBF1 mRNA expression in different tissues of silkworm B. mori L. was quantified using standardized SYBR Green I RT-PCR. The results suggested MBF1 gene was expressed in all investigated organs but highly expressed in the silk gland, showing its relation to biosynthesis of silk proteins.

Bone regeneration by polyhedral microcrystals from silkworm virus

PubMed Central

Matsumoto, Goichi; Ueda, Takayo; Shimoyama, Junko; Ijiri, Hiroshi; Omi, Yasushi; Yube, Hisato; Sugita, Yoshihiko; Kubo, Katsutoshi; Maeda, Hatsuhiko; Kinoshita, Yukihiko; Arias, Duverney Gaviria; Shimabukuro, Junji; Kotani, Eiji; Kawamata, Shin; Mori, Hajime

2012-01-01

Bombyx mori cypovirus is a major pathogen which causes significant losses in silkworm cocoon harvests because the virus particles are embedded in micrometer-sized protein crystals called polyhedra and can remain infectious in harsh environmental conditions for years. But the remarkable stability of polyhedra can be applied on slow-release carriers of cytokines for tissue engineering. Here we show the complete healing in critical-sized bone defects by bone morphogenetic protein-2 (BMP-2) encapsulated polyhedra. Although absorbable collagen sponge (ACS) safely and effectively delivers recombinant human BMP-2 (rhBMP-2) into healing tissue, the current therapeutic regimens release rhBMP-2 at an initially high rate after which the rate declines rapidly. ACS impregnated with BMP-2 polyhedra had enough osteogenic activity to promote complete healing in critical-sized bone defects, but ACS with a high dose of rhBMP-2 showed incomplete bone healing, indicating that polyhedral microcrystals containing BMP-2 promise to advance the state of the art of bone healing. PMID:23226833

Zygotic amplification of secondary piRNAs during silkworm embryogenesis

PubMed Central

Kawaoka, Shinpei; Arai, Yuji; Kadota, Koji; Suzuki, Yutaka; Hara, Kahori; Sugano, Sumio; Shimizu, Kentaro; Tomari, Yukihide; Shimada, Toru; Katsuma, Susumu

2011-01-01

PIWI-interacting RNAs (piRNAs) are 23–30-nucleotide-long small RNAs that act as sequence-specific silencers of transposable elements in animal gonads. In flies, genetics and deep sequencing data have led to a hypothesis for piRNA biogenesis called the ping-pong cycle, where antisense primary piRNAs initiate an amplification loop to generate sense secondary piRNAs. However, to date, the process of the ping-pong cycle has never been monitored at work. Here, by large-scale profiling of piRNAs from silkworm ovary and embryos of different developmental stages, we demonstrate that maternally inherited antisense-biased piRNAs trigger acute amplification of secondary sense piRNA production in zygotes, at a time coinciding with zygotic transcription of sense transposon mRNAs. These results provide on-site evidence for the ping-pong cycle. PMID:21628432

MicroRNA-281 regulates the expression of ecdysone receptor (EcR) isoform B in the silkworm, Bombyx mori

USDA-ARS?s Scientific Manuscript database

Hundreds of Bombyx mori miRNAs had been identified in recent years, but their function in vivo remains poorly understood. The silkworm EcR gene (BmEcR) has three transcriptional isoforms, A, B1 and B2. Isoform sequences are different in the 3’UTR region of the gene, which is the case only in insects...

Annotation and expression of carboxylesterases in the silkworm, Bombyx mori.

PubMed

Yu, Quan-You; Lu, Cheng; Li, Wen-Le; Xiang, Zhong-Huai; Zhang, Ze

2009-11-24

Carboxylesterase is a multifunctional superfamily and ubiquitous in all living organisms, including animals, plants, insects, and microbes. It plays important roles in xenobiotic detoxification, and pheromone degradation, neurogenesis and regulating development. Previous studies mainly used Dipteran Drosophila and mosquitoes as model organisms to investigate the roles of the insect COEs in insecticide resistance. However, genome-wide characterization of COEs in phytophagous insects and comparative analysis remain to be performed. Based on the newly assembled genome sequence, 76 putative COEs were identified in Bombyx mori. Relative to other Dipteran and Hymenopteran insects, alpha-esterases were significantly expanded in the silkworm. Genomics analysis suggested that BmCOEs showed chromosome preferable distribution and 55% of which were tandem arranged. Sixty-one BmCOEs were transcribed based on cDNA/ESTs and microarray data. Generally, most of the COEs showed tissue specific expressions and expression level between male and female did not display obvious differences. Three main patterns could be classified, i.e. midgut-, head and integument-, and silk gland-specific expressions. Midgut is the first barrier of xenobiotics peroral toxicity, in which COEs may be involved in eliminating secondary metabolites of mulberry leaves and contaminants of insecticides in diet. For head and integument-class, most of the members were homologous to odorant-degrading enzyme (ODE) and antennal esterase. RT-PCR verified that the ODE-like esterases were also highly expressed in larvae antenna and maxilla, and thus they may play important roles in degradation of plant volatiles or other xenobiotics. B. mori has the largest number of insect COE genes characterized to date. Comparative genomic analysis suggested that the gene expansion mainly occurred in silkworm alpha-esterases. Expression evidence indicated that the expanded genes were specifically expressed in midgut, integument and

Annotation and expression of carboxylesterases in the silkworm, Bombyx mori

PubMed Central

2009-01-01

Background Carboxylesterase is a multifunctional superfamily and ubiquitous in all living organisms, including animals, plants, insects, and microbes. It plays important roles in xenobiotic detoxification, and pheromone degradation, neurogenesis and regulating development. Previous studies mainly used Dipteran Drosophila and mosquitoes as model organisms to investigate the roles of the insect COEs in insecticide resistance. However, genome-wide characterization of COEs in phytophagous insects and comparative analysis remain to be performed. Results Based on the newly assembled genome sequence, 76 putative COEs were identified in Bombyx mori. Relative to other Dipteran and Hymenopteran insects, alpha-esterases were significantly expanded in the silkworm. Genomics analysis suggested that BmCOEs showed chromosome preferable distribution and 55% of which were tandem arranged. Sixty-one BmCOEs were transcribed based on cDNA/ESTs and microarray data. Generally, most of the COEs showed tissue specific expressions and expression level between male and female did not display obvious differences. Three main patterns could be classified, i.e. midgut-, head and integument-, and silk gland-specific expressions. Midgut is the first barrier of xenobiotics peroral toxicity, in which COEs may be involved in eliminating secondary metabolites of mulberry leaves and contaminants of insecticides in diet. For head and integument-class, most of the members were homologous to odorant-degrading enzyme (ODE) and antennal esterase. RT-PCR verified that the ODE-like esterases were also highly expressed in larvae antenna and maxilla, and thus they may play important roles in degradation of plant volatiles or other xenobiotics. Conclusion B. mori has the largest number of insect COE genes characterized to date. Comparative genomic analysis suggested that the gene expansion mainly occurred in silkworm alpha-esterases. Expression evidence indicated that the expanded genes were specifically

Effects of hypervitaminosis of vitamin B3 on silkworm biology.

PubMed

Etebari, Kayvan; Matindoost, Leila

2004-12-01

A high-dose of vitamin B(3) in silkworm diet interrupts larval feeding and normal growth. High mortality of larvae occurs during molting and they cannot complete this process normally. Also the larvae exhibit nicotinamide hypervitaminosis symptoms such as immobility, dyspepsia, darkening of the skin, inability to excrete normally, exerting brownish fluid from anus and swelling of rectal muscles. Maximum larval weights in 1, 2 and 3 g/l treatments were 2.9, 1.6 and 1.2 g respectively, while maximum larval weight in the control was 5.6 g. Larval stage compared to control had increased 18, 26 and 31 days respectively. The concentration increase of uric acid in haemolymph demonstrates the hyperuricemia, while other measured biochemical compounds show significant decrease; sodium and potassium did not change significantly.

Cloning and analysis of DnaJ family members in the silkworm, Bombyx mori.

PubMed

Li, Yinü; Bu, Cuiyu; Li, Tiantian; Wang, Shibao; Jiang, Feng; Yi, Yongzhu; Yang, Huipeng; Zhang, Zhifang

2016-01-15

Heat shock proteins (Hsps) are involved in a variety of critical biological functions, including protein folding, degradation, and translocation and macromolecule assembly, act as molecular chaperones during periods of stress by binding to other proteins. Using expressed sequence tag (EST) and silkworm (Bombyx mori) transcriptome databases, we identified 27 cDNA sequences encoding the conserved J domain, which is found in DnaJ-type Hsps. Of the 27 J domain-containing sequences, 25 were complete cDNA sequences. We divided them into three types according to the number and presence of conserved domains. By analyzing the gene structures, intron numbers, and conserved domains and constructing a phylogenetic tree, we found that the DnaJ family had undergone convergent evolution, obtaining new domains to expand the diversity of its family members. The acquisition of the new DnaJ domains most likely occurred prior to the evolutionary divergence of prokaryotes and eukaryotes. The expression of DnaJ genes in the silkworm was generally higher in the fat body. The tissue distribution of DnaJ1 proteins was detected by western blotting, demonstrating that in the fifth-instar larvae, the DnaJ1 proteins were expressed at their highest levels in hemocytes, followed by the fat body and head. We also found that the DnaJ1 transcripts were likely differentially translated in different tissues. Using immunofluorescence cytochemistry, we revealed that in the blood cells, DnaJ1 was mainly localized in the cytoplasm. Copyright © 2015 Elsevier B.V. All rights reserved.

Regioselective formation of quercetin 5-O-glucoside from orally administered quercetin in the silkworm, Bombyx mori.

PubMed

Hirayama, Chikara; Ono, Hiroshi; Tamura, Yasumori; Konno, Kotaro; Nakamura, Masatoshi

2008-03-01

The cocoons of some races of the silkworm, Bombyx mori, have been shown to contain 5-O-glucosylated flavonoids, which do not occur naturally in the leaves of their host plant, mulberry (Morus alba). Thus, dietary flavonoids could be biotransformed in this insect. In this study, we found that after feeding silkworms a diet rich in the flavonol quercetin, quercetin 5-O-glucoside was the predominant metabolite in the midgut tissue, while quercetin 5,4'-di-O-glucoside was the major constituent in the hemolymph and silk glands. UDP-glucosyltransferase (UGT) in the midgut could transfer glucose to each of the hydroxyl groups of quercetin, with a preference for formation of 5-O-glucoside, while quercetin 5,4'-di-O-glucoside was predominantly produced if the enzyme extracts of either the fat body or silk glands were incubated with quercetin 5-O-glucoside and UDP-glucose. These results suggest that dietary quercetin was glucosylated at the 5-O position in the midgut as the first-pass metabolite of quercetin after oral absorption, then glucosylated at the 4'-O position in the fat body or silk glands. The 5-O-glucosylated flavonoids retained biological activity in the insect, since the total free radical scavenging capacity of several tissues increased after oral administration of quercetin.

Altered expression of testis-specific genes, piRNAs, and transposons in the silkworm ovary masculinized by a W chromosome mutation

PubMed Central

2012-01-01

Background In the silkworm, Bombyx mori, femaleness is strongly controlled by the female-specific W chromosome. Originally, it was presumed that the W chromosome encodes female-determining gene(s), accordingly called Fem. However, to date, neither Fem nor any protein-coding gene has been identified from the W chromosome. Instead, the W chromosome is occupied with numerous transposon-related sequences. Interestingly, the silkworm W chromosome is a source of female-enriched PIWI-interacting RNAs (piRNAs). piRNAs are small RNAs of 23-30 nucleotides in length, which are required for controlling transposon activity in animal gonads. A recent study has identified a novel mutant silkworm line called KG, whose mutation in the W chromosome causes severe female masculinization. However, the molecular nature of KG line has not been well characterized yet. Results Here we molecularly characterize the KG line. Genomic PCR analyses using currently available W chromosome-specific PCR markers indicated that no large deletion existed in the KG W chromosome. Genetic analyses demonstrated that sib-crosses within the KG line suppressed masculinization. Masculinization reactivated when crossing KG females with wild type males. Importantly, the KG ovaries exhibited a significantly abnormal transcriptome. First, the KG ovaries misexpressed testis-specific genes. Second, a set of female-enriched piRNAs was downregulated in the KG ovaries. Third, several transposons were overexpressed in the KG ovaries. Conclusions Collectively, the mutation in the KG W chromosome causes broadly altered expression of testis-specific genes, piRNAs, and transposons. To our knowledge, this is the first study that describes a W chromosome mutant with such an intriguing phenotype. PMID:22452797

Determination of Albendazole and Metabolites in Silkworm Bombyx mori Hemolymph by Ultrafast Liquid Chromatography Tandem Triple Quadrupole Mass Spectrometry

PubMed Central

Li, Li; Xing, Dong-Xu; Li, Qing-Rong; Xiao, Yang; Ye, Ming-Qiang; Yang, Qiong

2014-01-01

Albendazole is a broad-spectrum parasiticide with high effectiveness and low host toxicity. No method is currently available for measuring albendazole and its metabolites in silkworm hemolymph. This study describes a rapid, selective, sensitive, synchronous and reliable detection method for albendazole and its metabolites in silkworm hemolymph using ultrafast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-MS/MS). The method is liquid-liquid extraction followed by UFLC separation and quantification in an MS/MS system with positive electrospray ionization in multiple reaction monitoring mode. Precursor-to-product ion transitions were monitored at 266.100 to 234.100 for albendazole (ABZ), 282.200 to 208.100 for albendazole sulfoxide (ABZSO), 298.200 to 159.100 for albendazole sulfone (ABZSO2) and 240.200 to 133.100 for albendazole amino sulfone (ABZSO2-NH2). Calibration curves had good linearities with R2 of 0.9905–0.9972. Limits of quantitation (LOQs) were 1.32 ng/mL for ABZ, 16.67 ng/mL for ABZSO, 0.76 ng/mL for ABZSO2 and 5.94 ng/mL for ABZSO2-NH2. Recoveries were 93.12%–103.83% for ABZ, 66.51%–108.51% for ABZSO, 96.85%–105.6% for ABZSO2 and 96.46%–106.14% for ABZSO2-NH2, (RSDs <8%). Accuracy, precision and stability tests showed acceptable variation in quality control (QC) samples. This analytical method successfully determined albendazole and its metabolites in silkworm hemolymph in a pharmacokinetic study. The results of single-dose treatment suggested that the concentrations of ABZ, ABZSO and ABZSO2 increased and then fell, while ABZSO2-NH2 level was low without obvious change. Different trends were observed for multi-dose treatment, with concentrations of ABZSO and ABZSO2 rising over time. PMID:25255321

Determination of albendazole and metabolites in silkworm Bombyx mori hemolymph by ultrafast liquid chromatography tandem triple quadrupole mass spectrometry.

PubMed

Li, Li; Xing, Dong-Xu; Li, Qing-Rong; Xiao, Yang; Ye, Ming-Qiang; Yang, Qiong

2014-01-01

Albendazole is a broad-spectrum parasiticide with high effectiveness and low host toxicity. No method is currently available for measuring albendazole and its metabolites in silkworm hemolymph. This study describes a rapid, selective, sensitive, synchronous and reliable detection method for albendazole and its metabolites in silkworm hemolymph using ultrafast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-MS/MS). The method is liquid-liquid extraction followed by UFLC separation and quantification in an MS/MS system with positive electrospray ionization in multiple reaction monitoring mode. Precursor-to-product ion transitions were monitored at 266.100 to 234.100 for albendazole (ABZ), 282.200 to 208.100 for albendazole sulfoxide (ABZSO), 298.200 to 159.100 for albendazole sulfone (ABZSO2) and 240.200 to 133.100 for albendazole amino sulfone (ABZSO2-NH2). Calibration curves had good linearities with R2 of 0.9905-0.9972. Limits of quantitation (LOQs) were 1.32 ng/mL for ABZ, 16.67 ng/mL for ABZSO, 0.76 ng/mL for ABZSO2 and 5.94 ng/mL for ABZSO2-NH2. Recoveries were 93.12%-103.83% for ABZ, 66.51%-108.51% for ABZSO, 96.85%-105.6% for ABZSO2 and 96.46%-106.14% for ABZSO2-NH2, (RSDs <8%). Accuracy, precision and stability tests showed acceptable variation in quality control (QC) samples. This analytical method successfully determined albendazole and its metabolites in silkworm hemolymph in a pharmacokinetic study. The results of single-dose treatment suggested that the concentrations of ABZ, ABZSO and ABZSO2 increased and then fell, while ABZSO2-NH2 level was low without obvious change. Different trends were observed for multi-dose treatment, with concentrations of ABZSO and ABZSO2 rising over time.

A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai: cloning and expression pattern in pharate first instar larvae in relation to diapause.

PubMed

Yang, Ping; Tanaka, Hiromasa; Kuwano, Eiichi; Suzuki, Koichi

2008-03-01

A new cytochrome P450 gene, CYP4G25, was identified as a differentially expressed gene between the diapausing and post-diapausing pharate first instar larvae of the wild silkmoth Antheraea yamamai, using subtractive cDNA hybridization. The cDNA sequence of CYP4G25 has an open reading frame of 1674 nucleotides encoding 557 amino acid residues. Sequence analysis of the putative CYP4G25 protein disclosed the motif FXXGXRXCXG that is essential for heme binding in P450 cytochromes. Hybridization in situ demonstrated predominant expression of CYP4G25 in the integument of pharate first instar larvae. Northern blotting analysis showed an intensive signal after the initiation of diapause and no or weak expression throughout the periods of pre-diapause and post-diapause, including larval development. These results indicate that CYP4G25 is strongly associated with diapause in pharate first instar larvae.

BmICE-2 is a novel pro-apoptotic caspase involved in apoptosis in the silkworm, Bombyx mori.

PubMed

Yi, Hua-Shan; Pan, Cai-Xia; Pan, Chun; Song, Juan; Hu, Yan-Fen; Wang, La; Pan, Min-Hui; Lu, Cheng

2014-02-28

In this study we identified a potential pro-apoptotic caspase gene, Bombyx mori(B. mori)ICE-2 (BmICE-2) which encoded a polypeptide of 284 amino acid residues, including a (169)QACRG(173) sequence which surrounded the catalytic site and contained a p20 and a p10 domain. BmICE-2 expressed in Escherichia coli (E. coli) exhibited high proteolytic activity for the synthetic human initiator caspase-9 substrates Ac-LEHD-pNA, but little activity towards the effector caspase-3 substrates Ac-DEVD-pNA. When BmICE-2 was transiently expressed in BmN-SWU1 silkworm B. mori cells, we found that the high proteolytic activity for Ac-LEHD-pNA triggered caspase-3-like protease activity resulting in spontaneous cleavage and apoptosis in these cells. This effect was not replicated in Spodoptera frugiperda 9 cells. In addition, spontaneous cleavage of endogenous BmICE-2 in BmN-SWU1 cells could be induced by actinomycin D. These results suggest that BmICE-2 may be a novel pro-apoptotic gene with caspase-9 activity which is involved apoptotic processes in BmN-SWU1 silkworm B. mori cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Improved isolation and purification of functional human Fas receptor extracellular domain using baculovirus-silkworm expression system.

PubMed

Muraki, Michiro; Honda, Shinya

2011-11-01

To achieve an efficient isolation of human Fas receptor extracellular domain (hFasRECD), a fusion protein of hFasRECD with human IgG1 heavy chain Fc domain containing thrombin cleavage sequence at the junction site was overexpressed using baculovirus-silkworm larvae expression system. The hFasRECD part was separated from the fusion protein by the effective cleavage of the recognition site with bovine thrombin. Protein G column treatment of the reaction mixture and the subsequent cation-exchange chromatography provided purified hFasRECD with a final yield of 13.5mg from 25.0 ml silkworm hemolymph. The functional activity of the product was examined by size-exclusion chromatography analysis. The isolated hFasRECD less strongly interacted with human Fas ligand extracellular domain (hFasLECD) than the Fc domain-bridged counterpart, showing the contribution of antibody-like avidity in the latter case. The purified glycosylated hFasRECD presented several discrete bands in the disulphide-bridge non-reducing SDS-PAGE analysis, and virtually all of the components were considered to participate in the binding to hFasLECD. The attached glycans were susceptible to PNGase F digestion, but mostly resistant to Endo Hf digestion under denaturing conditions. One of the components exhibited a higher susceptibility to PNGase F digestion under non-denaturing conditions. Copyright © 2011 Elsevier Inc. All rights reserved.

Self-assembled silk sericin/poloxamer nanoparticles as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery

NASA Astrophysics Data System (ADS)

Mandal, Biman B.; Kundu, S. C.

2009-09-01

In recent times self-assembled micellar nanoparticles have been successfully employed in tissue engineering for targeted drug delivery applications. In this review, silk sericin protein from non-mulberry Antheraea mylitta tropical tasar silk cocoons was blended with pluronic F-127 and F-87 in the presence of solvents to achieve self-assembled micellar nanostructures capable of carrying both hydrophilic (FITC-inulin) and hydrophobic (anticancer drug paclitaxel) drugs. The fabricated nanoparticles were subsequently characterized for their size distribution, drug loading capability, cellular uptake and cytotoxicity. Nanoparticle sizes ranged between 100 and 110 nm in diameter as confirmed by dynamic light scattering. Rapid uptake of these particles into cells was observed in in vitro cellular uptake studies using breast cancer MCF-7 cells. In vitro cytotoxicity assay using paclitaxel-loaded nanoparticles against breast cancer cells showed promising results comparable to free paclitaxel drugs. Drug-encapsulated nanoparticle-induced apoptosis in MCF-7 cells was confirmed by FACS and confocal microscopic studies using Annexin V staining. Up-regulation of pro-apoptotic protein Bax, down-regulation of anti-apoptotic protein Bcl-2 and cleavage of regulatory protein PARP through Western blot analysis suggested further drug-induced apoptosis in cells. This study projects silk sericin protein as an alternative natural biomaterial for fabrication of self-assembled nanoparticles in the presence of poloxamer for successful delivery of both hydrophobic and hydrophilic drugs to target sites.

Self-assembled silk sericin/poloxamer nanoparticles as nanocarriers of hydrophobic and hydrophilic drugs for targeted delivery.

PubMed

Mandal, Biman B; Kundu, S C

2009-09-02

In recent times self-assembled micellar nanoparticles have been successfully employed in tissue engineering for targeted drug delivery applications. In this review, silk sericin protein from non-mulberry Antheraea mylitta tropical tasar silk cocoons was blended with pluronic F-127 and F-87 in the presence of solvents to achieve self-assembled micellar nanostructures capable of carrying both hydrophilic (FITC-inulin) and hydrophobic (anticancer drug paclitaxel) drugs. The fabricated nanoparticles were subsequently characterized for their size distribution, drug loading capability, cellular uptake and cytotoxicity. Nanoparticle sizes ranged between 100 and 110 nm in diameter as confirmed by dynamic light scattering. Rapid uptake of these particles into cells was observed in in vitro cellular uptake studies using breast cancer MCF-7 cells. In vitro cytotoxicity assay using paclitaxel-loaded nanoparticles against breast cancer cells showed promising results comparable to free paclitaxel drugs. Drug-encapsulated nanoparticle-induced apoptosis in MCF-7 cells was confirmed by FACS and confocal microscopic studies using Annexin V staining. Up-regulation of pro-apoptotic protein Bax, down-regulation of anti-apoptotic protein Bcl-2 and cleavage of regulatory protein PARP through Western blot analysis suggested further drug-induced apoptosis in cells. This study projects silk sericin protein as an alternative natural biomaterial for fabrication of self-assembled nanoparticles in the presence of poloxamer for successful delivery of both hydrophobic and hydrophilic drugs to target sites.

Chitin in the Silk Gland Ducts of the Spider Nephila edulis and the Silkworm Bombyx mori

PubMed Central

Davies, Gwilym J. G.; Knight, David P.; Vollrath, Fritz

2013-01-01

Here we report the detection and localisation of chitin in the cuticle of the spinning ducts of both the spider Nephila edulis and the silkworm Bombyx mori. Our observations demonstrate that the duct walls of both animals contain chitin notwithstanding totally independent evolutionary pathways of the systems. We conclude that chitin may well be an essential component for the construction of spinning ducts; we further conclude that in both species chitin may indicate the evolutionary origin of the spinning ducts. PMID:24015298

Identification of plasmalogen in the gut of silkworm (Bombyx mori).

PubMed

Aboshi, Takako; Nishida, Ritsuo; Mori, Naoki

2012-08-01

Herbivorous insect species are constantly challenged with endogenous and exogenous oxidative stress. Consequently, they possess an array of antioxidant enzymes and small molecular weight antioxidants. Lipid-soluble small molecular antioxidants, such as tocopherols, have not been well studied in insects but may play important antioxidant roles. In this study, we identified plasmalogen phosphatidylethanolamines (pPEs) as well as α-, β/γ-, δ-tocopherol in the larvae of the silkworm Bombyx mori by LCMS analyses and examined their distribution. Plasmalogen are reported to inhibit the metal ion induced oxidation. The composition of tocopherols was the same among gut contents, gut tissues, and the other tissues. However, plasmalogens, a unique class of glycerophospholipids rich in polyunsaturated fatty acids and containing a vinyl ether bond at the sn-1 position, were mainly distributed in gut tissues. Plasmalogens might protect gut tissues from oxidation stress. Copyright © 2012 Elsevier Ltd. All rights reserved.

Correlation between fibroin amino acid sequence and physical silk properties.

PubMed

Fedic, Robert; Zurovec, Michal; Sehnal, Frantisek

2003-09-12

The fiber properties of lepidopteran silk depend on the amino acid repeats that interact during H-fibroin polymerization. The aim of our research was to relate repeat composition to insect biology and fiber strength. Representative regions of the H-fibroin genes were sequenced and analyzed in three pyralid species: wax moth (Galleria mellonella), European flour moth (Ephestia kuehniella), and Indian meal moth (Plodia interpunctella). The amino acid repeats are species-specific, evidently a diversification of an ancestral region of 43 residues, and include three types of regularly dispersed motifs: modifications of GSSAASAA sequence, stretches of tripeptides GXZ where X and Z represent bulky residues, and sequences similar to PVIVIEE. No concatenations of GX dipeptide or alanine, which are typical for Bombyx silkworms and Antheraea silk moths, respectively, were found. Despite different repeat structure, the silks of G. mellonella and E. kuehniella exhibit similar tensile strength as the Bombyx and Antheraea silks. We suggest that in these latter two species, variations in the repeat length obstruct repeat alignment, but sufficiently long stretches of iterated residues get superposed to interact. In the pyralid H-fibroins, interactions of the widely separated and diverse motifs depend on the precision of repeat matching; silk is strong in G. mellonella and E. kuehniella, with 2-3 types of long homogeneous repeats, and nearly 10 times weaker in P. interpunctella, with seven types of shorter erratic repeats. The high proportion of large amino acids in the H-fibroin of pyralids has probably evolved in connection with the spinning habit of caterpillars that live in protective silk tubes and spin continuously, enlarging the tubes on one end and partly devouring the other one. The silk serves as a depot of energetically rich and essential amino acids that may be scarce in the diet.

Genome-Wide Identification, Characterization and Expression Analysis of the Solute Carrier 6 Gene Family in Silkworm (Bombyx mori)

PubMed Central

Tang, Xin; Liu, Huawei; Chen, Quanmei; Wang, Xin; Xiong, Ying; Zhao, Ping

2016-01-01

The solute carrier 6 (SLC6) gene family, initially known as the neurotransmitter transporters, plays vital roles in the regulation of neurotransmitter signaling, nutrient absorption and motor behavior. In this study, a total of 16 candidate genes were identified as SLC6 family gene homologs in the silkworm (Bombyx mori) genome. Spatio-temporal expression patterns of silkworm SLC6 gene transcripts indicated that these genes were highly and specifically expressed in midgut, brain and gonads; moreover, these genes were expressed primarily at the feeding stage or adult stage. Levels of expression for most midgut-specific and midgut-enriched gene transcripts were down-regulated after starvation but up-regulated after re-feeding. In addition, we observed that expression levels of these genes except for BmSLC6-15 and BmGT1 were markedly up-regulated by a juvenile hormone analog. Moreover, brain-enriched genes showed differential expression patterns during wandering and mating processes, suggesting that these genes may be involved in modulating wandering and mating behaviors. Our results improve our understanding of the expression patterns and potential physiological functions of the SLC6 gene family, and provide valuable information for the comprehensive functional analysis of the SLC6 gene family. PMID:27706106

Genome-Wide Identification, Characterization and Expression Analysis of the Solute Carrier 6 Gene Family in Silkworm (Bombyx mori).

PubMed

Tang, Xin; Liu, Huawei; Chen, Quanmei; Wang, Xin; Xiong, Ying; Zhao, Ping

2016-10-03

The solute carrier 6 (SLC6) gene family, initially known as the neurotransmitter transporters, plays vital roles in the regulation of neurotransmitter signaling, nutrient absorption and motor behavior. In this study, a total of 16 candidate genes were identified as SLC6 family gene homologs in the silkworm (Bombyx mori) genome. Spatio-temporal expression patterns of silkworm SLC6 gene transcripts indicated that these genes were highly and specifically expressed in midgut, brain and gonads; moreover, these genes were expressed primarily at the feeding stage or adult stage. Levels of expression for most midgut-specific and midgut-enriched gene transcripts were down-regulated after starvation but up-regulated after re-feeding. In addition, we observed that expression levels of these genes except for BmSLC6-15 and BmGT1 were markedly up-regulated by a juvenile hormone analog. Moreover, brain-enriched genes showed differential expression patterns during wandering and mating processes, suggesting that these genes may be involved in modulating wandering and mating behaviors. Our results improve our understanding of the expression patterns and potential physiological functions of the SLC6 gene family, and provide valuable information for the comprehensive functional analysis of the SLC6 gene family.

Directing osteogenesis of stem cells with hydroxyapatite precipitated electrospun eri-tasar silk fibroin nanofibrous scaffold.

PubMed

Panda, N; Bissoyi, A; Pramanik, K; Biswas, A

2014-01-01

Stimulating stem cell differentiation without growth factor supplement offers a potent and cost-effective scaffold for tissue regeneration. We hypothesise that surface precipitation of nano-hydroxyapatite (nHAp) over blends of non-mulberry silk fibroin with better hydrophilicity and RGD amino acid sequences can direct the stem cell towards osteogenesis. This report focuses on the fabrication of a blended eri-tasar silk fibroin nanofibrous scaffold (ET) followed by nHAp deposition by a surface precipitation (alternate soaking in calcium and phosphate solution) method. Morphology, hydrophilicity, composition, and the thermal and mechanical properties of ET/nHAp were examined by field emission scanning electron microscopy, TEM, FT-IR, X-ray diffraction, TGA and contact angle measurement and compared with ET. The composite scaffold demonstrated improved thermal stability and surface hydrophilicity with an increase in stiffness and elastic modulus (778 ± 2.4 N/m and 13.1 ± 0.36 MPa) as compared to ET (160.6 ± 1.34 N/m and 8.3 ± 0.4 MPa). Mineralisation studies revealed an enhanced and more uniform surface deposition of HAp-like crystals, while significant differences in cellular viability and attachment were observed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and confocal microscopy study. The cell viability and expression of adhesion molecules (CD 44 and CD 29) are found to be optimum for subsequent stages of growth proliferation and differentiation. The rates of proliferation have been observed to decrease owing to the transition of MSC from a state of proliferation to a state of differentiation. The confirmation of improved osteogenic differentiation was finally verified through the alkaline phosphatase assay, pattern of gene expression related to osteogenic differentiation and morphological observations of differentiated cord blood human mesenchymal stem cells under fluorescence microscope. The results

Developing an Onboard Traffic-Aware Flight Optimization Capability for Near-Term Low-Cost Implementation

NASA Technical Reports Server (NTRS)

Wing, David J.; Ballin, Mark G.; Koczo, Stefan, Jr.; Vivona, Robert A.; Henderson, Jeffrey M.

2013-01-01

The concept of Traffic Aware Strategic Aircrew Requests (TASAR) combines Automatic Dependent Surveillance Broadcast (ADS-B) IN and airborne automation to enable user-optimal in-flight trajectory replanning and to increase the likelihood of Air Traffic Control (ATC) approval for the resulting trajectory change request. TASAR is designed as a near-term application to improve flight efficiency or other user-desired attributes of the flight while not impacting and potentially benefiting ATC. Previous work has indicated the potential for significant benefits for each TASAR-equipped aircraft. This paper will discuss the approach to minimizing TASAR's cost for implementation and accelerating readiness for near-term implementation.

Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

PubMed

Xxxx, Patmawati; Minamihata, Kosuke; Tatsuke, Tsuneyuki; Lee, Jae Man; Kusakabe, Takahiro; Kamiya, Noriho

2018-06-01

Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure of Bombyx mori densovirus 1, a silkworm pathogen.

PubMed

Kaufmann, Bärbel; El-Far, Mohamed; Plevka, Pavel; Bowman, Valorie D; Li, Yi; Tijssen, Peter; Rossmann, Michael G

2011-05-01

Bombyx mori densovirus 1 (BmDNV-1), a major pathogen of silkworms, causes significant losses to the silk industry. The structure of the recombinant BmDNV-1 virus-like particle has been determined at 3.1-Å resolution using X-ray crystallography. It is the first near-atomic-resolution structure of a virus-like particle within the genus Iteravirus. The particles consist of 60 copies of the 55-kDa VP3 coat protein. The capsid protein has a β-barrel "jelly roll" fold similar to that found in many diverse icosahedral viruses, including archaeal, bacterial, plant, and animal viruses, as well as other parvoviruses. Most of the surface loops have little structural resemblance to other known parvovirus capsid proteins. In contrast to vertebrate parvoviruses, the N-terminal β-strand of BmDNV-1 VP3 is positioned relative to the neighboring 2-fold related subunit in a "domain-swapped" conformation, similar to findings for other invertebrate parvoviruses, suggesting domain swapping is an evolutionarily conserved structural feature of the Densovirinae.

Novel tryptophan metabolic pathways in auxin biosynthesis in silkworm.

PubMed

Yokoyama, Chiaki; Takei, Mami; Kouzuma, Yoshiaki; Nagata, Shinji; Suzuki, Yoshihito

2017-08-01

In the course of our study of the biosynthetic pathway of auxin, a class of phytohormones, in insects, we proposed the biosynthetic pathway tryptophan (Trp)→indole-3-acetaldoxime (IAOx)→indole-3-acetadehyde (IAAld)→indole-3-acetic acid (IAA). In this study, we identified two branches in the metabolic pathways in the silkworm, possibly affecting the efficiency of IAA production: Trp→indole-3-pyruvic acid→indole-3-lactic acid and IAAld→indole-3-ethanol. We also determined the apparent conversion activities (2.05×10 -7 UmL -1 for Trp→IAA, 1.30×10 -5 UmL -1 for IAOx→IAA, and 3.91×10 -1 UmL -1 for IAAld→IAA), which explain why IAOx and IAAld are barely detectable as either endogenous compounds or metabolites of their precursors. The failure to detect IAAld, even in the presence of an inhibitor of the conversion IAAld→IAA, is explained by a switch in the conversion from IAAld→IAA to IAAld→IEtOH. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inhibitors of Eicosanoid Biosynthesis Influencing the Transcripts Level of sHSP21.4 Gene Induced by Pathogen Infections, in Antheraea pernyi

PubMed Central

Zhang, Congfen; Dai, Lishang; Wang, Lei; Qian, Cen; Wei, Guoqing; Li, Jun; Zhu, Baojian; Liu, Chaoliang

2015-01-01

Small heat shock proteins (sHSPs) can regulate protein folding and protect cells from stress. To investigate the role of sHSPs in the silk-producing insect Antheraea pernyi response to microorganisms, a sHsp gene termed as Ap-sHSP21.4, was identified. This gene encoded a 21.4 kDa protein which shares the conserved structure of insect sHsps and belongs to sHSP21.4 family. Ap-sHSP21.4 was highly expressed in fat body and up-regulated in midgut and fat body of A. pernyi challenged with Escherichia coli, Beauveria bassiana and nuclear polyhedrosis virus (NPV), which was determined by quantitative real-time PCR. Meanwhile, knock down of Ap-sHSP21.4 with dsRNA result in the decrease at the expression levels of several immune response-related genes (defensin, Dopa decarboxylase, Toll1, lysozyme and Kazal-type serine protease inhibitor). Additionally, the impact of eicosanoid biosynthesis on the expression of Ap-sHSP21.4 response to NPV was determined using qPCR, inhibitors of eicosanoid biosynthesis significantly suppress Ap-HSP21.4 expression upon NPV challenge. All together, Ap-sHSP21.4 was involved in the immunity of A. pernyi against microorganism and possibly mediated by eicosanoids pathway. These results will shed light in the understanding of the pathogen-host interaction in A. pernyi. PMID:25844646

Ecdysone has an effect on the regeneration of midgut epithelial cells that is distinct from 20-hydroxyecdysone in the silkworm Bombyx mori.

PubMed

Tanaka, Y; Yukuhiro, F

1999-12-01

We investigated the effects of two ecdysteroids, ecdysone (E) and 20-hydroxyecdysone (20E), on silkworm larval development. Silkworm larvae, Bombyx mori, were fed an artificial diet supplemented with 20E during the fourth instar to promote premature molting. At the onset of the fifth instar, these precocious fifth-instar larvae were fed diets supplemented with either E or 20E to determine the effects of the two ecdysteroids on the morphology of midgut epithelial cells. Regeneration of midgut epithelial cells normally occurs only during the molting period. However, in larvae fed E, complete replacement of midgut epithelial cells was observed 24 h before the larvae entered apolysis. In larvae fed 20E, the morphology of midgut epithelial cells was disrupted, leading to death of the larvae during the fifth instar. We also observed similar differences in the effects of the two ecdysteroids in an in vitro experiment. These results suggest that E has a specific effect on the morphological change of midgut epithelial cells in precocious fifth-instar larvae that is distinct from 20E. Copyright 1999 Academic Press.

Identification of a functional element in the promoter of the silkworm (Bombyx mori) fat body-specific gene Bmlp3.

PubMed

Xu, Hanfu; Deng, Dangjun; Yuan, Lin; Wang, Yuancheng; Wang, Feng; Xia, Qingyou

2014-08-01

30K proteins are a group of structurally related proteins that play important roles in the life cycle of the silkworm Bombyx mori and are largely synthesized and regulated in a time-dependent manner in the fat body. Little is known about the upstream regulatory elements associated with the genes encoding these proteins. In the present study, the promoter of Bmlp3, a fat body-specific gene encoding a 30K protein family member, was characterized by joining sequences containing the Bmlp3 promoter with various amounts of 5' upstream sequences to a luciferase reporter gene. The results indicated that the sequences from -150 to -250bp and -597 to -675bp upstream of the Bmlp3 transcription start site were necessary for high levels of luciferase activity. Further analysis showed that a 21-bp sequence located between -230 and -250 was specifically recognized by nuclear factors from silkworm fat bodies and BmE cells, and could enhance luciferase reporter-gene expression 2.8-fold in BmE cells. This study provides new insights into the Bmlp3 promoter and contributes to the further clarification of the function and developmental regulation of Bmlp3. Copyright © 2014. Published by Elsevier B.V.

Starvation-responsive glycine-rich protein gene in the silkworm Bombyx mori.

PubMed

Taniai, Kiyoko; Hirayama, Chikara; Mita, Kazuei; Asaoka, Kiyoshi

2014-10-01

Four glycine-rich protein (GRP) genes were identified from expressed sequence tags of the maxillary galea of the silkworm. All four genes were expressed in the maxillary pulp, antenna, labrum, and labium, but none of the genes were expressed in most internal organs. Expression of one of the genes, termed bmSIGRP, was further increased approximately fivefold in the mouth region (including the maxilla, antenna, labrum, labium, and mandible) after 24 h of starvation. bmSIGRP expression peaked at 24 h and gradually declined during the subsequent 2 days. When a synthetic diet not containing proteins was fed, bmSIGRP expression increased significantly in the mouth region to levels similar to that observed in starved larvae. Synthetic diets that lacked vitamins or salts but contained amino acids did not significantly affect bmSIGRP expression. These results suggest that amino acid depletion increases bmSIGRP expression.

Downregulation of aldose reductase is responsible for developmental abnormalities of the silkworm purple quail-like mutant (q-lp).

PubMed

Wang, Pingyang; Bi, Simin; Wei, Weiyang; Qiu, Zhiyong; Xia, Dingguo; Shen, Xingjia; Zhao, Qiaoling

2018-05-03

Aldose reductase (AR) is a rate-limiting enzyme in the polyol pathway and is also the key enzyme involved in diabetic complications. The silkworm purple quail-like mutant (q-l p ) exhibits pigmented dots on its epidermis. The q-l p mutant also shows developmental abnormalities and decreased vitality. In this study, fat bodies from the q-l p mutant and the wildtype 932VR strain were subjected to two-dimensional gel electrophoresis (2-DE) analysis, and the Bombyx mori AR (BmAR) protein was found to be significantly downregulated in the q-l p mutant. The expression of BmAR at the mRNA level was also significantly downregulated, as verified through quantitative reverse transcription PCR (qRT-PCR). Knockdown of the expression of BmAR via RNAi resulted in a reduction of silkworm weight. The sorbitol level in q-l p was significantly lower than in the wildtype. These results suggested that the BmAR gene is closely related to the development of the q-l p mutant. Investigation of the cause of BmAR downregulation in the q-l p mutant could contribute to revealing the function of AR in insects and offers a new method of identifying AR inhibitors for the treatment of diabetic complications. Copyright © 2017. Published by Elsevier B.V.

A novel angiotensin-І converting enzyme (ACE) inhibitory peptide from gastrointestinal protease hydrolysate of silkworm pupa (Bombyx mori) protein: Biochemical characterization and molecular docking study.

PubMed

Wu, Qiongying; Jia, Junqiang; Yan, Hui; Du, Jinjuan; Gui, Zhongzheng

2015-06-01

Silkworm pupa (Bombyx mori) protein was hydrolyzed using gastrointestinal endopeptidases (pepsin, trypsin and α-chymotrypsin). Then, the hydrolysate was purified sequentially by ultrafiltration, gel filtration chromatography and RP-HPLC. A novel ACE inhibitory peptide, Ala-Ser-Leu, with the IC50 value of 102.15μM, was identified by IT-MS/MS. This is the first report of Ala-Ser-Leu from natural protein. Lineweaver-Burk plots suggest that the peptide is a competitive inhibitor against ACE. The molecular docking studies revealed that the ACE inhibition of Ala-Ser-Leu is mainly attributed to forming very strong hydrogen bonds with the S1 pocket (Ala354) and the S2 pocket (Gln281 and His353). The results indicate that silkworm pupa (B. mori) protein or its gastrointestinal protease hydrolysate could be used as a functional ingredient in auxiliary therapeutic foods against hypertension. Copyright © 2014 Elsevier Inc. All rights reserved.

Stage-dependent and temperature-controlled expression of the gene encoding the precursor protein of diapause hormone and pheromone biosynthesis activating neuropeptide in the silkworm, Bombyx mori.

PubMed

Xu, W H; Sato, Y; Ikeda, M; Yamashita, O

1995-02-24

Embryonic diapause and sex pheromone biosynthesis in the silkworm, Bombyx mori, are, respectively, induced by diapause hormone (DH) and pheromone biosynthesis activating neuropeptide (PBAN), which are produced in the subesophageal ganglion from a common polyprotein precursor (DH-PBAN precursor) encoded by a single gene (DH-PBAN gene). Using DH-PBAN cDNA as a probe, we quantitatively measured DH-PBAN mRNA content throughout embryonic and postembryonic development and observed the effects of incubation temperature, which is a key factor for determination of diapause, on DH-PBAN gene expression. The silkworm, which is programmed to lay diapause eggs by being incubated at 25 degrees C, showed peaks of DH-PBAN mRNA content at five different stages throughout the life cycle: at the late embryonic stage, at the middle of the fourth and the fifth larval instars, and at early and late stages of pupal-adult development. In the non-diapause type silkworms programmed by a 15 degrees C incubation, only the last peak of DH-PBAN mRNA in pupal-adult development was found, and the other peaks were absent. Furthermore, interruption of the incubation period at 25 degrees C by incubation at 15 degrees C decreased both DH-PBAN mRNA content in mature embryos and in subesophageal ganglia of day 3 pupae and the incidence of diapause eggs. Thus, there were two types of regulatory mechanisms for DH-PBAN gene expression. One is a temperature-controlled expression that is responsible for diapause induction, and the other is a temperature-independent, stage-dependent expression related to pheromone production.

Structure of Bombyx mori Densovirus 1, a Silkworm Pathogen▿‡

PubMed Central

Kaufmann, Bärbel; El-Far, Mohamed; Plevka, Pavel; Bowman, Valorie D.; Li, Yi; Tijssen, Peter; Rossmann, Michael G.

2011-01-01

Bombyx mori densovirus 1 (BmDNV-1), a major pathogen of silkworms, causes significant losses to the silk industry. The structure of the recombinant BmDNV-1 virus-like particle has been determined at 3.1-Å resolution using X-ray crystallography. It is the first near-atomic-resolution structure of a virus-like particle within the genus Iteravirus. The particles consist of 60 copies of the 55-kDa VP3 coat protein. The capsid protein has a β-barrel “jelly roll” fold similar to that found in many diverse icosahedral viruses, including archaeal, bacterial, plant, and animal viruses, as well as other parvoviruses. Most of the surface loops have little structural resemblance to other known parvovirus capsid proteins. In contrast to vertebrate parvoviruses, the N-terminal β-strand of BmDNV-1 VP3 is positioned relative to the neighboring 2-fold related subunit in a “domain-swapped” conformation, similar to findings for other invertebrate parvoviruses, suggesting domain swapping is an evolutionarily conserved structural feature of the Densovirinae. PMID:21367906

Bombyx mori E26 transformation-specific 2 (BmEts2), an Ets family protein, represses Bombyx mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in the silkworm Bombyx mori.

PubMed

Tanaka, H; Sagisaka, A; Suzuki, N; Yamakawa, M

2016-10-01

E26 transformation-specific (Ets) family transcription factors are known to play roles in various biological phenomena, including immunity, in vertebrates. However, the mechanisms by which Ets proteins contribute to immunity in invertebrates remain poorly understood. In this study, we identified a cDNA encoding BmEts2, which is a putative orthologue of Drosophila Yan and human translocation-ets-leukemia/Ets-variant gene 6, from the silkworm Bombyx mori. Expression of the BmEts2 gene was significantly increased in the fat bodies of silkworm larvae in response to injection with Escherichia coli and Staphylococcus aureus. BmEts2 overexpression dramatically repressed B. mori Rels (BmRels)-mediated promoter activation of antimicrobial peptide genes in silkworm cells. Conversely, gene knockdown of BmEts2 significantly enhanced BmRels activity. In addition, two κB sites located on the 5' upstream region of cecropin B1 were found to be involved in the repression of BmRels-mediated promoter activation. Protein-competition analysis further demonstrated that BmEts2 competitively inhibited binding of BmRels to κB sites. Overall, BmEts2 acts as a repressor of BmRels-mediated transactivation of antimicrobial protein genes by inhibiting the binding of BmRels to κB sites. © 2016 The Royal Entomological Society.

Horizontal gene transfer in silkworm, Bombyx mori.

PubMed

Zhu, Bo; Lou, Miao-Miao; Xie, Guan-Lin; Zhang, Guo-Qing; Zhou, Xue-Ping; Li, Bin; Jin, Gu-Lei

2011-05-19

The domesticated silkworm, Bombyx mori, is the model insect for the order Lepidoptera, has economically important values, and has gained some representative behavioral characteristics compared to its wild ancestor. The genome of B. mori has been fully sequenced while function analysis of BmChi-h and BmSuc1 genes revealed that horizontal gene transfer (HGT) maybe bestow a clear selective advantage to B. mori. However, the role of HGT in the evolutionary history of B. mori is largely unexplored. In this study, we compare the whole genome of B. mori with those of 382 prokaryotic and eukaryotic species to investigate the potential HGTs. Ten candidate HGT events were defined in B. mori by comprehensive sequence analysis using Maximum Likelihood and Bayesian method combining with EST checking. Phylogenetic analysis of the candidate HGT genes suggested that one HGT was plant-to- B. mori transfer while nine were bacteria-to- B. mori transfer. Furthermore, functional analysis based on expression, coexpression and related literature searching revealed that several HGT candidate genes have added important characters, such as resistance to pathogen, to B. mori. Results from this study clearly demonstrated that HGTs play an important role in the evolution of B. mori although the number of HGT events in B. mori is in general smaller than those of microbes and other insects. In particular, interdomain HGTs in B. mori may give rise to functional, persistent, and possibly evolutionarily significant new genes.

Horizontal gene transfer in silkworm, Bombyx mori

PubMed Central

2011-01-01

Background The domesticated silkworm, Bombyx mori, is the model insect for the order Lepidoptera, has economically important values, and has gained some representative behavioral characteristics compared to its wild ancestor. The genome of B. mori has been fully sequenced while function analysis of BmChi-h and BmSuc1 genes revealed that horizontal gene transfer (HGT) maybe bestow a clear selective advantage to B. mori. However, the role of HGT in the evolutionary history of B. mori is largely unexplored. In this study, we compare the whole genome of B. mori with those of 382 prokaryotic and eukaryotic species to investigate the potential HGTs. Results Ten candidate HGT events were defined in B. mori by comprehensive sequence analysis using Maximum Likelihood and Bayesian method combining with EST checking. Phylogenetic analysis of the candidate HGT genes suggested that one HGT was plant-to- B. mori transfer while nine were bacteria-to- B. mori transfer. Furthermore, functional analysis based on expression, coexpression and related literature searching revealed that several HGT candidate genes have added important characters, such as resistance to pathogen, to B. mori. Conclusions Results from this study clearly demonstrated that HGTs play an important role in the evolution of B. mori although the number of HGT events in B. mori is in general smaller than those of microbes and other insects. In particular, interdomain HGTs in B. mori may give rise to functional, persistent, and possibly evolutionarily significant new genes. PMID:21595916

Mechanical features of various silkworm crystalline considering hydration effect via molecular dynamics simulations.

PubMed

Kim, Yoonjung; Lee, Myeongsang; Choi, Hyunsung; Baek, Inchul; Kim, Jae In; Na, Sungsoo

2018-04-01

Silk materials are receiving significant attention as base materials for various functional nanomaterials and nanodevices, due to its exceptionally high mechanical properties, biocompatibility, and degradable characteristics. Although crystalline silk regions are composed of various repetitive motifs with differing amino acid sequences, how the effect of humidity works differently on each of the motifs and their structural characteristics remains unclear. We report molecular dynamics (MD) simulations on various silkworm fibroins composed of major motifs (i.e. (GAGAGS) n , (GAGAGA) n , and (GAGAGY) n ) at varying degrees of hydration, and reveal how each major motifs of silk fibroins change at each degrees of hydration using MD simulations and their structural properties in mechanical perspective via steered molecular dynamics simulations. Our results explain what effects humidity can have on nanoscale materials and devices consisting of crystalline silk materials.

Molecular Characterization and Functional Analysis of a Ferritin Heavy Chain Subunit from the Eri-Silkworm, Samia cynthia ricini

PubMed Central

Yu, Hai-Zhong; Zhang, Shang-Zhi; Ma, Yan; Fei, Dong-Qiong; Li, Bing; Yang, Li-Ang; Wang, Jie; Li, Zhen; Muhammad, Azharuddin; Xu, Jia-Ping

2017-01-01

Ferritins are conserved iron-binding proteins that are primarily involved in iron storage, detoxification and the immune response. Despite the importance of ferritin in organisms, little is known about their roles in the eri-silkworm (Samia cynthia ricini). We previously identified a ferritin heavy chain subunit named ScFerHCH in the S. c. ricini transcriptome database. The full-length S. c. ricini ferritin heavy chain subunit (ScFerHCH) was 1863 bp and encoded a protein of 231 amino acids with a deduced molecular weight of 25.89 kDa. Phylogenetic analysis revealed that ScFerHCH shared a high amino acid identity with the Bombyx mori and Danaus plexippus heavy chain subunits. Higher ScFerHCH expression levels were found in the silk gland, fat body and midgut of S. c. ricini by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Injection of Staphylococcus aureus and Pseudomonas aeruginosa was associated with an upregulation of ScFerHCH in the midgut, fat body and hemolymph, indicating that ScFerHCH may contribute to the host’s defense against invading pathogens. In addition, the anti-oxidation activity and iron-binding capacity of recombinant ScFerHCH protein were examined. Taken together, our results suggest that the ferritin heavy chain subunit from eri-silkworm may play critical roles not only in innate immune defense, but also in organismic iron homeostasis. PMID:29036914

Two Adenine Nucleotide Translocase Paralogues Involved in Cell Proliferation and Spermatogenesis in the Silkworm Bombyx mori

PubMed Central

Sugahara, Ryohei; Jouraku, Akiya; Nakakura, Takayo; Kusakabe, Takahiro; Yamamoto, Takenori; Shinohara, Yasuo; Miyoshi, Hideto; Shiotsuki, Takahiro

2015-01-01

Mitochondrial adenine nucleotide translocase (ANT) specifically acts in ADP/ATP exchange through the mitochondrial inner membrane. This transporter protein thereby plays a significant role in energy metabolism in eukaryotic cells. Most mammals have four paralogous ANT genes (ANT1-4) and utilize these paralogues in different types of cells. The fourth paralogue of ANT (ANT4) is present only in mammals and reptiles and is exclusively expressed in testicular germ cells where it is required for meiotic progression in the spermatocytes. Here, we report that silkworms harbor two ANT paralogues, the homeostatic paralogue (BmANTI1) and the testis-specific paralogue (BmANTI2). The BmANTI2 protein has an N-terminal extension in which the positions of lysine residues in the amino acid sequence are distributed as in human ANT4. An expression analysis showed that BmANTI2 transcripts were restricted to the testis, suggesting the protein has a role in the progression of spermatogenesis. By contrast, BmANTI1 was expressed in all tissues tested, suggesting it has an important role in homeostasis. We also observed that cultured silkworm cells required BmANTI1 for proliferation. The ANTI1 protein of the lepidopteran Plutella xylostella (PxANTI1), but not those of other insect species (or PxANTI2), restored cell proliferation in BmANTI1-knockdown cells suggesting that ANTI1 has similar energy metabolism functions across the Lepidoptera. Our results suggest that BmANTI2 is evolutionarily divergent from BmANTI1 and has developed a specific role in spermatogenesis similar to that of mammalian ANT4. PMID:25742135

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

PubMed Central

Patnaik, Bharat Bhusan; Kim, Dong Hyun; Oh, Seung Han; Song, Yong-Su; Chanh, Nguyen Dang Minh; Kim, Jong Sun; Jung, Woo-jin; Saha, Atul Kumar; Bindroo, Bharat Bhushan; Han, Yeon Soo

2012-01-01

Background Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens. Methodology/Principal Findings Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense. Conclusions/Significance The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found

Isolation, Purification, and Identification of an Important Pigment, Sepiapterin, from Integument of the lemon Mutant of the Silkworm, Bombyx mori

PubMed Central

Wang, Jing; Wang, Wenjing; Liu, Chaoliang; Meng, Yan

2013-01-01

Sepiapterin is the precursor of tetrahydrobiopterin, an important coenzyme of aromatic amino acid hydroxylases, the lack of which leads to a variety of physiological metabolic diseases or neurological syndromes in humans. Sepiapterin is a main pigment component in the integument of the lemon mutant of the silkworm, Bombyx mori (L.) (Lepidoptera: Bombycidae), and is present there in extremely high content, so lemon is a valuable genetic resource to extract sepiapterin. In this study, an effective experimental system was set up for isolation and purification of sepiapterin from lemon silkworms by optimizing homogenization solvent, elution buffer, and separation chromatographic column. The results showed that ethanol was the most suitable solvent to homogenize the integument, with a concentration of 50% and solid:liquid ratio of 1:20 (g/mL). Sepiapterin was purified successively by column chromatography of cellulose Ecteola, sephadex G-25-150, and cellulose phosphate, and was identified by ultraviolet-visible absorption spectrometry. A stable and accurate high performance liquid chromatography method was constructed to identify sepiapterin and conduct qualitative and quantitative analyses. Sepiapterin of high purity was achieved, and the harvest reached about 40 ug/g of integument in the experiments. This work helps to obtaining natural sepiapterin in large amounts in order to use the lemon B. mori mutant to produce BH4 in vitro. PMID:24773269

Carbohydrate metabolism during starvation in the silkworm Bombyx mori.

PubMed

Satake, S; Kawabe, Y; Mizoguchi, A

2000-06-01

The effect of starvation on carbohydrate metabolism in the last instar larvae of the silkworm Bombyx mori was examined. Trehalose concentration in the hemolymph increased slightly during the first 6 h of starvation and decreased thereafter, whereas glucose concentration decreased rapidly immediately after diet deprivation. Starvation-induced hypertrehalosemia was completely inhibited by neck ligation, suggesting that starvation stimulates the release of a hypertrehalosemic factor(s) from the head. The percentage of active glycogen phosphorylase in the fat body increased within 3 h of starvation and its glycogen content decreased gradually. These observations suggest that production of trehalose from glycogen is enhanced in starved larvae. However, hypertrehalosemia during starvation cannot be explained by the increased supply of trehalose into hemolymph alone, as similar changes in phosphorylase activity and glycogen content in the fat body were observed in neck-ligated larvae, in which hemolymph trehalose concentration did not increase but decreased gradually. When injected into larvae, trehalose disappeared from hemolymph at a rate about 40% lower in starved larvae than neck-ligated larvae. The hemolymph lipid concentration increased during starvation, suggesting that an increased supply of lipids to tissues suppresses the consumption of hemolymph trehalose and this is an important factor in hypertrehalosemia. Copyright 2000 Wiley-Liss, Inc.

Two hemocyte lineages exist in silkworm larval hematopoietic organ.

PubMed

Nakahara, Yuichi; Kanamori, Yasushi; Kiuchi, Makoto; Kamimura, Manabu

2010-07-28

Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster. To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids. From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori.

APA-style human milk fat analogue from silkworm pupae oil: Enzymatic production and improving storage stability using alkyl caffeates.

PubMed

Liu, Xi; Wang, Xudong; Pang, Na; Zhu, Weijie; Zhao, Xingyu; Wang, Fangqin; Wu, Fuan; Wang, Jun

2015-12-08

Silkworm pupae oil derived from reeling waste is a rich source of α-linolenic acid (ALA), which has multipal applications. ALAs were added in sn-1, 3 positions in a triacylglycerol (TAG) to produce an APA-human milk fat analogues (APA-HMFAs, A: α-linolenic acid, P: palmitic acid). The optimum condition is that tripalmitin to free fatty acids of 1:12 (mole ratio) at 65 °C for 48 h using lipase Lipozyme RM IM. Results show that, the major TAG species that comprised APA-HMFAs were rich in ALA and palmitic acid, which contained 64.52% total unsaturated fatty acids (UFAs) and 97.05% PA at the sn-2 position. The melting point of APA was -27.5 °C which is much lower than tripalmitin (40.5 °C) indicating more plastic character. In addition, the practical application of alkyl caffeates as liposoluble antioxidants in APA was developed. Alkyl caffeate showed a superior IC50 (1.25-1.66 μg/mL) compared to butyl hydroxy anisd (1.67 μg/mL) and L-ascorbic acid-6-palmitate (L-AP) (1.87 μg/mL) in DPPH analysis. The addition of ethyl caffeate to oil achieved a higher UFAs content (73.58%) at high temperatures. Overall, APA was obtained from silkworm pupae oil successfully, and the addition of caffeates extended storage ranges for APA-HMFAs.

APA-style human milk fat analogue from silkworm pupae oil: Enzymatic production and improving storage stability using alkyl caffeates

PubMed Central

Liu, Xi; Wang, Xudong; Pang, Na; Zhu, Weijie; Zhao, Xingyu; Wang, Fangqin; Wu, Fuan; Wang, Jun

2015-01-01

Silkworm pupae oil derived from reeling waste is a rich source of α-linolenic acid (ALA), which has multipal applications. ALAs were added in sn-1, 3 positions in a triacylglycerol (TAG) to produce an APA-human milk fat analogues (APA-HMFAs, A: α-linolenic acid, P: palmitic acid). The optimum condition is that tripalmitin to free fatty acids of 1:12 (mole ratio) at 65 °C for 48 h using lipase Lipozyme RM IM. Results show that, the major TAG species that comprised APA-HMFAs were rich in ALA and palmitic acid, which contained 64.52% total unsaturated fatty acids (UFAs) and 97.05% PA at the sn-2 position. The melting point of APA was −27.5 °C which is much lower than tripalmitin (40.5 °C) indicating more plastic character. In addition, the practical application of alkyl caffeates as liposoluble antioxidants in APA was developed. Alkyl caffeate showed a superior IC50 (1.25–1.66 μg/mL) compared to butyl hydroxy anisd (1.67 μg/mL) and L-ascorbic acid-6-palmitate (L-AP) (1.87 μg/mL) in DPPH analysis. The addition of ethyl caffeate to oil achieved a higher UFAs content (73.58%) at high temperatures. Overall, APA was obtained from silkworm pupae oil successfully, and the addition of caffeates extended storage ranges for APA-HMFAs. PMID:26643045

Robust composite silk fibers pulled out of silkworms directly fed with nanoparticles.

PubMed

Wu, GuoHua; Song, Peng; Zhang, DongYang; Liu, ZeYu; Li, Long; Huang, HuiMing; Zhao, HongPing; Wang, NanNan; Zhu, YanQiu

2017-11-01

This paper reports the impacts of direct feeding silkworms with different nanoparticles (Cu, Fe, and TiO 2 ) on the morphology, structures, and mechanical properties of the resulting silk fiber (SF). The contents of the Cu nanoparticles were 38 times higher in the posterior silk glands and only 2-3 times higher in the SF and in the middle silk glands compared with the controlled groups. Significant changes of the surface morphology, structures, and diameter of the Cu nanoparticle fed SF have been observed, which are attributed to a slight SF protein reconstruction or conformational change in the mixture of silk fibroin and sericin in the silk glands. The resulting Cu-containing SF exhibits good tensile strength of 360MPa and reaches a strain of 38%, which are 89% and 36% higher than those of the natural SF. This study offers a new green strategy for the easy modification to achieve robust composite SF. Copyright © 2017 Elsevier B.V. All rights reserved.

Ligand binding turns moth pheromone-binding protein into a pH sensor: effect on the Antheraea polyphemus PBP1 conformation.

PubMed

Katre, Uma V; Mazumder, Suman; Prusti, Rabi K; Mohanty, Smita

2009-11-13

In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the "open" (bound) conformation at high pH but switches to the "closed" (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of approximately 1:1. Mutation of His(70) and His(95) to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.

Functional characterization of the vitellogenin promoter in the silkworm, Bombyx mori.

PubMed

Xu, J; Wang, Y Q; Li, Z Q; Ling, L; Zeng, B S; You, L; Chen, Y Z; Aslam, A F M; Huang, Y P; Tan, A J

2014-10-01

Genetic transformation and genome editing technologies have been successfully established in the lepidopteran insect model, the domesticated silkworm, Bombyx mori, providing great potential for functional genomics and practical applications. However, the current lack of cis-regulatory elements in B. mori gene manipulation research limits further exploitation in functional gene analysis. In the present study, we characterized a B. mori endogenous promoter, Bmvgp, which is a 798-bp DNA sequence adjacent to the 5'-end of the vitellogenin gene (Bmvg). PiggyBac-based transgenic analysis shows that Bmvgp precisely directs expression of a reporter gene, enhanced green fluorescent protein (EGFP), in a sex-, tissue- and stage-specific manner. In transgenic animals, EGFP expression can be detected in the female fat body from larval-pupal ecdysis to the following pupal and adult stage. Furthermore, in vitro and in vivo experiments revealed that EGFP expression can be activated by 20-hydroxyecdysone, which is consistent with endogenous Bmvg expression. These data indicate that Bmvgp is an effective endogenous cis-regulatory element in B. mori. © 2014 The Royal Entomological Society.

RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

PubMed

Singh, Chabungbam Orville; Xin, Hu-hu; Chen, Rui-ting; Wang, Mei-xian; Liang, Shuang; Lu, Yan; Cai, Zi-zheng; Zhang, Deng-pan; Miao, Yun-gen

2015-06-01

Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C-terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth-instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L. © 2015 Wiley Periodicals, Inc.

Oral immunization with Escherichia coli K-12 of the fifth instar larvae of the silkworm, Bombyx mori, reared on an artificial diet under completely aseptic conditions.

PubMed

Ichimori, H; Yuhki, T; Mori, H; Matsubara, F; Sumida, M

1992-01-01

1. Effect of oral administration of live or formalin-treated Escherichia coli (E. coli) K-12 to the fifth instar, days 1 and 3 larvae of the silkworm, Bombyx mori, on induction of antibacterial activity in the haemolymph was investigated using the silkworms reared on an artificial diet under completely aseptic conditions. 2. When live E. coli was administered to the male day 1 larvae, low but significant antibacterial activity of 3.8 mm was detectable in the haemolymph of one individual at 48 hr after immunization. The proportion of the larvae to express antibacterial activity increased thereafter and at 120 hr after immunization, all three individuals showed antibacterial activity. In day 3 male larvae, activity was detectable at 48 and 72 hr after immunization. 3. When formalin-treated E. coli was orally administered to days 1 and 3 male larvae, no activity was detectable at any time post-immunization. 4. In the second experiment, when day 1 larvae, females and males were orally immunized with live E. coli, only females showed antibacterial activity in the haemolymph, beginning from 24 hr after immunization and up to 96 hr. 5. Removal of an antibiotic, chloramphenicol, from ingredients of an artificial diet was required for induction of antibacterial activity with oral administration of live E. coli. 6. When live E. coli that grows at pH 9.0 was selected and used for oral immunization, antibacterial activity was induced both in females and males at 72 hr after immunization and the activity was observed at 96 hr. 7. These results suggest that establishment of oral immunization with live E. coli in the silkworm larvae requires multiplication of E. coli in the midgut lumen and possibly its colonization on the luminal surface.

The Silkworm (Bombyx mori) microRNAs and Their Expressions in Multiple Developmental Stages

PubMed Central

Luo, Qibin; Cai, Yimei; Lin, Wen-chang; Chen, Huan; Yang, Yue; Hu, Songnian; Yu, Jun

2008-01-01

Background MicroRNAs (miRNAs) play crucial roles in various physiological processes through post-transcriptional regulation of gene expressions and are involved in development, metabolism, and many other important molecular mechanisms and cellular processes. The Bombyx mori genome sequence provides opportunities for a thorough survey for miRNAs as well as comparative analyses with other sequenced insect species. Methodology/Principal Findings We identified 114 non-redundant conserved miRNAs and 148 novel putative miRNAs from the B. mori genome with an elaborate computational protocol. We also sequenced 6,720 clones from 14 developmental stage-specific small RNA libraries in which we identified 35 unique miRNAs containing 21 conserved miRNAs (including 17 predicted miRNAs) and 14 novel miRNAs (including 11 predicted novel miRNAs). Among the 114 conserved miRNAs, we found six pairs of clusters evolutionarily conserved cross insect lineages. Our observations on length heterogeneity at 5′ and/or 3′ ends of nine miRNAs between cloned and predicted sequences, and three mature forms deriving from the same arm of putative pre-miRNAs suggest a mechanism by which miRNAs gain new functions. Analyzing development-related miRNAs expression at 14 developmental stages based on clone-sampling and stem-loop RT PCR, we discovered an unusual abundance of 33 sequences representing 12 different miRNAs and sharply fluctuated expression of miRNAs at larva-molting stage. The potential functions of several stage-biased miRNAs were also analyzed in combination with predicted target genes and silkworm's phenotypic traits; our results indicated that miRNAs may play key regulatory roles in specific developmental stages in the silkworm, such as ecdysis. Conclusions/Significance Taking a combined approach, we identified 118 conserved miRNAs and 151 novel miRNA candidates from the B. mori genome sequence. Our expression analyses by sampling miRNAs and real-time PCR over multiple developmental

Preliminary Assessment of Operational Hazards and Safety Requirements for Airborne Trajectory Management (ABTM) Roadmap Applications

NASA Technical Reports Server (NTRS)

Cotton, William B.; Hilb, Robert; Koczo, Stefan, Jr.; Wing, David J.

2016-01-01

A set of five developmental steps building from the NASA TASAR (Traffic Aware Strategic Aircrew Requests) concept are described, each providing incrementally more efficiency and capacity benefits to airspace system users and service providers, culminating in a Full Airborne Trajectory Management capability. For each of these steps, the incremental Operational Hazards and Safety Requirements are identified for later use in future formal safety assessments intended to lead to certification and operational approval of the equipment and the associated procedures. Two established safety assessment methodologies that are compliant with the FAA's Safety Management System were used leading to Failure Effects Classifications (FEC) for each of the steps. The most likely FEC for the first three steps, Basic TASAR, Digital TASAR, and 4D TASAR, is "No effect". For step four, Strategic Airborne Trajectory Management, the likely FEC is "Minor". For Full Airborne Trajectory Management (Step 5), the most likely FEC is "Major".

RIP-seq of BmAgo2-associated small RNAs reveal various types of small non-coding RNAs in the silkworm, Bombyx mori

PubMed Central

2013-01-01

Background Small non-coding RNAs (ncRNAs) are important regulators of gene expression in eukaryotes. Previously, only microRNAs (miRNAs) and piRNAs have been identified in the silkworm, Bombyx mori. Furthermore, only ncRNAs (50-500nt) of intermediate size have been systematically identified in the silkworm. Results Here, we performed a systematic identification and analysis of small RNAs (18-50nt) associated with the Bombyx mori argonaute2 (BmAgo2) protein. Using RIP-seq, we identified various types of small ncRNAs associated with BmAGO2. These ncRNAs showed a multimodal length distribution, with three peaks at ~20nt, ~27nt and ~33nt, which included tRNA-, transposable element (TE)-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. The tRNA-derived fragments (tRFs) were found at an extremely high abundance and accounted for 69.90% of the BmAgo2-associated small RNAs. Northern blotting confirmed that many tRFs were expressed or up-regulated only in the BmNPV-infected cells, implying that the tRFs play a prominent role by binding to BmAgo2 during BmNPV infection. Additional evidence suggested that there are potential cleavage sites on the D, anti-codon and TψC loops of the tRNAs. TE-derived small RNAs and piRNAs also accounted for a significant proportion of the BmAgo2-associated small RNAs, suggesting that BmAgo2 could be involved in the maintenance of genome stability by suppressing the activities of transposons guided by these small RNAs. Finally, Northern blotting was also used to confirm the Bombyx 5.8 s rRNA-derived small RNAs, demonstrating that various novel small RNAs exist in the silkworm. Conclusions Using an RIP-seq method in combination with Northern blotting, we identified various types of small RNAs associated with the BmAgo2 protein, including tRNA-, TE-, rRNA-, snoRNA- and snRNA-derived small RNAs as well as miRNAs and piRNAs. Our findings provide new clues for future functional studies of the role of small RNAs in insect

Recycling of urea associated with the host plant urease in the silkworm larvae, Bombyx mori.

PubMed

Hirayama, C; Sugimura, M; Shinbo, H

1999-01-01

Urea concentration and urease activity in the midgut content were compared between larvae of the silkworm, Bombyx mori fed an artificial diet and those fed fresh mulberry leaves. A considerable amount of urea was found in the midgut content of the both larvae, however it was significantly lower in the larvae fed fresh mulberry leaves than in the larvae fed the artificial diet; average urea concentrations in the midgut content of the larvae fed fresh mulberry leaves and the artificial diet were 2.9 and 4.6 &mgr;mol/g, respectively. Urea in the midgut content seems to be secreted from the insect itself since the amount of urea in both diets were negligibly small. Urease activity was detected only in the midgut content of the larvae fed fresh mulberry leaves but not in other tissues of the larvae. On the other hand, no urease activity was detected in the midgut content of the larvae fed the artificial diet. Subsequently, to elucidate the role of mulberry leaf urease in the midgut lumen, larvae that had been reared on the artificial diet were switched to fresh mulberry leaves. The diet switch caused a rapid decrease in urea concentration in the midgut content and an increase in ammonia concentration in the midgut content, suggesting that secreted urea could be hydrolyzed to ammonia by mulberry leaf urease in the midgut lumen. Furthermore, to investigate the physiological significance of mulberry leaf urease on urea metabolism of the silkworm, (15)N-urea was injected into the hemocoel, and after 12 h the larvae were dissected for (15)N analysis. A considerable amount of (15)N was found to be incorporated into the silk-protein of the larvae fed fresh mulberry leaves, but there was little incorporation of (15)N into the silk-protein of the larvae fed the artificial diet. These data indicate that urea is converted into ammonia by the action of mulberry leaf urease in the midgut lumen and used as a nitrogen source in larvae fed mulberry leaves.

Partial deletions of the W chromosome due to reciprocal translocation in the silkworm Bombyx mori.

PubMed

Abe, H; Seki, M; Ohbayashi, F; Tanaka, N; Yamashita, J; Fujii, T; Yokoyama, T; Takahashi, M; Banno, Y; Sahara, K; Yoshido, A; Ihara, J; Yasukochi, Y; Mita, K; Ajimura, M; Suzuki, M G; Oshiki, T; Shimada, T

2005-08-01

In the silkworm, Bombyx mori (female, ZW; male, ZZ), femaleness is determined by the presence of a single W chromosome, irrespective of the number of autosomes or Z chromosomes. The W chromosome is devoid of functional genes, except the putative female-determining gene (Fem). However, there are strains in which chromosomal fragments containing autosomal markers have been translocated on to W. In this study, we analysed the W chromosomal regions of the Zebra-W strain (T(W;3)Ze chromosome) and the Black-egg-W strain (T(W;10)+(w-2) chromosome) at the molecular level. Initially, we undertook a project to identify W-specific RAPD markers, in addition to the three already established W-specific RAPD markers (W-Kabuki, W-Samurai and W-Kamikaze). Following the screening of 3648 arbitrary 10-mer primers, we obtained nine W-specific RAPD marker sequences (W-Bonsai, W-Mikan, W-Musashi, W-Rikishi, W-Sakura, W-Sasuke, W-Yukemuri-L, W-Yukemuri-S and BMC1-Kabuki), almost all of which contained the border regions of retrotransposons, namely portions of nested retrotransposons. We confirmed the presence of eleven out of twelve W-specific RAPD markers in the normal W chromosomes of twenty-five silkworm strains maintained in Japan. These results indicate that the W chromosomes of the strains in Japan are almost identical in type. The Zebra-W strain (T(W;3)Ze chromosome) lacked the W-Samurai and W-Mikan RAPD markers and the Black-egg-W strain (T(W;10)+(w-2) chromosome) lacked the W-Mikan RAPD marker. These results strongly indicate that the regions containing the W-Samurai and W-Mikan RAPD markers or the W-Mikan RAPD marker were deleted in the T(W;3)Ze and T(W;10)+(w-2) chromosomes, respectively, due to reciprocal translocation between the W chromosome and the autosome. This deletion apparently does not affect the expression of Fem; therefore, this deleted region of the W chromosome does not contain the putative Fem gene.

Two Hemocyte Lineages Exist in Silkworm Larval Hematopoietic Organ

PubMed Central

Nakahara, Yuichi; Kanamori, Yasushi; Kiuchi, Makoto; Kamimura, Manabu

2010-01-01

Background Insects have multiple hemocyte morphotypes with different functions as do vertebrates, however, their hematopoietic lineages are largely unexplored with the exception of Drosophila melanogaster. Methodology/Principal Findings To study the hematopoietic lineage of the silkworm, Bombyx mori, we investigated in vivo and in vitro differentiation of hemocyte precursors in the hematopoietic organ (HPO) into the four mature hemocyte subsets, namely, plasmatocytes, granulocytes, oenocytoids, and spherulocytes. Five days after implantation of enzymatically-dispersed HPO cells from a GFP-expressing transgenic line into the hemocoel of normal larvae, differentiation into plasmatocytes, granulocytes and oenocytoids, but not spherulocytes, was observed. When the HPO cells were cultured in vitro, plasmatocytes appeared rapidly, and oenocytoids possessing prophenol oxidase activity appeared several days later. HPO cells were also able to differentiate into a small number of granulocytes, but not into spherulocytes. When functionally mature plasmatocytes were cultured in vitro, oenocytoids were observed 10 days later. These results suggest that the hemocyte precursors in HPO first differentiate into plasmatocytes, which further change into oenocytoids. Conclusions/Significance From these results, we propose that B. mori hemocytes can be divided into two major lineages, a granulocyte lineage and a plasmatocyte-oenocytoid lineage. The origins of the spherulocytes could not be determined in this study. We construct a model for the hematopoietic lineages at the larval stage of B. mori. PMID:20676370

Genome-Wide Identification and Immune Response Analysis of Serine Protease Inhibitor Genes in the Silkworm, Bombyx mori

PubMed Central

Duan, Jun; Wang, Genhong; Wang, Lingyan; Li, Youshan; Xiang, Zhonghuai; Xia, Qingyou

2012-01-01

In most insect species, a variety of serine protease inhibitors (SPIs) have been found in multiple tissues, including integument, gonad, salivary gland, and hemolymph, and are required for preventing unwanted proteolysis. These SPIs belong to different families and have distinct inhibitory mechanisms. Herein, we predicted and characterized potential SPI genes based on the genome sequences of silkworm, Bombyx mori. As a result, a total of eighty SPI genes were identified in B. mori. These SPI genes contain 10 kinds of SPI domains, including serpin, Kunitz_BPTI, Kazal, TIL, amfpi, Bowman-Birk, Antistasin, WAP, Pacifastin, and alpha-macroglobulin. Sixty-three SPIs contain single SPI domain while the others have at least two inhibitor units. Some SPIs also contain non-inhibitor domains for protein-protein interactions, including EGF, ADAM_spacer, spondin_N, reeler, TSP_1 and other modules. Microarray analysis showed that fourteen SPI genes from lineage-specific TIL family and Group F of serpin family had enriched expression in the silk gland. The roles of SPIs in resisting pathogens were investigated in silkworms when they were infected by four pathogens. Microarray and qRT-PCR experiments revealed obvious up-regulation of 8, 4, 3 and 3 SPI genes after infection with Escherichia coli, Bacillus bombysepticus, Beauveria bassiana or B. mori nuclear polyhedrosis virus (BmNPV), respectively. On the contrary, 4, 11, 7 and 9 SPI genes were down-regulated after infection with E. coli, B. bombysepticus, B. bassiana or BmNPV, respectively. These results suggested that these SPI genes may be involved in resistance to pathogenic microorganisms. These findings may provide valuable information for further clarifying the roles of SPIs in the development, immune defence, and efficient synthesis of silk gland protein. PMID:22348050

Expression of a functional intrabody against hepatitis C virus core protein in Escherichia coli and silkworm pupae.

PubMed

Kato, Tatsuya; Hasegawa, Moeko; Yamamoto, Takeshi; Miyazaki, Takatsugu; Suzuki, Ryosuke; Wakita, Takaji; Suzuki, Tetsuro; Park, Enoch Y

2018-10-01

It has been shown that the single-domain intrabody 2H9-L against the hepatitis C virus (HCV) capsid (core) protein inhibits the viral propagation and NF-κB promoter activity induced by the HCV core. In this study, 2H9-L fused with the FLAG tag sequence was expressed in both Escherichia coli and silkworm pupae and then purified. In addition, the full-length and its C terminal deletions of the HCV core protein, i.e., 1-123 amino acid residues (C123), 1-152 amino acid residues (C152), 1-177 amino acid residues (C177) and 1-191 amino acid residues (C191), were expressed as fusion proteins with a 6 × His tag at their N-terminus in E. coli and then purified. Approximately 175 and 132 μg of the intrabody were purified from 100 ml of E. coli culture and 10 silkworm pupae, respectively, by affinity chromatography. The C123, C152, C177 and C191 HCV core protein variants were purified to approximately 152, 127, 103 and 155 μg, respectively, from 100 ml of E. coli culture. An ELISA in which the intrabodies were immobilized revealed that the intrabodies purified from both hosts were bound to all HCV core protein variants. However, their binding to the C191 appeared to be weak compared to their bindings to the other HCV core protein variants. When C152 was immobilized in the ELISA, the binding of each intrabody to the core protein was also observed. These purified intrabodies can be used in biochemical analyses of the inhibitory mechanism of HCV propagation and as protein interference reagents, thus providing a potential pathway to developing a new type of antiviral drug. Copyright © 2018 Elsevier Inc. All rights reserved.

DNA replication events during larval silk gland development in the silkworm, Bombyx mori.

PubMed

Zhang, Chun-Dong; Li, Fang-Fang; Chen, Xiang-Yun; Huang, Mao-Hua; Zhang, Jun; Cui, Hongjuan; Pan, Min-Hui; Lu, Cheng

2012-07-01

The silk gland is an important organ in silkworm as it synthesizes silk proteins and is critical to spinning. The genomic DNA content of silk gland cells dramatically increases 200-400 thousand times for the larval life span through the process of endomitosis. Using in vitro culture, DNA synthesis was measured using BrdU labeling during the larval molt and intermolt periods. We found that the cell cycle of endomitosis was activated during the intermolt and was inhibited during the molt phase. The anterior silk gland, middle silk gland, and posterior silk gland cells asynchronously exit the endomitotic cycle after day 6 in 5th instar larvae, which correlated with the reduced expression of the cell cycle-related cdt1, pcna, cyclin E, cdk2 and cdk1 mRNAs in the wandering phase. Additional starvation had no effect on the initiation of silk gland DNA synthesis of the freshly ecdysed larvae. Copyright © 2012 Elsevier Ltd. All rights reserved.

Structural Insights into the Ligand Binding and Releasing Mechanism of Antheraea polyphemus PBP1: Role of the C-terminal Tail

PubMed Central

Katre, Uma V.; Mazumder, Suman; Mohanty, Smita

2013-01-01

Pheromone-binding proteins (PBPs) in lepidopteran moths selectively transport the hydrophobic pheromone molecules across the sensillar lymph to trigger the neuronal response. Moth PBPs are known to bind ligand at physiological pH and release it at acidic pH while undergoing a conformational change. Two molecular switches are considered to play a role in this mechanism: (i) Protonation of His70 and His95 situated at one end of binding pocket, and (ii) Switch of the unstructured C-terminus at the other end of the binding pocket to a helix that enters the pocket. We have reported previously the role of the histidine-driven switch in ligand release for Antheraea polyphemus PBP1 (ApolPBP1). Here we show that the C-terminus plays a role in ligand release and binding mechanism of ApolPBP1. The C-terminus truncated mutants of ApolPBP1 (ApolPBP1ΔP129-V142 and ApolPBP1H70A/H95AΔP129-V142) exist only in the bound conformation at all pH levels, and they fail to undergo pH- or ligand- dependent conformational switch. Although these proteins could bind ligands even at acidic pH unlike the wild-type ApolPBP1, they had ~4 fold reduced affinity towards the ligand at both acidic and physiological pH than that of ApolPBP1wt and ApolPBP1H70A/H95A. Thus, apart from helping in the ligand-release at acidic pH, the C-terminus in ApolPBP1 also plays an important role in ligand binding and/or locking the ligand in the binding pocket. Our results are in stark contrast to those reported for BmorPBP and AtraPBP, where C-terminus truncated proteins had similar or increased pheromone-binding affinity at any pH. PMID:23327454

Evaluation of Target Specificity of Antibacterial Agents Using Staphylococcus aureus ddlA Mutants and d-Cycloserine in a Silkworm Infection Model▿

PubMed Central

Kurokawa, Kenji; Hamamoto, Hiroshi; Matsuo, Miki; Nishida, Satoshi; Yamane, Noriko; Lee, Bok Luel; Murakami, Kazuhisa; Maki, Hideki; Sekimizu, Kazuhisa

2009-01-01

The availability of a silkworm larva infection model to evaluate the therapeutic effectiveness of antibiotics was examined. The 50% effective doses (ED50) of d-cycloserine against the Staphylococcus aureus ddlA mutant-mediated killing of larvae were remarkably lower than those against the parental strain-mediated killing of larvae. Changes in MICs and ED50 of other antibiotics were negligible, suggesting that these alterations are d-cycloserine selective. Therefore, this model is useful for selecting desired compounds based on their therapeutic effectiveness during antibiotic development. PMID:19546371

Differentially expressed genes in the ovary of the sixth day of pupal "Ming" lethal egg mutant of silkworm, Bombyx mori.

PubMed

Gao, Peng; Chen, An-Li; Zhao, Qiao-Ling; Shen, Xing-Jia; Qiu, Zhi-Yong; Xia, Ding-Guo; Tang, Shun-Ming; Zhang, Guo-Zheng

2013-09-15

The "Ming" lethal egg mutant (l-em) is a vitelline membrane mutant in silkworm, Bombyx mori. The eggs laid by the l-em mutant lose water, ultimately causing death within an hour. Previous studies have shown that the deletion of BmEP80 is responsible for the l-em mutation in silkworm, B. mori. In the current study, digital gene expression (DGE) was performed to investigate the difference of gene expression in ovaries between wild type and l-em mutant on the sixth day of the pupal stage to obtain a global view of gene expression profiles using the ovaries of three l-em mutants and three wild types. The results showed a total of 3,463,495 and 3,607,936 clean tags in the wild type and the l-em mutant libraries, respectively. Compared with those of wild type, 239 differentially expressed genes were detected in the l-em mutant, wherein 181 genes are up-regulated and 58 genes are down-regulated in the mutant strain. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that no pathway was significantly enriched and three pathways are tightly related to protein synthesis among the five leading pathways. Moreover, the expression profiles of eight important differentially expressed genes related to oogenesis changed. These results provide a comprehensive gene expression analysis of oogenesis and vitellogenesis in B. mori which facilitates understanding of both the specific molecular mechanism of the 1-em mutant and Lepidopteran oogenesis in general. Copyright © 2013 Elsevier B.V. All rights reserved.

The FOXO transcription factor controls insect growth and development by regulating juvenile hormone degradation in the silkworm, Bombyx mori.

PubMed

Zeng, Baosheng; Huang, Yuping; Xu, Jun; Shiotsuki, Takahiro; Bai, Hua; Palli, Subba Reddy; Huang, Yongping; Tan, Anjiang

2017-07-14

Forkhead box O (FOXO) functions as the terminal transcription factor of the insulin signaling pathway and regulates multiple physiological processes in many organisms, including lifespan in insects. However, how FOXO interacts with hormone signaling to modulate insect growth and development is largely unknown. Here, using the transgene-based CRISPR/Cas9 system, we generated and characterized mutants of the silkworm Bombyx mori FOXO ( BmFOXO ) to elucidate its physiological functions during development of this lepidopteran insect. The BmFOXO mutant (FOXO-M) exhibited growth delays from the first larval stage and showed precocious metamorphosis, pupating at the end of the fourth instar (trimolter) rather than at the end of the fifth instar as in the wild-type (WT) animals. However, different from previous reports on precocious metamorphosis caused by juvenile hormone (JH) deficiency in silkworm mutants, the total developmental time of the larval period in the FOXO-M was comparable with that of the WT. Exogenous application of 20-hydroxyecdysone (20E) or of the JH analog rescued the trimolter phenotype. RNA-seq and gene expression analyses indicated that genes involved in JH degradation but not in JH biosynthesis were up-regulated in the FOXO-M compared with the WT animals. Moreover, we identified several FOXO-binding sites in the promoter of genes coding for JH-degradation enzymes. These results suggest that FOXO regulates JH degradation rather than its biosynthesis, which further modulates hormone homeostasis to control growth and development in B. mori In conclusion, we have uncovered a pivotal role for FOXO in regulating JH signaling to control insect development. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Silk sericin-alginate-chitosan microcapsules: hepatocytes encapsulation for enhanced cellular functions.

PubMed

Nayak, Sunita; Dey, Sanchareeka; Kundu, Subhas C

2014-04-01

The encapsulation based technology permits long-term delivery of desired therapeutic products in local regions of body without the need of immunosuppressant drugs. In this study microcapsules composed of sericin and alginate micro bead as inner core and with an outer chitosan shell are prepared. This work is proposed for live cell encapsulation for potential therapeutic applications. The sericin protein is obtained from cocoons of non-mulberry silkworm Antheraea mylitta. The sericin-alginate micro beads are prepared via ionotropic gelation under high applied voltage. The beads further coated with chitosan and crosslinked with genipin. The microcapsules developed are nearly spherical in shape with smooth surface morphology. Alamar blue assay and confocal microscopy indicate high cell viability and uniform encapsulated cell distribution within the sericin-alginate-chitosan microcapsules indicating that the microcapsules maintain favourable microenvironment for the cells. The functional analysis of encapsulated cells demonstrates that the glucose consumption, urea secretion rate and intracellular albumin content increased in the microcapsules. The study suggests that the developed sericin-alginate-chitosan microcapsule contributes towards the development of cell encapsulation model. It also offers to generate enriched population of metabolically and functionally active cells for the future therapeutics especially for hepatocytes transplantation in acute liver failure. Copyright © 2014 Elsevier B.V. All rights reserved.

Juvenile Hormone Analogues, Methoprene and Fenoxycarb Dose-Dependently Enhance Certain Enzyme Activities in the Silkworm Bombyx Mori (L)

PubMed Central

Mamatha, Devi M.; Kanji, Vijaya K.; Cohly, Hari H.P.; Rao, M. Rajeswara

2008-01-01

Use of Juvenile Hormone Analogues (JHA) in sericulture practices has been shown to boost good cocoon yield; their effect has been determined to be dose-dependent. We studied the impact of low doses of JHA compounds such as methoprene and fenoxycarb on selected key enzymatic activities of the silkworm Bombyx mori. Methoprene and fenoxycarb at doses of 1.0 μg and 3.0fg/larvae/48 hours showed enhancement of the 5th instar B. mori larval muscle and silkgland protease, aspartate aminotransaminase (AAT) and alanine aminotransaminase (ALAT), adenosine triphosphate synthase (ATPase) and cytochrome-c-oxidase (CCO) activity levels, indicating an upsurge in the overall oxidative metabolism of the B.mori larval tissues. PMID:18678927

N-acetyltransferase (nat) is a critical conjunct of photoperiodism between the circadian system and endocrine axis in Antheraea pernyi.

PubMed

Mohamed, Ahmed A M; Wang, Qiushi; Bembenek, Jadwiga; Ichihara, Naoyuki; Hiragaki, Susumu; Suzuki, Takeshi; Takeda, Makio

2014-01-01

Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16:8 (LD) and LD12:12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4 °C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNA(NAT) caused dysfunction of photoperiodism. dsRNA(PER) upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNA(NAT) decreased melatonin while dsRNA(PER) increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNA(NAT), to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism.

N-acetyltransferase (nat) Is a Critical Conjunct of Photoperiodism between the Circadian System and Endocrine Axis in Antheraea pernyi

PubMed Central

Bembenek, Jadwiga; Hiragaki, Susumu; Suzuki, Takeshi; Takeda, Makio

2014-01-01

Since its discovery in 1923, the biology of photoperiodism remains a mystery in many ways. We sought the link connecting the circadian system to an endocrine switch, using Antheraea pernyi. PER-, CLK- and CYC-ir were co-expressed in two pairs of dorsolateral neurons of the protocerebrum, suggesting that these are the circadian neurons that also express melatonin-, NAT- and HIOMT-ir. The results suggest that a melatonin pathway is present in the circadian neurons. Melatonin receptor (MT2 or MEL-1B-R)-ir in PTTH-ir neurons juxtaposing clock neurons suggests that melatonin gates PTTH release. RIA showed a melatonin rhythm with a peak four hours after lights off in adult brain both under LD16∶8 (LD) and LD12∶12 (SD), and both the peak and the baseline levels were higher under LD than SD, suggesting a photoperiodic influence. When pupae in diapause were exposed to 10 cycles of LD, or stored at 4°C for 4 months under constant darkness, an increase of NAT activity was observed when PTTH released ecdysone. DNA sequence upstream of nat contained E-boxes to which CYC/CLK could bind, and nat transcription was turned off by clk or cyc dsRNA. dsRNANAT caused dysfunction of photoperiodism. dsRNAPER upregulated nat transcription as anticipated, based on findings in the Drosophila melanogaster circadian system. Transcription of nat, cyc and clk peaked at ZT12. RIA showed that dsRNANAT decreased melatonin while dsRNAPER increased melatonin. Thus nat, a clock controlled gene, is the critical link between the circadian clock and endocrine switch. MT-binding may release PTTH, resulting in termination of diapause. This study thus examined all of the basic functional units from the clock: a photoperiodic counter as an accumulator of mRNANAT, to endocrine switch for photoperiodism in A. pernyi showing this system is self-complete without additional device especially for photoperiodism. PMID:24667367

Silkworm Gut Fiber of Bombyx mori as an Implantable and Biocompatible Light-Diffusing Fiber

PubMed Central

Cenis, Jose Luis; Aznar-Cervantes, Salvador D.; Lozano-Pérez, Antonio Abel; Rojo, Marta; Muñoz, Juan; Meseguer-Olmo, Luis; Arenas, Aurelio

2016-01-01

This work describes a new approach to the delivery of light in deeper tissues, through a silk filament that is implantable, biocompatible, and biodegradable. In the present work, silkworm gut fibers (SGFs) of Bombyx mori L., are made by stretching the silk glands. Morphological, structural, and optical properties of the fibers have been characterized and the stimulatory effect of red laser light diffused from the fiber was assayed in fibroblast cultures. SGFs are formed by silk fibroin (SF) mainly in a β-sheet conformation, a stable and non-soluble state in water or biological fluids. The fibers showed a high degree of transparency to visible and infrared radiation. Using a red laser (λ = 650 nm) as source, the light was efficiently diffused along the fiber wall, promoting a significant increment in the cell metabolism 5 h after the irradiation. SGFs have shown their excellent properties as light-diffusing optical fibers with a stimulatory effect on cells. PMID:27438824

Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System.

PubMed

Dong, Zhanqi; Dong, Feifan; Yu, Xinbo; Huang, Liang; Jiang, Yaming; Hu, Zhigang; Chen, Peng; Lu, Cheng; Pan, Minhui

2018-01-01

The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 ( ie-1 ) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System

PubMed Central

Dong, Zhanqi; Dong, Feifan; Yu, Xinbo; Huang, Liang; Jiang, Yaming; Hu, Zhigang; Chen, Peng; Lu, Cheng; Pan, Minhui

2018-01-01

The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 (ie-1) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment. PMID:29503634

Isolation, purification, crystallization and preliminary X-ray studies of two 30 kDa proteins from silkworm haemolymph.

PubMed

Pietrzyk, Agnieszka J; Bujacz, Anna; Łochyńska, Małgorzata; Jaskólski, Mariusz; Bujacz, Grzegorz

2011-03-01

Juvenile hormone-binding protein (JHBP) and the low-molecular-mass lipoprotein PBMHP-12 belong to a group of 30 kDa proteins that comprise the major protein component of the haemolymph specific to the fifth-instar larvae stage of the mulberry silkworm Bombyx mori L. Proteins from this group are often essential for the development of the insect. In a project aimed at crystallographic characterization of B. mori JHBP (BmJHBP), it was copurified together with PBMHP-12. Eventually, the two proteins were isolated and crystallized separately. The BmJHBP crystals were orthorhombic (space group C222(1)) and the PBMHP-12 crystals were triclinic. The crystals diffracted X-rays to 2.9 Å (BmJHBP) and 1.3 Å (PBMHP-12) resolution.

A novel sodium N-fatty acyl amino acid surfactant using silkworm pupae as stock material

PubMed Central

Wu, Min-Hui; Wan, Liang-Ze; Zhang, Yu-Qing

2014-01-01

A novel sodium N-fatty acyl amino acid (SFAAA) surfactant was synthesized using pupa oil and pupa protein hydrolysates (PPH) from a waste product of the silk industry. The aliphatic acids from pupa oil were modified into N-fatty acyl chlorides by thionyl chloride (SOCl2). SFAAA was synthesized using acyl chlorides and PPH. GC-MS analysis showed fatty acids from pupa oil consist mainly of unsaturated linolenic and linoleic acids and saturated palmitic and stearic acids. SFAAA had a low critical micelle concentration, great efficiency in lowering surface tension and strong adsorption at an air/water interface. SFAAA had a high emulsifying power, as well as a high foaming power. The emulsifying power of PPH and SFAAA in an oil/water emulsion was better with ethyl acetate as the oil phase compared to n-hexane. The environment-friendly surfactant made entirely from silkworm pupae could promote sustainable development of the silk industry. PMID:24651079

Effect of Induced Oxidative Stress and Herbal Extracts on Acid Phosphatase Activity in Lysosomal and Microsomal Fractions of Midgut Tissue of the Silkworm, Bombyx mori

PubMed Central

Gaikwad, Y. B.; Gaikwad, S. M.; Bhawane, G. P.

2010-01-01

Lysosomal and microsomal acid phosphatase activity was estimated in midgut tissue of silkworm larvae, Bombyx mori L. (Lepidoptera: Bombycidae), after induced oxidative stress by D-galactose. The larvae were simultaneously were treated with ethanolic extracts of Bacopa monniera and Lactuca sativa to study their antioxidant properties. Lipid peroxidation and fluorescence was measured to analyze extent of oxidative stress. The ethanolic extract of Lactuca sativa was found to be more effective in protecting membranes against oxidative stress than Bacopa monniera. PMID:20874583

Caspase-1 from the silkworm, Bombyx mori, is involved in Bombyx mori nucleopolyhedrovirus infection.

PubMed

Wang, Qiang; Ju, Xiaoli; Chen, Liang; Chen, Keping

2017-03-01

Caspase-1 is one of the effector caspases in mammals that plays a central role in apoptosis. However, the lepidopteran caspase-1, especially the Bombyx mori caspase-1 (Bm-caspase-1), has not been investigated in detail. In this study, Bm-caspase-1 was identified from an expressed sequence tag database in B. mori by BLAST search. The open reading frame of Bm-caspase-1 contained 879 nucleotides and encoded 293 amino acids with a predicted molecular mass of 33 kDa. Bm-caspase-1 contained two consensus amino acid motifs of caspase cleavage sites, DEGDA and TETDG. Caspase activity assays revealed significant proteolytic activity of the Ac-DEVD-pNA substrate. Bm-caspase-1 can be detected in all tissues and developmental stages by a semi quantitative polymerase chain reaction assay. More importantly, the expression level of Bm-caspase-1 is increased upon baculovirus infection and up-regulated in BmNPV-resistant silkworms. Taken together, these results indicate that Bm-caspase-1 plays an important role during baculovirus infection.

Proteins in the Cocoon of Silkworm Inhibit the Growth of Beauveria bassiana

PubMed Central

Zhang, Yan; Li, Youshan; Liu, Huawei; Xia, Qingyou; Zhao, Ping

2016-01-01

Silk cocoons are composed of fiber proteins (fibroins) and adhesive glue proteins (sericins), which provide a physical barrier to protect the inside pupa. Moreover, other proteins were identified in the cocoon silk, many of which are immune related proteins. In this study, we extracted proteins from the silkworm cocoon by Tris-HCl buffer (pH7.5), and found that they had a strong inhibitory activity against fungal proteases and they had higher abundance in the outer cocoon layers than in the inner cocoon layers. Moreover, we found that extracted cocoon proteins can inhibit the germination of Beauveria bassiana spores. Consistent with the distribution of protease inhibitors, we found that proteins from the outer cocoon layers showed better inhibitory effects against B. bassiana spores than proteins from the inner layers. Liquid chromatography-tandem mass spectrometry was used to reveal the extracted components in the scaffold silk, the outermost cocoon layer. A total of 129 proteins were identified, 30 of which were annotated as protease inhibitors. Protease inhibitors accounted for 89.1% in abundance among extracted proteins. These protease inhibitors have many intramolecular disulfide bonds to maintain their stable structure, and remained active after being boiled. This study added a new understanding to the antimicrobial function of the cocoon. PMID:27032085

[Effect of white spot syndrome virus envelope protein Vp28 expressed in silkworm (Bombyx mori) pupae on disease resistence in Procambarus clarkii].

PubMed

Wei, Ke Qiang; Xu, Zi Rong

2005-06-01

The vaccine made of recombinant envelope protein (rVp28) of white spot syndrome virus (WSSV) expressed in silkworm (Bombyx mori) pupae using a baculovirus vector was used to investigate the efficacy of oral administration on WSSV disease resistance of Procambarus clarkii. Vaccine was mixed with diet at a ratio of 2% (w/w), and Procambarus clarkii were orally administered throughout 75 days. Vaccination with rVP28 showed the significantly higher cumulative survival compared with positive and negative control (P < 0.05) following an oral challenge on the 35th day post-vaccination (dpv), with PRP values 54.16% and 59.26%, respectively. rVP28 induced higher resistance via IM (intramuscular) injection challenge with WSSV stock, with PRP value of 46.12% and 49.99%, respectively. The survivors were subsequently re-challenged on the 55th dpv. rVP28 induced the significantly higher resistance to oral re-challenge (P < 0.05), with both PRP values 55.80% and 63.16%, respectively. rVP28 induced higher resistance to IM injection re-challenge, with both PRP values 31.25%. A DIG labeled WSSV DNA probe was used to detect WSSV by in situ hybridization. The positive cells were observed in epithelial cells of stomach, hepatopancreas and gut of the infected control crayfish, while negative reaction were observed in the tissues of survivors-vaccinated. These results indicated that vaccination of crayfish with recombinant protein had significant effect on oral infection, and had higher resistance against intramuscular injection challenge. This suggested the protection against WSSV could be induced in crayfish by recombinant protein rVp28 expressed in silkworm pupae.

Effect of 1-(4-phenoxyphenoxypropyl)imidazole (KS-175) on larval growth in the silkworm Bombyx mori.

PubMed

Shiotsuki, T; Yukuhiro, F; Kiuchi, M; Kuwano, E

1999-12-01

1-(4-Phenoxyphenoxypropyl)imidazole (KS-175), which has two types of characteristic moieties of insect growth regulators (IGRs), the phenoxyphenoxyalkyl group of juvenile hormone analogs (JHAs) and imidazole of 1,5-disubstituted imidazole such as KK-42, was tested for its biological activity on the silkworm, Bombyx mori. Penultimate (4th) instar larvae topically treated with KS-175 did not molt for more than 20 days. This activity was different from that reported for any IGRs. After the treatment, ecdysteroid levels in the hemolymph did not increase and the cells of the prothoracic gland had shrunk. When the treated penultimate larvae were fed an artificial diet supplemented with 20 ppm of 20-hydroxyecdysone, the larvae molted to the ultimate (5th) instar with a timing similar to that of control larvae fed a diet with or without 20-hydroxyecdysone. These results suggest that topical application of KS-175 irreversibly damages ecdysone biosynthesis in the prothoracic glands.

The POU homeodomain transcription factor POUM2 and broad complex isoform 2 transcription factor induced by 20-hydroxyecdysone collaboratively regulate vitellogenin gene expression and egg formation in the silkworm Bombyx mori.

PubMed

Lin, Y; Liu, H; Yang, C; Gu, J; Shen, G; Zhang, H; Chen, E; Han, C; Zhang, Y; Xu, Y; Wu, J; Xia, Q

2017-10-01

Vitellogenin (Vg) is a source of nutrition for embryo development. Our previous study showed that the silkworm (Bombyx mori) transcription factor broad complex isoform 2 (BmBrC-Z2) regulates gene expression of the Vg gene (BmVg) by induction with 20-hydroxyecdysone (20E). However, the mechanism by which 20E regulates BmVg expression was not clarified. In this study, cell transfection experiments showed that the BmVg promoter containing the POU homeodomain transcription factor POUM2 (POUM2) and BrC-Z2 cis-response elements (CREs) showed a more significant response to 20E than that harbouring only the BrC-Z2 or POUM2 CRE. An electrophoretic mobility shift assay and chromatin immunoprecipitation assay showed that BmPOUM2 could bind to the POUM2 CRE of the BmVg promoter. Over-expression of BmPOUM2 and BmBrC-Z2 in B. mori embryo-derived cell line (BmE) could enhance the activity of the BmVg promoter carrying both the POUM2 and BrC-Z2 CREs following 20E induction. Quantitative PCR and immunofluorescence histochemistry showed that the expression pattern and tissue localization of BmPOUM2 correspond to those of BmVg. Glutathione S-transferase pull-down and co-immunoprecipitation assays confirmed that BmPOUM2 interacts only with BmBrC-Z2 to regulate BmVg expression. Down-regulation of BmPOUM2 in female silkworm by RNA interference significantly reduced BmVg expression, leading to abnormal egg formation. In summary, these results indicate that BmPOUM2 binds only to BmBrC-Z2 to collaboratively regulate BmVg expression by 20E induction to control vitellogenesis and egg formation in the silkworm. Moreover, these findings suggest that homeodomain protein POUM2 plays a novel role in regulating insect vitellogenesis. © 2017 The Royal Entomological Society.

Equilibrium and kinetic adsorption study of a cationic dye by a natural adsorbent--silkworm pupa.

PubMed

Noroozi, B; Sorial, G A; Bahrami, H; Arami, M

2007-01-02

In this work the use of silkworm pupa, which is the waste of silk spinning industries has been investigated as an adsorbent for the removal of C.I. Basic Blue 41. The amino acid nature of the pupa provided a reasonable capability for dye removal. Equilibrium adsorption isotherms and kinetics were investigated. The adsorption equilibrium data were analyzed by using various adsorption isotherm models and the results have shown that adsorption behavior of the dye could be described reasonably well by either Langmuir or Freundlich models. The characteristic parameters for each isotherm have been determined. The monolayer adsorption capacity was determined to be 555 mg/g. Kinetic studies indicated that the adsorption follows pseudo-second-order kinetics with a rate constant of 0.0434 and 0.0572 g/min mg for initial dye concentration of 200 mg/l at 20 and 40 degrees C, respectively. Kinetic studies showed that film diffusion and intra-particle diffusion were simultaneously operating during the adsorption process. The rate constant for intra-particle diffusion was estimated to be 1.985 mg/g min(0.5).

Effect of TiO2 nanoparticles on the reproduction of silkworm.

PubMed

Ni, Min; Li, Fanchi; Wang, Binbin; Xu, Kaizun; Zhang, Hua; Hu, Jingsheng; Tian, Jianghai; Shen, Weide; Li, Bing

2015-03-01

Silkworm (Bombyx mori) is an important economic insect and the model insect of Lepidoptera. Because of its high fecundity and short reproduction cycle, it has been widely used in reproduction and development research. The high concentrations of titanium dioxide nanoparticles (TiO2 NPs) show reproductive toxicity, while low concentrations of TiO2 NPs have been used as feed additive and demonstrated significant biological activities. However, whether the low concentrations of TiO2 NPs affect the reproduction of B. mori has not been reported. In this study, the growth and development of gonad of B. mori fed with a low concentration of TiO2 NPs (5 mg/L) were investigated by assessing egg production and expression of reproduction-related genes. The results showed that the low concentration of TiO2 NPs resulted in faster development of the ovaries and testes and more gamete differentiation and formation, with an average increase of 51 eggs per insect and 0.34 × 10(-4) g per egg after the feeding. The expressions of several reproduction-related genes were upregulated, such as the yolk-development-related genes Ovo-781 and vitellogenin (Vg) were increased by 5.33- and 6.77-folds, respectively. This study shows that TiO2 NPs feeding at low concentration can enhance the reproduction of B. mori, and these results are useful in developing new methods to improve fecundity in B. mori and providing new clues for its broad biological applications.

Transcriptomic analysis of two Beauveria bassiana strains grown on cuticle extracts of the silkworm uncovers their different metabolic response at early infection stage.

PubMed

Wang, Jing-Jie; Bai, Wen-Wen; Zhou, Wei; Liu, Jing; Chen, Jie; Liu, Xiao-Yuan; Xiang, Ting-Ting; Liu, Ren-Hua; Wang, Wen-Hui; Zhang, Bao-Ling; Wan, Yong-Ji

2017-05-01

Beauveria bassiana is an important entomopathogenic fungus which not only widely distributes in the environment but also shows phenotypic diversity. However, the mechanism of pathogenic differences among natural B. bassiana strains has not been revealed at transcriptome-wide level. In the present study, in order to explore the mechanism, two B. bassiana strains with different pathogenicity were isolated from silkworms (Bombyx mori L.) and selected to analyze the gene expression of early stage by culturing on cuticle extracts of the silkworm and using RNA-sequencing technique. A total of 2108 up-regulated and 1115 down-regulated genes were identified in B. bassiana strain GXsk1011 (hyper-virulent strain) compared with B. bassiana strain GXtr1009 (hypo-virulent strain), respectively. The function categorization of differential expressed genes (DEGs) showed that most of them involved in metabolic process, biosynthesis of secondary metabolites, catalytic activity, and some involved in nutrition uptake, adhesion and host defense were also noted. Based on our data, distinct pathogenicity among different strains of B. bassiana may largely attribute to unique gene expression pattern which differed at very early infection process. Most of the genes involved in conidia adhesion, cuticle degradation and fungal growth were up-regulated in hyper-virulent B. bassiana strain GXsk1011. Furthermore, in combination with fungal growth analysis, our research provided a clue that fungal growth may also play an important role during early infection process. The results will help to explain why different B. bassiana strains show distinct pathogenicity on the same host even under same condition. Moreover, the transcriptome data were also useful for screening potential virulence factors. Copyright © 2017 Elsevier Inc. All rights reserved.

Silkworm (Bombyx mori) hemolymph unable to substitute fetal bovine serum in insect cell culture

NASA Astrophysics Data System (ADS)

Suparto, Irma H.; Khalam, Chandra Nur; Praira, Willy; Sajuthi, Dondin

2014-03-01

Fetal Bovine Serum (FBS) in animal cell culture media is an important source of nutrients for cell growth. However, the harvest and collection of FBS cause bioethical concerns. Efforts to reduce and preferably replace FBS with synthetic or other natural alternatives are continually being explored. Hemolymph silkworm (Bombyx mori) contains many nutrients needed for the process of metamorphosis. Therefore, there is possibility as an alternative nutritional supplement for cell culture to reduce the use of FBS. The objective of this study was to evaluate the macrocomponent of hemolymph and the possibility as medium supplement for Spodoptera fugiperda (Sf9) cell culture. Proximate analyses showed that hemolymph contains 89.76% of water, 2.52 mg/mL carbohydrate, 2.35% fat and 55.61 mg/mL protein. Further protein analysis, it consists of 15 fractions containing molecular weight of 22 - 152 kDa. The use of hemolymph as FBS substitution in Sf9 cell culture with various concentrations was unable to maintain and support cell growth. Further research still needed by prior adaptation of the tissue culture to minimal nutrition media before introduction of the hemolymph as supplement.

Midgut epithelium in molting silkworm: A fine balance among cell growth, differentiation, and survival.

PubMed

Franzetti, Eleonora; Casartelli, Morena; D'Antona, Paola; Montali, Aurora; Romanelli, Davide; Cappellozza, Silvia; Caccia, Silvia; Grimaldi, Annalisa; de Eguileor, Magda; Tettamanti, Gianluca

2016-07-01

The midgut of insects has attracted great attention as a system for studying intestinal stem cells (ISCs) as well as cell death-related processes, such as apoptosis and autophagy. Among insects, Lepidoptera represent a good model to analyze these cells and processes. In particular, larva-larva molting is an interesting developmental phase since the larva must deal with nutrient starvation and its organs are subjected to rearrangements due to proliferation and differentiation events. Several studies have analyzed ISCs in vitro and characterized key factors involved in their division and differentiation during molt. However, in vivo studies performed during larva-larva transition on these cells, and on the whole midgut epithelium, are fragmentary. In the present study, we analyzed the larval midgut epithelium of the silkworm, Bombyx mori, during larva-larva molting, focusing our attention on ISCs. Moreover, we investigated the metabolic changes that occur in the epithelium and evaluated the intervention of autophagy. Our data on ISCs proliferation and differentiation, autophagy activation, and metabolic and functional activities of the midgut cells shed light on the complexity of this organ during the molting phase. Copyright © 2016 Elsevier Ltd. All rights reserved.

Durable grafting of silkworm pupa protein onto the surface of polyethylene terephthalate fibers.

PubMed

Zhou, Jianfeng; Zheng, Dandan; Zhang, Fengxiu; Zhang, Guangxian

2016-12-01

In this paper, reactive -NH2 groups (8.36×10(-6)mol/g fabric) were introduced to the surface of polyethylene terephthalate (PET) fabrics by a nitration and reduction method, and epoxy groups were introduced to silkworm pupa protein (SPP) by reaction with epoxy chloropropane. PET-SPP composite fabrics were then prepared by reaction of these two precursors. The results showed that the SPP was firmly grafted onto the PET fabric surface and that the hydrophilicity of the fabric was markedly improved by the grafting of SPP. SEM images revealed a layer of substance covering the surface of the PET fibers, and XPS investigation showed that the nitrogen content of the PET-SPP fabric was higher than that of the original PET fabric (2.32% vs 0%). ATR-FTIR adsorption bands at 1653 and 1543cm(-1) suggested the successful grafting of SPP onto the PET fabric surface. The DSC and TG of the PET fibers demonstrated that the thermal stability of the original PET fibers was maintained well by the SPP-grafted PET fibers. The breaking strength, bending rigidity, air permeability, and crease recovery angle of the original PET fabric were also retained by the SPP-grafted PET fabric. Copyright © 2016 Elsevier B.V. All rights reserved.

Advanced technologies for genetically manipulating the silkworm Bombyx mori, a model Lepidopteran insect

PubMed Central

Xu, Hanfu; O'Brochta, David A.

2015-01-01

Genetic technologies based on transposon-mediated transgenesis along with several recently developed genome-editing technologies have become the preferred methods of choice for genetically manipulating many organisms. The silkworm, Bombyx mori, is a Lepidopteran insect of great economic importance because of its use in silk production and because it is a valuable model insect that has greatly enhanced our understanding of the biology of insects, including many agricultural pests. In the past 10 years, great advances have been achieved in the development of genetic technologies in B. mori, including transposon-based technologies that rely on piggyBac-mediated transgenesis and genome-editing technologies that rely on protein- or RNA-guided modification of chromosomes. The successful development and application of these technologies has not only facilitated a better understanding of B. mori and its use as a silk production system, but also provided valuable experiences that have contributed to the development of similar technologies in non-model insects. This review summarizes the technologies currently available for use in B. mori, their application to the study of gene function and their use in genetically modifying B. mori for biotechnology applications. The challenges, solutions and future prospects associated with the development and application of genetic technologies in B. mori are also discussed. PMID:26108630

A study on effects of glutathione s-transferase from silkworm on CCL4-induced mouse liver injury.

PubMed

Yan, Hui; Gui, Zhongzheng; Wang, Bochu

2011-01-01

To assess the hepatoprotective activity of Glutathione S-transferase(GSTsw), extracted and purified from silkworm, in experimental acute mice liver injury and explore mechanisms. Mice were divided into five groups: control group, carbon tetrachloride (CCl4) group, and three treatment groups that received CCl4 and GSTsw at doses of 0.083 mg•g(-1), 0.0415 mg•g(-1) and 0.0207 mg•g(-1) for 3 days. ALT in serum, GST, SOD and T-AOC in liver tissue homogenate, and changes in liver pathology in the five groups were studied. CCl4 administration led to pathological and biochemical evidence of liver injury as compared to untreated controls. GSTsw administration led to significant protection against CCl4-induced changes in liver pathology. It was also associatedwith significantly lower serum ALT levels, higher GST-SOD and T-AOC level in live tissue homogenate. Thus, GSTsw showed protective activity against CCl4-induced hepatotoxicity in mice.

The dipteran parasitoid Exorista bombycis induces pro- and anti-oxidative reactions in the silkworm Bombyx mori: Enzymatic and genetic analysis.

PubMed

Makwana, Pooja; Pradeep, Appukuttan Nair R; Hungund, Shambhavi P; Ponnuvel, Kangayam M; Trivedy, Kanika

2017-02-01

Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro-oxidative reactions and activation of anti-oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione-S-transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro- and anti-oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology. © 2017 Wiley Periodicals, Inc.

Genetically-determined polymorphism of nonspecific esterases and phosphoglucomutase in eight introduced breeds of the silkworm, Bombyx mori, raised in Bulgaria.

PubMed

Staykova, Teodora

2008-01-01

Isoenzymes are very suitable markers for the study of the inter-breed diversity of the silkworm Bombyx mon L. (Lepidoptera: Bombycidae). More than 250 breeds are raised in Bulgaria, which are not very well studied with regard to their isoenzymic polymorphism. Polymorphism of nonspecific esterases from pupal haemolymph was analyzed, as well as of phosphoglucomutase from different organs of larvae, pupae and imago, from eight introduced breeds. Electrophoresis in polyacrylamide gels was used. A polylocus control of nonspecific esterases, and possible monolocus control of phosphoglucomutase was ascertained. Biallele and triallele polymorphism of phosphoglucomutase locus and in three of the esterase loci was determined. The allelic frequencies of the polymorphic loci in each breed were analyzed. Inter-breed differences were found in different allelic frequencies, different heterozygosity and polymorphism.

Molecular and enzymatic characterization of two enzymes BmPCD and BmDHPR involving in the regeneration pathway of tetrahydrobiopterin from the silkworm Bombyx mori.

PubMed

Li, Wentian; Gong, Meixia; Shu, Rui; Li, Xin; Gao, Junshan; Meng, Yan

2015-08-01

Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases and nitric oxide synthase so that BH4 plays a key role in many biological processes. BH4 deficiency is associated with numerous metabolic syndromes and neuropsychological disorders. BH4 concentration in mammals is maintained through a de novo synthesis pathway and a regeneration pathway. Previous studies showed that the de novo pathway of BH4 is similar between insects and mammals. However, knowledge about the regeneration pathway of BH4 (RPB) is very limited in insects. Several mutants in the silkworm Bombyx mori have been approved to be associated with BH4 deficiency, which are good models to research on the RPB in insects. In this study, homologous genes encoding two enzymes, pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) involving in RPB have been cloned and identified from B. mori. Enzymatic activity of DHPR was found in the fat body of wild type silkworm larvae. Together with the transcription profiles, it was indicated that BmPcd and BmDhpr might normally act in the RPB of B. mori and the expression of BmDhpr was activated in the brain and sexual glands while BmPcd was expressed in a wider special pattern when the de novo pathway of BH4 was lacked in lemon. Biochemical analyses showed that the recombinant BmDHPR exhibited high enzymatic activity and more suitable parameters to the coenzyme of NADH in vitro. The results in this report give new information about the RPB in B. mori and help in better understanding insect BH4 biosynthetic networks. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular characterization and phylogenetic relationships among microsporidian isolates infecting silkworm, Bombyx mori using small subunit rRNA (SSU-rRNA) gene sequence analysis.

PubMed

Nath, B Surendra; Gupta, S K; Bajpai, A K

2012-12-01

The life cycle, spore morphology, pathogenicity, tissue specificity, mode of transmission and small subunit rRNA (SSU-rRNA) gene sequence analysis of the five new microsporidian isolates viz., NIWB-11bp, NIWB-12n, NIWB-13md, NIWB-14b and NIWB-15mb identified from the silkworm, Bombyx mori have been studied along with type species, NIK-1s_mys. The life cycle of the microsporidians identified exhibited the sequential developmental cycles that are similar to the general developmental cycle of the genus, Nosema. The spores showed considerable variations in their shape, length and width. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIWB-15mb was found to be more virulent than other isolates. All of the microsporidians were found to infect most of the tissues examined and showed gonadal infection and transovarial transmission in the infected silkworms. SSU-rRNA sequence based phylogenetic tree placed NIWB-14b, NIWB-12n and NIWB-11bp in a separate branch along with other Nosema species and Nosema bombycis; while NIWB-15mb and NIWB-13md together formed another cluster along with other Nosema species. NIK-1s_mys revealed a signature sequence similar to standard type species, N. bombycis, indicating that NIK-1s_mys is similar to N. bombycis. Based on phylogenetic relationships, branch length information based on genetic distance and nucleotide differences, we conclude that the microsporidian isolates identified are distinctly different from the other known species and belonging to the genus, Nosema. This SSU-rRNA gene sequence analysis method is found to be more useful approach in detecting different and closely related microsporidians of this economically important domestic insect.

Phylogenetic relationships of three new microsporidian isolates from the silkworm, Bombyx mori.

PubMed

Nageswara Rao, S; Muthulakshmi, M; Kanginakudru, S; Nagaraju, J

2004-07-01

The pathogenicity, mode of transmission, tissue specificity of infection and the small subunit rRNA (SSU-rRNA) gene sequences of the three new microsporidian isolates from the silkworm Bombyx mori were studied. Out of the three, NIK-2r revealed life cycle features and SSU-rRNA gene sequence similar to Nosema bombycis, suggesting that it is N. bombycis. The other two, NIK-4m and NIK-3h, differed from each other as well as from N. bombycis. NIK-4m was highly pathogenic and did not show any vertical transmission, in accordance with the apparent lack of gonadal infection, whereas NIK-3h was less pathogenic and vertical transmission was not detected but could not be excluded. Phylogenetic analysis based on SSU-rRNA gene sequence placed NIK-3h and NIK-4m in a distinct clade that included almost all the Vairimorpha species and Nosema species that infect lepidopteran and non-lepidopteran hosts, while NIK-2r was included in a clade containing almost all the Nosema isolates that infect only lepidopteran hosts. Thus, we have presented molecular evidence that one of the three isolates is in fact the type species N. bombycis, while the other two isolates are Vairimorpha spp. There was distinct separation of microsporidian isolates infecting only lepidopteran hosts and those infecting lepidopteran and non-lepidopteran hosts, reflecting possible co-evolution of hosts and microsporidian isolates.

Genetic mapping of a food preference gene in the silkworm, Bombyx mori, using restriction fragment length polymorphisms (RFLPs).

PubMed

Mase, Keisuke; Iizuka, Tetsuya; Yamamoto, Toshio; Okada, Eiji; Hara, Wajirou

2007-06-01

The domesticated silkworm, Bombyx mori, has strict food preferences and grows by feeding on mulberry leaves. However, "Sawa-J", an abnormal feeding habit strain selected from the genetic stock, feeds on an artificial diet without mulberry leaf powder. In this study, the food preference gene in Sawa-J was genetically identified using restriction fragment length polymorphisms (RFLPs) of a cDNA clone on each linkage group. Taking advantage of a lack of genetic recombination in females, reciprocal backcrossed F1 (BC1) progenies were independently prepared using a non-feeding strain, C108, as a mating partner of Sawa-J. Our results of linkage analysis and mapping proved that the feeding behavior is primarily controlled by a major recessive gene mapped at 20.2 cM on RFLP linkage group 9 (RFLG9), and clone e73 at a distance of 4.2 cM was found as the first linked molecular marker.

BmNPV resistance of silkworm larvae resulting from the ingestion of TiO₂ nanoparticles.

PubMed

Li, Bing; Xie, Yi; Cheng, Zhe; Cheng, Jie; Hu, Rengping; Gui, Suxin; Sang, Xuezi; Sun, Qingqing; Zhao, Xiaoyang; Sheng, Lei; Shen, Weide; Hong, Fashui

2012-12-01

Bombyx mori nucleopolyhedrovirus (BmNPV) causes infection in the silkworm that is often lethal. The infection is hard to prevent, partly because of the nature of the virus particles and partly because of the different strains of B. mori. Titanium dioxide nanoparticles (TiO₂ NPs) have been demonstrated to have antimicrobial properties. The present study investigated whether TiO₂ NPs added to an artificial diet can increase the resistance of B. mori larvae to BmNPV and examined the molecular mechanism behind any resistance shown. The results indicated that ingested TiO₂ NPs decreased reactive oxygen species and NO accumulation in B. mori larvae under BmNPV infection, which in turn led to a decrease in their growth inhibition and mortality. In addition, the TiO₂ NPs significantly promoted the expression of resistance-related genes, including those encoding superoxide dismutase, catalase, glutathione peroxidase, acetylcholine esterase, carboxylesterase, heat shock protein 21, glutathione S transferase o1, P53, and transferring and of genes encoding cytochrome p302 and nitric oxide synthase. These findings are a useful addition to the understanding of the mechanism of BmNPV resistance of B. mori larvae in response to TiO₂ NPs addition. Such information also provides a theoretical basis for the use of TiO₂ NPs in sericulture.

[Chromatin in diapause of the silkworm Bombyx mori L.: thermal parthenogenesis and normal development].

PubMed

Klimenko, V V; Khaoiuan', Lian

2012-01-01

Having used hematoxylin as a stain, some features of silkworm embryo chromatin in diapause have been studied in normal and parthenogenetic development. With found direct correlation between the number of interphase chromatin grains and the number of chromosomes in the nucleus, we examined cell polyploidization in the embryo at diapause stage. Polyploidization by parthenogenesis is not reducible to endomitotic doubling of the chromosome set because it comprises 6n-nuclei. Explanation of more diverse range of polyploid cells in parthenogenesis needs to consider the fusion of cleavage nuclei that is carried out by the cytoplasmic karyogamic mechanism in the absence of fertilization. For the first time on squash preparations, in diapausing embryo, we have identified primary germ cells (PGC) that are characterized by less compact chromatin, especially in the zygotic form of development, a larger size of the nucleus and cytoplasm, and irregular number and size of nucleoli. Evaluation of PGC ploidy in parthenogenesis by calculation of "loose" chromatin grains in diapause is possible and testifies polyploidization in embryo germ-line. This explains the inevitable admixture of tetraploid eggs in diploid parthenoclone grain and its absence in normal development. Cytological method used has revealed a spiral arrangement of chromatin grains on the inner surface of the nucleus at different levels of ploidy.

Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori.

PubMed

Yang, P-J; Zhan, M-Y; Ye, C; Yu, X-Q; Rao, X-J

2017-12-01

Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern-recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N-acetylmuramoyl-L-alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP-S4, a short-form PGRP from the domesticated silkworm, Bombyx mori. The PGRP-S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP-S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP-S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn 2+ . Scanning electron microscopy showed that PGRP-S4 disrupted the bacterial cell surface. PGRP-S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP-S4 has multiple functions in immunity. © 2017 The Royal Entomological Society.

Antibacterial activity inducible in the haemolymph of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 during the fifth larval instar and pharate adult development.

PubMed

Sumida, M; Ichimori, H; Johchi, S; Takaoka, A; Yuhki, T; Mori, H; Matsubara, F

1992-01-01

1. Antibacterial activity inducible in the haemolymph of the silkworm, Bombyx mori, by immunization, i.e. by injection of formalin-treated Escherichia coli (E. coli) K-12 during the fifth larval instar and pharate adult development that was reared aseptically on an artificial diet was determined by inhibition zone assay using the same bacterium as a test organism. 2. A peak of antibacterial activity was observed in each development stage; approximately 8 mm in diameter of a clear zone at days 3 or 4 in the fifth larval instar and approximately 5 mm at day 1 in the pharate adults. 3. Acid polyacrylamide gel electrophoresis of immunized haemolymph followed by overlay assay showed that an activity band was associated with two peptide bands that were similar to the cecropin-like peptides A and B that were reported in the silkworm (Morishima et al., 1988, Agri. Biol. Chem. 52, 929-934). Any other activity bands were not observed. No activity band was detectable from the haemolymph of non-immunized insects. 4. Fractionation of antibacterial peptides in immunized haemolymph on a CM-cellulose column resulted in separation of two groups of activity, both in the fifth instar larvae and in the pharate adults with a slight difference in elution conditions. 5. Duration of high antibacterial activity induced by a single immunization was approximately 12 hr in the fifth instar day 3 larvae and 48 hr in the day 2 pharate adults.

Deficiency of gustatory sensitivity to some deterrent compounds in "polyphagous" mutant strains of the silkworm, Bombyx mori.

PubMed

Asaoka, K

2000-11-01

Sawa-J, CSJ02 and N601 x C601 are selected mutant strains of Bombyx mori, which grow on various artificial diets or temporarily ingest various plant leaves. To examine the mechanisms mediating diet breadth of caterpillars, gustatory spike responses of the silkworms, called 'polyphagous" strains, were compared with normal strains, N137 x C146 and C02. There were notable differences in their feeding habits and in their sensitivity to salicin in deterrent cells in the maxillary medial styloconic sensilla and the epipharyngeal sensilla. By contrast, the deterrent cells of all strains responded similarly to strychnine nitrate in a dose-dependent manner. In additional comparisons of Sawa-J and N137 x C146, Sawa-J maxillary deterrent cells were significantly less sensitive to phloridzin, amygdalin and arbutin, but responded to some alkaloids and 20-hydroxyecdysone with similar or even higher firing rate. These results suggest that the deficiency of the sensitivity to some deterrent compounds on the deterrent cell of the polyphagous strains may be caused by mutant genes and affects the diet breadth of caterpillars.

Physiological requirements for 20-hydroxyecdysone-induced rectal sac distention in the pupa of the silkworm, Bombyx mori.

PubMed

Suzuki, Takumi; Sakurai, Sho; Iwami, Masafumi

2010-06-01

Successful insect development is achieved via appropriate fluctuation of ecdysteroid levels. When an insect's ecdysteroid level is disrupted, physiological and developmental defects occur. In the pupa of the silkworm, Bombyx mori, the rectal sac is an essential organ that operates as a repository for degraded ecdysteroids, and it can be distended by administration of 20-hydroxyecdysone (20E). Our previous study showed that rectal sac distention appears 4 days after 20E administration. Hemolymph ecdysteroid levels, however, decrease to lower level during this period. Thus, the timing of the rectal sac distention does not match with that of ecdysteroid elevation. Here, we examine how 20E induces rectal sac distention. A ligature experiment and ecdysteroid quantification showed that continuous 20E stimulation induces rectal sac distention. Thorax tissue contributed to the continuous 20E stimulation needed to induce distention. Ecdysteroid released from the thorax tissue may be converted to 20E by ecdysone 20-hydroxylase to produce continuous 20E stimulation. Thus, the ecdysone metabolic pathway plays a critical role in rectal sac distention. Copyright 2010 Elsevier Ltd. All rights reserved.

Differential gene expression during early embryonic development in diapause and non-diapause eggs of multivoltine silkworm Bombyx mori.

PubMed

Ponnuvel, Kangayam M; Murthy, Geetha N; Awasthi, Arvind K; Rao, Guruprasad; Vijayaprakash, Nanjappa B

2010-11-01

Quantification of the differential expression of metabolic enzyme and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm B. mori was carried out by semi-quantitative RT-PCR. Data analysis revealed that, the phosphofructokinase (PFK) expression started at a higher level in the early stage (6 h after oviposition) in non-diapause eggs, while in diapause induced eggs, it started at a lower level. However, the PFK gene expression in diapause eggs was comparatively higher than in non-diapause eggs. PFK facilitates use of carbohydrate reserves. The lower level of PFK gene expression in the early stage of diapause induced eggs but comparatively higher level of expression than in non-diapause eggs is due to enzyme inactivation via protein phosphorylation during early embryogenesis followed by de-phosphorylation in later stage. The sorbitol dehydrogenase-2 (SDH-2) gene was down regulated in diapause induced eggs up to 24 h and its expression levels in diapause induced eggs coincided with that of PFK gene at 48h in non-diapause eggs. During carbohydrate metabolism, there is an initial temporary accumulation of sorbitol which acts as protectant. The down regulation of SDH-2 gene during the first 24 hours in diapause induced eggs was due to the requirement of sorbitol as protectant. However, since the diapause process culminates by 48 h, the SDH-2 gene expression increased and coincided with that of PFK gene expression. The trehalase (Tre) gene expression was at a lower level in diapause induced eggs compared to non-diapausing eggs. The induction of Tre activity is to regulate uptake and use of sugar by the tissues. The non-diapause eggs revealed maximum expression of GPase gene with major fluctuations as well as an overall higher expression compared to diapause induced eggs. The diapause process requires less energy source which reflects lower activity of the gene. Heat shock protein (Hsp) genes (Hsp20.4, 40, 70, and 90

Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes.

PubMed

Kikuchi, Akira; Nakazato, Takeru; Ito, Katsuhiko; Nojima, Yosui; Yokoyama, Takeshi; Iwabuchi, Kikuo; Bono, Hidemasa; Toyoda, Atsushi; Fujiyama, Asao; Sato, Ryoichi; Tabunoki, Hiroko

2017-01-13

Various insect species have been added to genomic databases over the years. Thus, researchers can easily obtain online genomic information on invertebrates and insects. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of subsequent functional analyses. To address this problem, we used a combination of dry and wet bench processes to select functional genes from public databases. Enolase is an important glycolytic enzyme in all organisms. We used a combination of dry and wet bench processes to identify functional enolases in the silkworm Bombyx mori (BmEno). First, we detected five annotated enolases from public databases using a Hidden Markov Model (HMM) search, and then through cDNA cloning, Northern blotting, and RNA-seq analysis, we revealed three functional enolases in B. mori: BmEno1, BmEno2, and BmEnoC. BmEno1 contained a conserved key amino acid residue for metal binding and substrate binding in other species. However, BmEno2 and BmEnoC showed a change in this key amino acid. Phylogenetic analysis showed that BmEno2 and BmEnoC were distinct from BmEno1 and other enolases, and were distributed only in lepidopteran clusters. BmEno1 was expressed in all of the tissues used in our study. In contrast, BmEno2 was mainly expressed in the testis with some expression in the ovary and suboesophageal ganglion. BmEnoC was weakly expressed in the testis. Quantitative RT-PCR showed that the mRNA expression of BmEno2 and BmEnoC correlated with testis development; thus, BmEno2 and BmEnoC may be related to lepidopteran-specific spermiogenesis. We identified and characterized three functional enolases from public databases with a combination of dry and wet bench processes in the silkworm B. mori. In addition, we determined that BmEno2 and BmEnoC had species-specific functions. Our strategy could be helpful for the detection of minor genes and functional genes in non-model organisms from public databases.

Diet-consumer nitrogen isotope fractionation for prolonged fasting arthropods.

PubMed

Mizota, Chitoshi; Yamanaka, Toshiro

2011-12-01

Nitrogen acquisition for cellular metabolism during diapause is a primary concern for herbivorous arthropods. Analyses of naturally occurring stable isotopes of nitrogen help elucidate the mechanism. Relevant articles have cited (58 times up to mid-June 2011) anomalously elevated δ(15)N (per mil deviation of (15)N/(14)N, relative to atmospheric nitrogen=0 ‰) values (diet-consumer nitrogen isotope fractionation; up to 12 ‰) for a prolonged fasting raspberry beetle (Byturus tomentosus Degeer (Coleoptera: Byturidae)), which feeds on red raspberries (Rubus idaeus: δ(15)N= ~ +2 ‰). Biologists have hypothesised that extensive recycling of amino acid nitrogen is responsible for the prolonged fasting. Since this hypothesis was proposed in 1995, scientists have integrated biochemical and molecular knowledge to support the mechanism of prolonged diapausing of animals. To test the validity of the recycling hypothesis, we analysed tissue nitrogen isotope ratios for four Japanese arthropods: the shield bug Parastrachia japonensis Scott (Hemiptera: Cydnidae), the burrower bug Canthophorus niveimarginatus Scott (Hemiptera: Cydnidae), leaf beetle Gastrophysa atrocyanea Motschulsky (Coleoptera: Chrysomelidae) and the Japanese oak silkworm Antheraea yamamai (Lepidoptera: Saturniidae), all of which fast for more than 6 months as part of their life-history strategy. Resulting diet-consumer nitrogen isotope discrimination during fasting ranged from 0 to 7‰, as in many commonly known terrestrial arthropods. We conclude that prolonged fasting of arthropods does not always result in anomalous diet-consumer nitrogen isotope fractionation, since the recycling process is closed or nearly closed with respect to nitrogen isotopes.

Development of a method for long-term preservation of Bombyx mori silkworm strains using frozen ovaries.

PubMed

Banno, Yutaka; Nagasaki, Kiyomi; Tsukada, Marino; Minohara, Yuko; Banno, Junko; Nishikawa, Kazuhiro; Yamamoto, Kazunori; Tamura, Kei; Fujii, Tsuguru

2013-06-01

Development of long-term preservation is essential for conservation of stocks of silkworm genetic resources. Thus far, a few methods have been reported, but more improvement is required for practical use. We have developed two effective modifications of a method for long-term preservation using frozen ovaries. One was slow cooling (1 °C per min) until -80 °C of the donor ovaries made possible by use of a BICELL freezing vessel. Using donor ovaries of 4th instar larvae, the average number of eggs laid per moth increased significantly from 110.7 ± 53.4 eggs per moth by slow cooling with the BICELL vessel vs 12.3 ± 10.3 eggs per moth by direct cooling in liquid nitrogen. A second improvement was connecting the thread bodies of the donor ovaries with those of the host in the transplantation step. Females operated on with the new method yielded a significantly higher percentage of moths that laid fertilized eggs than those transplanted with the standard procedure (70.4 ± 21.6% vs 22.9 ± 9.3%). Copyright © 2013 Elsevier Inc. All rights reserved.

Effect of degumming time on silkworm silk fibre for biodegradable polymer composites

NASA Astrophysics Data System (ADS)

Ho, Mei-po; Wang, Hao; Lau, Kin-tak

2012-02-01

Recently, many studies have been conducted on exploitation of natural materials for modern product development and bioengineering applications. Apart from plant-based materials (such as sisal, hemp, jute, bamboo and palm fibre), animal-based fibre is a kind of sustainable natural materials for making novel composites. Silkworm silk fibre extracted from cocoon has been well recognized as a promising material for bio-medical engineering applications because of its superior mechanical and bioresorbable properties. However, when producing silk fibre reinforced biodegradable/bioresorbable polymer composites, hydrophilic sericin has been found to cause poor interfacial bonding with most polymers and thus, it results in affecting the resultant properties of the composites. Besides, sericin layers on fibroin surface may also cause an adverse effect towards biocompatibility and hypersensitivity to silk for implant applications. Therefore, a proper pre-treatment should be done for sericin removal. Degumming is a surface modification process which allows a wide control of the silk fibre's properties, making the silk fibre possible to be used for the development and production of novel bio-composites with unique/specific mechanical and biodegradable properties. In this paper, a cleaner and environmentally friendly surface modification technique for tussah silk in polymer based composites is proposed. The effectiveness of different degumming parameters including degumming time and temperature on tussah silk is discussed through the analyses of their mechanical and morphological properties. Based on results obtained, it was found that the mechanical properties of tussah silk are affected by the degumming time due to the change of the fibre structure and fibroin alignment.

Positional cloning of a gene responsible for the cts mutation of the silkworm, Bombyx mori.

PubMed

Ito, Katsuhiko; Kidokoro, Kurako; Katsuma, Susumu; Shimada, Toru; Yamamoto, Kimiko; Mita, Kazuei; Kadono-Okuda, Keiko

2012-07-01

The larval head cuticle and anal plates of the silkworm mutant cheek and tail spot (cts) have chocolate-colored spots, unlike the entirely white appearance of the wild-type (WT) strain. We report the identification and characterization of the gene responsible for the cts mutation. Positional cloning revealed a cts candidate on chromosome 16, designated BmMFS, based on the high similarity of the deduced amino acid sequence between the candidate gene from the WT strain and the major facilitator superfamily (MFS) protein. BmMFS likely encodes a membrane protein with 11 putative transmembrane domains, while the putative structure deduced from the cts-type allele possesses only 10-pass transmembrane domains owing to a deletion in its coding region. Quantitative RT-PCR analysis showed that BmMFS mRNA was strongly expressed in the integument of the head and tail, where the cts phenotype is observed; expression markedly increased at the molting and newly ecdysed stages. These results indicate that the novel BmMFS gene is cts and the membrane structure of its protein accounts for the cts phenotype. These expression profiles and the cts phenotype are quite similar to those of melanin-related genes, such as Bmyellow-e and Bm-iAANAT, suggesting that BmMFS is involved in the melanin synthesis pathway.

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle

PubMed Central

Daimon, Takaaki; Uchibori, Miwa; Nakao, Hajime; Sezutsu, Hideki; Shinoda, Tetsuro

2015-01-01

Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor. PMID:26195792

Knockout silkworms reveal a dispensable role for juvenile hormones in holometabolous life cycle.

PubMed

Daimon, Takaaki; Uchibori, Miwa; Nakao, Hajime; Sezutsu, Hideki; Shinoda, Tetsuro

2015-08-04

Insect juvenile hormones (JHs) prevent precocious metamorphosis and allow larvae to undergo multiple rounds of status quo molts. However, the roles of JHs during the embryonic and very early larval stages have not been fully understood. We generated and characterized knockout silkworms (Bombyx mori) with null mutations in JH biosynthesis or JH receptor genes using genome-editing tools. We found that embryonic growth and morphogenesis are largely independent of JHs in Bombyx and that, even in the absence of JHs or JH signaling, pupal characters are not formed in first- or second-instar larvae, and precocious metamorphosis is induced after the second instar at the earliest. We also show by mosaic analysis that a pupal specifier gene broad, which is dramatically up-regulated in the late stage of the last larval instar, is essential for pupal commitment in the epidermis. Importantly, the mRNA expression level of broad, which is thought to be repressed by JHs, remained at very low basal levels during the early larval instars of JH-deficient or JH signaling-deficient knockouts. Therefore, our study suggests that the long-accepted paradigm that JHs maintain the juvenile status throughout larval life should be revised because the larval status can be maintained by a JH-independent mechanism in very early larval instars. We propose that the lack of competence for metamorphosis during the early larval stages may result from the absence of an unidentified broad-inducing factor, i.e., a competence factor.

Tetra-heteroatom self-doped carbon nanosheets derived from silkworm excrement for high-performance supercapacitors

NASA Astrophysics Data System (ADS)

Lei, Shuijin; Chen, Lianfu; Zhou, Wei; Deng, Peiqin; Liu, Yan; Fei, Linfeng; Lu, Wei; Xiao, Yanhe; Cheng, Baochang

2018-03-01

Carbon materials are deemed to be competitive candidate electrode materials for energy storage systems. It is still a great challenge to explore advanced carbon-based electrode materials for high-performance supercapacitors by a facile, economical and efficient method. In this work, N-, P-, S-, O-self-doped carbon nanosheets with high surface area and well-developed porosity are successfully prepared by pyrolysis carbonization and post KOH activation from silkworm excrement, a novel abundant, low-cost and eco-friendly agricultural waste. Thanks to their unique multi-heteroatom doping and porous structure, the obtained carbon materials exhibit high charge storage capacity with a specific capacitance of 401 F g-1 at a current density of 0.5 A g-1 in 6 M KOH and good cycling stability with a capacitance retention of 93.8% over 10000 cycles. A symmetric supercapacitor device using 1 M Na2SO4 aqueous solution as the electrolyte can deliver a specific capacitance of 41.7 F g-1 at a current density of 0.5 A g-1, and a high energy density of 23.17 Wh kg-1 at a power density of 500 W kg-1 with a wide voltage window of 2.0 V. This work develops a new strategy to produce favorable carbon-based electrode materials for supercapacitors with high electrochemical performances.

Molecular and biochemical responses in the midgut of the silkworm, Bombyx mori, infected with Nosema bombycis.

PubMed

Li, Zhi; Wang, Yu; Wang, Linling; Zhou, Zeyang

2018-03-06

Microsporidia are a group of eukaryotic intracellular parasites that infect almost all vertebrates and invertebrates. However, there is little information available of how microsporidia obtain nutrients and energy from host cells. The purpose of this study was to investigate the energy and material requirements of Nosema bombycis for the invasion procedure through analyzing the global variation of the gene expression, protein abundance, fatty acids level and ATP flux induced by the microsporidia N. bombycis infection in the midgut of the silkworm Bombyx mori. A suppression subtractive hybridization (SSH) and quantitative real-time PCR (qPCR) analysis were performed to identify the genes upregulated in the midgut of B. mori 48 h following N. bombycis infection. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to annotate and summarize the differentially expressed genes, according to the categories 'molecular function', 'cellular component' and 'biological process'. To evaluate the nutrition material and energy costs in B.mori infected by N. bombycis, biochemical analysis was performed to determine the variation of protein abundance, fatty acid levels and ATP flux with or without the microsporidia N. bombycis infection in the midgut of the silkworm B. mori. A total of 744 clones were obtained, 288 clones were randomly selected for sequencing, and 110 unigenes were generated. Amongst these, 49.21%, 30.16% and 14.29% genes were involved in 19 molecular functions, 19 biological processes and nine cellular components, respectively. A total of 11 oxidative phosphorylation- and eight proton-coupled ATP synthesis-related genes were upregulated. Seven protein degradation-, three fat degradation-related genes were upregulated, and no genes related to the de novo synthesis of amino acids and fatty acids were significantly upregulated. The data from the biochemical analysis showed the contents of total protein and ATP of B. mori

The optical properties of regenerated silk fibroin films obtained from different sources

NASA Astrophysics Data System (ADS)

Perotto, Giovanni; Zhang, Yuji; Naskar, Deboki; Patel, Nereus; Kaplan, David L.; Kundu, Subhas C.; Omenetto, Fiorenzo G.

2017-09-01

Silk fibroin possesses unique properties for bio-functional optical interfaces and has been attracting increasing interest as an optical material. Here, we report on the refractive index and absorption coefficient of silk fibroin extracted from Bombyx mori, Antheraea mylitta, Samia ricini, and Antheraea assamensis. The influence of protein molecular weight, residual water content, and crystallinity on refractive index was investigated. The parameters for the Cauchy dispersion law and Urbach absorption were determined for each of the silk fibroins. By exploiting the differences in refractive index between the different fibroins, an all-protein slab waveguide was fabricated.

BmRobo1a and BmRobo1b control axon repulsion in the silkworm Bombyx mori.

PubMed

Li, Xiao-Tong; Yu, Qi; Zhou, Qi-Sheng; Zhao, Xiao; Liu, Zhao-Yang; Cui, Wei-Zheng; Liu, Qing-Xin

2016-02-15

The development of the nervous system is based on the growth and connection of axons, and axon guidance molecules are the dominant regulators during this course. Robo, as the receptor of axon guidance molecule Slit, plays a key role as a conserved repellent cue for axon guidance during the development of the central nervous system. However, the function of Robo in the silkworm Bombyx mori is unknown. In this study, we cloned two novel robo genes in B. mori (Bmrobo1a and Bmrobo1b). BmRobo1a and BmRobo1b lack an Ig and a FNIII domain in the extracellular region and the CC0 and CC2 motifs in the intracellular region. BmRobo1a and BmRobo1b were colocalized with BmSlit in the neuropil. Knock-down of Bmrobo1a and Bmrobo1b by RNA interference (RNAi) resulted in abnormal development of axons. Our results suggest that BmRobo1a and BmRobo1b have repulsive function in axon guidance, even though their structures are different from Robo1 of other species. Copyright © 2015 Elsevier B.V. All rights reserved.

Consumption of Bt Rice Pollen Containing Cry1C or Cry2A Protein Poses a Low to Negligible Risk to the Silkworm Bombyx mori (Lepidoptera: Bombyxidae)

PubMed Central

Yang, Yan; Liu, Yue; Cao, Fengqin; Chen, Xiuping; Cheng, Lisheng; Romeis, Jörg; Li, Yunhe; Peng, Yufa

2014-01-01

By consuming mulberry leaves covered with pollen from nearby genetically engineered, insect-resistant rice lines producing Cry proteins derived from Bacillus thuringiensis (Bt), larvae of the domestic silkworm, Bombyx mori (Linnaeus) (Lepidoptera: Bombyxidae), could be exposed to insecticidal proteins. Laboratory experiments were conducted to assess the potential effects of Cry1C- or Cry2A-producing transgenic rice (T1C-19, T2A-1) pollen on B. mori fitness. In a short-term assay, B. mori larvae were fed mulberry leaves covered with different densities of pollen from Bt rice lines or their corresponding near isoline (control) for the first 3 d and then were fed mulberry leaves without pollen. No effect was detected on any life table parameter, even at 1800 pollen grains/cm2 leaf, which is much higher than the mean natural density of rice pollen on leaves of mulberry trees near paddy fields. In a long-term assay, the larvae were fed Bt and control pollen in the same way but for their entire larval stage (approximately 27 d). Bt pollen densities ≥150 grains/cm2 leaf reduced 14-d larval weight, increased larval development time, and reduced adult eclosion rate. ELISA analyses showed that 72.6% of the Cry protein was still detected in the pollen grains excreted with the feces. The low exposure of silkworm larvae to Cry proteins when feeding Bt rice pollen may be the explanation for the relatively low toxicity detected in the current study. Although the results demonstrate that B. mori larvae are sensitive to Cry1C and Cry2A proteins, the exposure levels that harmed the larvae in the current study are far greater than natural exposure levels. We therefore conclude that consumption of Bt rice pollen will pose a low to negligible risk to B. mori. PMID:25014054

In vivo effects of metal ions on conformation and mechanical performance of silkworm silks.

PubMed

Wang, Xin; Li, Yi; Liu, Qingsong; Chen, Quanmei; Xia, Qingyou; Zhao, Ping

2017-03-01

The mechanism of silk fiber formation is of particular interest. Although in vitro evidence has shown that metal ions affect conformational transitions of silks, the in vivo effects of metal ions on silk conformations and mechanical performance are still unclear. This study explored the effects of metal ions on silk conformations and mechanical properties of silk fibers by adding K + and Cu 2+ into the silk fibroin solutions or injecting them into the silkworms. Aimed by CD analysis, FTIR analysis, and mechanical testing, the conformational and mechanical changes of the silks were estimated. By using BION Web Server, the interactions of K + and N-terminal of silk fibroin were also simulated. We presented that K + and Cu 2+ induced the conformational transitions of silk fibroin by forming β-sheet structures. Moreover, the mechanical parameters of silk fibers, such as strength, toughness and Young's modulus, were also improved after K + or Cu 2+ injection. Using BION Web Server, we found that potassium ions may have strong electrostatic interactions with the negatively charged residues. We suggest that K + and Cu 2+ play crucial roles in the conformation and mechanical performances of silks and they are involved in the silk fiber formation in vivo. Our results are helpful for clarifying the mechanism of silk fiber formation, and provide insights for modifying the mechanical properties of silk fibers. Copyright © 2016 Elsevier B.V. All rights reserved.

The Expression, Purification, and Characterization of a Ras Oncogene (Bras2) in Silkworm (Bombyx mori).

PubMed

Lv, Zhengbing; Wang, Tao; Zhuang, Wenhua; Wang, Dan; Chen, Jian; Nie, Zuoming; Liu, Lili; Zhang, Wenping; Wang, Lisha; Wang, Deming; Wu, Xiangfu; Li, Jun; Qian, Lian; Zhang, Yaozhou

2013-01-01

The Ras oncogene of silkworm pupae (Bras2) may belong to the Ras superfamily. It shares 77% of its amino acid identity with teratocarcinoma oncogene 21 (TC21) related ras viral oncogene homolog-2 (R-Ras2) and possesses an identical core effector region. The mRNA of Bombyx mori Bras2 has 1412 bp. The open reading frame contains 603 bp, which encodes 200 amino acid residues. This recombinant BmBras2 protein was subsequently used as an antigen to raise a rabbit polyclonal antibody. Western blotting and real-time PCR analyses showed that BmBras2 was expressed during four developmental stages. The BmBras2 expression level was the highest in the pupae and was low in other life cycle stages. BmBras2 was expressed in all eight tested tissues, and it was highly expressed in the head, intestine, and epidermis. Subcellular localization studies indicated that BmBras2 was predominantly localized in the nuclei of Bm5 cells, although cytoplasmic staining was also observed to a lesser extent. A cell proliferation assay showed that rBmBras2 could stimulate the proliferation of hepatoma cells. The higher BmBras2 expression levels in the pupal stage, tissue expression patterns, and a cell proliferation assay indicated that BmBras2 promotes cell division and proliferation, most likely by influencing cell signal transduction.

Effect of cadmium-feeding on tissue concentrations of elements in germ-free silkworm (Bombyx mori) larvae and distribution of cadmium in the alimentary canal.

PubMed

Suzuki, K T; Aoki, Y; Nishikawa, M; Masui, H; Matsubara, F

1984-01-01

Silkworm (Bombyx mori) larvae were reared on an artificial diet containing cadmium (Cd) at concentrations of 5 and 80 micrograms/g wet diet from hatching to the fourth instar and then for 5 days at the fifth instar, respectively. Concentrations of Cd and other elements in the alimentary canal, Malpighian tubes, silk gland, fat body and other organs were determined simultaneously by inductively coupled argon plasma-atomic emission spectrometry. Cd was accumulated in the alimentary canal and Malpighian tubes at concentrations of 1100 and 470 micrograms/g dry wt, respectively. The distribution of Cd in the supernatants of the two highly accumulated organs were determined on an SW column by high performance liquid chromatography-atomic absorption spectrophotometry. Cd was primarily bound to inducible high molecular weight Cd-binding proteins.

Analyzing the promoters of two CYP9A genes in the silkworm Bombyx mori by dual-luciferase reporter assay.

PubMed

Zhao, Si-Si; Zhao, Guo-Dong; Di, Tian-Yuan; Ding, Hua; Wan, Xiao-Ling; Li, Bing; Chen, Yu-Hua; Xu, Ya-Xiang; Shen, Wei-De; Wei, Zheng-Guo

2013-02-01

Cytochrome P450s (CYPs) are widespread proteins that interact with exogenous chemicals from the diet or the environment. CYP9A subfamily genes are important in the silkworm Bombyx mori. We previously reported transcriptional levels of two CYP9A genes in different tissues and their responses to sodium fluoride (NaF). In this study, promoter truncation analysis using a dual-luciferase reporter assay in B. mori ovary cells (BmN) showed that the regions -1,496 to -1,102 bp for CYP9A19, and -1,630 to -1,210 bp for CYP9A22 were essential for basal transcriptional activity. Sequence analysis of these regions revealed several transcriptional regulatory elements but no typical promoter elements. Promoter activities were regulated after NaF induction and with an obvious dose effect. Although the dual-luciferase assay has been widely used to determine the activity of a given promoter in cell lines, problems with it still exist. Our results indicate that both plasmid size and construct protocols affect the experimental results.

Silk: Optical Properties over 12.6 Octaves THz-IR-Visible-UV Range

PubMed Central

Balčytis, Armandas; Ryu, Meguya; Wang, Xuewen; Novelli, Fabio; Seniutinas, Gediminas; Du, Shan; Wang, Xungai; Li, Jingliang; Davis, Jeffrey; Appadoo, Dominique; Morikawa, Junko; Juodkazis, Saulius

2017-01-01

Domestic (Bombyx mori) and wild (Antheraea pernyi) silk fibers were characterised over a wide spectral range from THz 8 cm−1 (λ= 1.25 mm, f= 0.24 THz) to deep-UV 50×103 cm−1 (λ= 200 nm, f= 1500 THz) wavelengths or over a 12.6 octave frequency range. Spectral features at β-sheet, α-coil and amorphous fibroin were analysed at different spectral ranges. Single fiber cross sections at mid-IR were used to determine spatial distribution of different silk constituents and revealed an α-coil rich core and more broadly spread β-sheets in natural silk fibers obtained from wild Antheraea pernyi moths. Low energy T-ray bands at 243 and 229 cm−1 were observed in crystalline fibers of domestic and wild silk fibers, respectively, and showed no spectral shift down to 78 K temperature. A distinct 20±4 cm−1 band was observed in the crystalline Antheraea pernyi silk fibers. Systematic analysis and assignment of the observed spectral bands is presented. Water solubility and biodegradability of silk, required for bio-medical and sensor applications, are directly inferred from specific spectral bands. PMID:28772716

Molecular tracing of white muscardine in the silkworm, Bombyx mori (Linn.) II. Silkworm white muscardine is not caused by artificial release or natural epizootic of Beauveria bassiana in China.

PubMed

Chen, Xue; Huang, Cui; He, Lingmin; Zhang, Shengli; Li, Zengzhi

2015-02-01

The fungal pathogen Beauveria bassiana causes serious economic losses in sericulture. Its origin is usually attributed to the release of B. bassiana insecticides against pine caterpillars (Dendrolimus punctuatus). In the present study, 488 B. bassiana isolates obtained from silkworm (Bombyx mori) collected from 13 Chinese provinces, and 327 B. bassiana isolates obtained from D. punctatus collected from 9 provinces, were analyzed for population genetic structure using the ISSR technique based on genetic distance. A UPGMA dendrogram clustered them into three independent clades: two B. mori clades and one D. punctatus clade. A 3-D principal component analysis further divided them into two completely independent host groups, revealing high host-specificity. This suggested that white muscardine occurring in B. mori populations throughout southern China was not caused by any B. bassiana strain either naturally prevailing in D. punctatus populations or by any strain artificially released as a fungal insecticide against D. punctatus. We further investigated the genetic differentiation coefficient Gst and gene flow between B. mori-pathogenic and D. punctatus-pathogenic B. bassiana isolates from across China and from five provinces inhabited by both B. mori and D. punctatus. The Gst value across China was computed as 0.410, while the values of the five provinces ranged from 0.508 to 0.689; all above 0.25, which is the threshold for significant genetic differentiation. This suggests that B. bassiana strains isolated from the two different hosts maintained their respective heredity without a convergent homogenization trend, and reduces the possibility that the host range of the caterpillar isolates could expand and enhance their virulence in B. mori. These findings indicate that the use of B. bassiana does not threaten the safety of sericulture. Copyright © 2014 Elsevier Inc. All rights reserved.

Transcriptome analysis of the brain of the silkworm Bombyx mori infected with Bombyx mori nucleopolyhedrovirus: A new insight into the molecular mechanism of enhanced locomotor activity induced by viral infection.

PubMed

Wang, Guobao; Zhang, Jianjia; Shen, Yunwang; Zheng, Qin; Feng, Min; Xiang, Xingwei; Wu, Xiaofeng

2015-06-01

Baculoviruses have been known to induce hyperactive behavior in their lepidopteran hosts for over a century. As a typical lepidopteran insect, the silkworm Bombyx mori displays enhanced locomotor activity (ELA) following infection with B. mori nucleopolyhedrovirus (BmNPV). Some investigations have focused on the molecular mechanisms underlying this abnormal hyperactive wandering behavior due to the virus; however, there are currently no reports about B. mori. Based on previous studies that have revealed that behavior is controlled by the central nervous system, the transcriptome profiles of the brains of BmNPV-infected and non-infected silkworm larvae were analyzed with the RNA-Seq technique to reveal the changes in the BmNPV-infected brain on the transcriptional level and to provide new clues regarding the molecular mechanisms that underlies BmNPV-induced ELA. Compared with the controls, a total of 742 differentially expressed genes (DEGs), including 218 up-regulated and 524 down-regulated candidates, were identified, of which 499, 117 and 144 DEGs could be classified into GO categories, KEGG pathways and COG annotations by GO, KEGG and COG analyses, respectively. We focused our attention on the DEGs that are involved in circadian rhythms, synaptic transmission and the serotonin receptor signaling pathway of B. mori. Our analyses suggested that these genes were related to the locomotor activity of B. mori via their essential roles in the regulations of a variety of behaviors and the down-regulation of their expressions following BmNPV infection. These results provide new insight into the molecular mechanisms of BmNPV-induced ELA. Copyright © 2015 Elsevier Inc. All rights reserved.

Protective Effect of Boiled and Freeze-dried Mature Silkworm Larval Powder Against Diethylnitrosamine-induced Hepatotoxicity in Mice.

PubMed

Cho, Jae-Min; Kim, Kee-Young; Ji, Sang-Deok; Kim, Eun-Hee

2016-09-01

Hepatocellular carcinoma (HCC) is a representative inflammation-associated cancer and known to be the most frequent tumors. HCC may also induce important pro- and anti-tumor immune reactions. However, the underlying mechanisms are unsatisfactorily identified. We investigated the protective effect of boiled and freeze-dried mature silkworm larval powder (BMSP) on diethylnitrosamine (DEN)-induced hepatotoxicity in mice. Mice were fed with diet containing BMSP (0.1, 1, and 10 g/kg) for two weeks and DEN (100 mg/kg, intraperitoneally) was injected 18 hours before the end of this experiment. Liver toxicity was determined in serum and histopathological examination was assessed in the liver tissues. Infiltration of immune cells and expressions of inflammatory cytokines and chemokines were also examined. Pretreatment with BMSP reduced necrotic and histopathological changes induced by DEN in the liver. Measurement of serum biochemical indicators, the levels of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase, showed that pretreatment with BMSP also decreased DEN-induced hepatotoxicity. In addition, BMSP inhibited the macrophage and CD31 infiltration in a dose-dependent manner. The expressions of interleukin-1β, IFN-γ and chemokines for T cell activation were decreased in BMSP pretreatment groups. BMSP may have a protective effect against acute liver injury by inhibiting necrosis and inflammatory response in DEN-treated mice.

Analysis of oligomeric transition of silkworm small heat shock protein sHSP20.8 using high hydrostatic pressure native PAGE

NASA Astrophysics Data System (ADS)

Fujisawa, Tetsuro; Ueda, Toshifumi; Kameyama, Keiichi; Aso, Yoichi; Ishiguro, Ryo

2013-06-01

The small heat shock proteins (sHSPs) solubilize thermo-denatured proteins without adenosine triphosphate energy consumption to facilitate protein refolding. sHSP20.8 is one of the silkworm (Bombyx mori) sHSPs having only one cystein in the N-terminal domain: Cys43. We report a simple measurement of oligomeric transition of sHSP20.8 using high hydrostatic pressure native polyacrylamide gel electrophoresis (high hydrostatic pressure (HP) native polyacrylamide gel electrophoresis (PAGE)). At ambient pressure under oxydative condition, the native PAGE of thermal transition of sHSP20.8 oligomer displayed a cooperative association. In contrast, HP native PAGE clearly demonstrated that sHSP20.8 dissociated at 80 MPa and 25°C, and the resultant molecular species gradually reassociated with time under that condition. In addition, the reassociation process was suppressed in the presence of the reductant. These results are consistent with the idea that sHSP20.8 oligomer temporally dissociates at the first thermo-sensing step and reassociates with the oxidation of Cys43.

A serine protease homologue Bombyx mori scarface induces a short and fat body shape in silkworm.

PubMed

Wang, R-X; Tong, X-L; Gai, T-T; Li, C-L; Qiao, L; Hu, H; Han, M-J; Xiang, Z-H; Lu, C; Dai, F-Y

2018-06-01

Body shape is one of the most prominent and basic characteristics of any organism. In insects, abundant variations in body shape can be observed both within and amongst species. However, the molecular mechanism underlying body shape fine-tuning is very complex and has been largely unknown until now. In the silkworm Bombyx mori, the tubby (tub) mutant has an abnormal short fat body shape and the abdomen of tub larvae expands to form a fusiform body shape. Morphological investigation revealed that the body length was shorter and the body width was wider than that of the Dazao strain. Thus, this mutant is a good model for studying the molecular mechanisms of body shape fine-tuning. Using positional cloning, we identified a gene encoding the serine protease homologue, B. mori scarface (Bmscarface), which is associated with the tub phenotype. Sequence analysis revealed a specific 312-bp deletion from an exon of Bmscarface in the tub strain. In addition, recombination was not observed between the tub and Bmscarface loci. Moreover, RNA interference of Bmscarface resulted in the tub-like phenotype. These results indicate that Bmscarface is responsible for the tub mutant phenotype. This is the first study to report that mutation of a serine protease homologue can induce an abnormal body shape in insects. © 2018 The Royal Entomological Society.

Precocious Metamorphosis in the Juvenile Hormone–Deficient Mutant of the Silkworm, Bombyx mori

PubMed Central

Daimon, Takaaki; Kozaki, Toshinori; Niwa, Ryusuke; Kobayashi, Isao; Furuta, Kenjiro; Namiki, Toshiki; Uchino, Keiro; Banno, Yutaka; Katsuma, Susumu; Tamura, Toshiki; Mita, Kazuei; Sezutsu, Hideki; Nakayama, Masayoshi; Itoyama, Kyo; Shimada, Toru; Shinoda, Tetsuro

2012-01-01

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several “moltinism” mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval–larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval–pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH–deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis. PMID:22412378

Differential effects of sugar-mimic alkaloids in mulberry latex on sugar metabolism and disaccharidases of Eri and domesticated silkworms: enzymatic adaptation of Bombyx mori to mulberry defense.

PubMed

Hirayama, Chikara; Konno, Kotaro; Wasano, Naoya; Nakamura, Masatoshi

2007-12-01

Mulberry leaves (Morus spp.) exude latex rich in sugar-mimic alkaloids, 1,4-dideoxy-1,4-imino-d-arabinitol (d-AB1) and 1-deoxynojirimycin (DNJ), as a defense against herbivorous insects. Sugar-mimic alkaloids are inhibitors of sugar-metabolizing enzymes, and are toxic to the Eri silkworm, Samia ricini, a generalist herbivore, but not at all to the domesticated silkworm, Bombyx mori, a mulberry specialist. To address the phenomena, we fed both larvae diets containing different sugar sources (sucrose, glucose or none) with or without sugar-mimic alkaloids from mulberry latex. In S. ricini, addition of sugar-mimic alkaloids to the sucrose (the major sugar in mulberry leaves) diet reduced both growth and the absorption ratio of sugar, but it reduced neither in B. mori. The midgut soluble sucrase activity of S. ricini was low and inhibited by very low concentrations of sugar-mimic alkaloids (IC(50)=0.9-8.2microM), but that of B. mori was high and not inhibited even by very high concentrations (IC(50)>1000microM) of sugar-mimic alkaloids. In S. ricini, the addition of sugar-mimic alkaloids to the glucose diet still had considerable negative effects on growth, although it did not reduce the absorption ratio of glucose. The hemolymph of S. ricini fed sugar-mimic alkaloids contained sugar-mimic alkaloids. The trehalose concentration in the hemolymph increased significantly in S. ricini fed sugar-mimic alkaloids, but not in B. mori. The trehalase activities of S. ricini were lower and inhibited by lower concentrations of sugar-mimic alkaloids than those of B. mori. These results suggest that sugar-mimic alkaloids in mulberry latex exert toxicity to S. ricini larvae first by inhibiting midgut sucrase and digestion of sucrose, and secondly, after being absorbed into hemolymph, by inhibiting trehalase and utilization of trehalose, the major blood sugar. Further, our results reveal that B. mori larvae evolved enzymatic adaptation to mulberry defense by developing sucrase and

Selection of reference genes for tissue/organ samples on day 3 fifth-instar larvae in silkworm, Bombyx mori.

PubMed

Wang, Genhong; Chen, Yanfei; Zhang, Xiaoying; Bai, Bingchuan; Yan, Hao; Qin, Daoyuan; Xia, Qingyou

2018-06-01

The silkworm, Bombyx mori, is one of the world's most economically important insect. Surveying variations in gene expression among multiple tissue/organ samples will provide clues for gene function assignments and will be helpful for identifying genes related to economic traits or specific cellular processes. To ensure their accuracy, commonly used gene expression quantification methods require a set of stable reference genes for data normalization. In this study, 24 candidate reference genes were assessed in 10 tissue/organ samples of day 3 fifth-instar B. mori larvae using geNorm and NormFinder. The results revealed that, using the combination of the expression of BGIBMGA003186 and BGIBMGA008209 was the optimum choice for normalizing the expression data of the B. mori tissue/organ samples. The most stable gene, BGIBMGA003186, is recommended if just one reference gene is used. Moreover, the commonly used reference gene encoding cytoplasmic actin was the least appropriate reference gene of the samples investigated. The reliability of the selected reference genes was further confirmed by evaluating the expression profiles of two cathepsin genes. Our results may be useful for future studies involving the quantification of relative gene expression levels of different tissue/organ samples in B. mori. © 2018 Wiley Periodicals, Inc.

Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

PubMed Central

Ciufo, Leonora F.; Murray, Patricia A.; Thompson, Anu; Rigden, Daniel J.; Rees, Huw H.

2011-01-01

Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction. Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation. PMID:21738635

BmDredd is an initiator caspase and participates in Emodin-induced apoptosis in the silkworm, Bombyx mori.

PubMed

Wang, La; Song, Juan; Bao, Xi-Yan; Chen, Peng; Yi, Hua-Shan; Pan, Min-Hui; Lu, Cheng

2016-10-15

The identification and analysis of the caspases is essential to research into apoptosis in lepidoptera insects. The domesticated silkworm, Bombyx mori, is the model system for lepidopterans. In this study, we cloned and characterized a B. mori Dredd gene, BmDredd, the proposed insect homologue of human caspase-8, which encoded a polypeptide of 543 amino acids. BmDredd possesses a long N-terminal prodomain, a p20 domain, and a p10 domain. When transiently expressed in Escherichia coli cells, BmDredd underwent spontaneous cleavage and exhibited high proteolytic activity for caspase-8 substrate but relatively low for caspase-3 or -9 substrate. In addition, BmDredd induced apoptosis when transiently expressed in BmN-SWU1 cells, an ovarian cell line of B. mori. Moreover, after the treatment of Emodin, a novel apoptosis inducer, endogenous BmDredd expression level, the caspase-8 activity and the apoptotic rate increased notably in BmN-SWU1 cells. When BmDredd was subjected to interference in BmN-SWU1 cells and Emodin treatment, BmDredd expression levels decreased and the apoptotic rate also decreased significantly. These results suggest BmDredd is the homologue of human caspase-8 and plays a role in Emodin-induced apoptosis in BmN-SWU1 cells of B. mori. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of TiO2 NPs on Silkworm Growth and Feed Efficiency.

PubMed

Li, YangYang; Ni, Min; Li, FanChi; Zhang, Hua; Xu, KaiZun; Zhao, XiaoMing; Tian, JiangHai; Hu, JingSheng; Wang, BinBin; Shen, WeiDe; Li, Bing

2016-02-01

Silkworm (Bombyx mori) (B. mori) is an economically important insect and a model species for Lepidoptera. It has been reported that feeding of low concentrations of titanium dioxide nanoparticles (TiO2 NPs) can improve feed efficiency and increase cocoon mass, cocoon shell mass, and the ratio of cocoon shell. However, high concentrations of TiO2 NPs are toxic. In this study, we fed B. mori with different concentrations of TiO2 NPs (5, 10, 20, 40, 80, and 160 mg/L) and investigated B. mori growth, feed efficiency, and cocoon quality. We found that low concentrations of TiO2 NPs (5 and 10 mg/L) were more effective for weight gains, with significant weight gain being obtained at 72 h (P < 0.05). TiO2 NPs at 20 mg/L or higher had certain inhibitory effects, with significant inhibition to B. mori growth being observed at 48 h. The feed efficiency was significantly improved at low concentrations of 5 and 10 mg/L for 14.6 and 13.1 %, respectively (P < 0.05). All B. mori fed with TiO2 NPs showed increased cocoon mass and cocoon shell mass; at 5 and 10 mg/L TiO2 NPs, cocoon mass was significantly increased by 8.29 and 9.39 %, respectively (P < 0.05). We also found that low concentrations (5 and 10 mg/L) of TiO2 NPs promoted B. mori growth and development, improved feed efficiency, and increased cocoon production, while high concentrations (20 mg/L or higher) of TiO2 NPs showed inhibitory effect to the B. mori. Consecutive feeding of high concentrations of TiO2 NPs led to some degrees of adaptability. This study provides a reference for the research on TiO2 NPs toxicity and the basis for the development of TiO2 NPs as a feed additive for B. mori.

Flufenoxuron, an insect growth regulator, promotes peroral infection by nucleopolyhedrovirus (BmNPV) budded particles in the silkworm, Bombyx mori L.

PubMed

Arakawa, Toru; Furuta, Yoji; Miyazawa, Mitsuhiro; Kato, Masao

2002-02-01

A novel method was developed to infect perorally the silkworm Bombyx mori L. with budded particles of nucleopolyhedrovirus (BmNPV) using flufenoxuron, an insect growth regulator. NPV vectors are used to obtain proteins that occur naturally in minute amounts. NPV vectors are constructed conventionally by replacing the polyhedrin gene with the foreign gene of interest. These vectors thus do not produce polyhedra. The budded virus particle suspension of these vectors is produced in a cell culture and used as the stock inoculum. Budded NPV particles do not infect their host perorally. The inoculum is injected manually into the individual host using a syringe. It was found that B. mori L. fed on the insect growth regulator flufenoxuron were sensitive to BmNPV budded particles given perorally. Over 90% of B. mori L. ingesting BmNPV budded particles (1.3 x 10(6) TCID(50) units per larva) after consuming an artificial diet for 24 h, containing 0.1% (w/w) flufenoxuron died of the viral infection. The peroral inoculation of BmNPV budded particles by flufenoxuron may thus lead to industrial pharmaceutical production using a baculovirus vector for large numbers of insect hosts.

Regulation of Silk Genes by Hox and Homeodomain Proteins in the Terminal Differentiated Silk Gland of the Silkworm Bombyx mori

PubMed Central

Takiya, Shigeharu; Tsubota, Takuya; Kimoto, Mai

2016-01-01

The silk gland of the silkworm Bombyx mori is a long tubular organ that is divided into several subparts along its anteroposterior (AP) axis. As a trait of terminal differentiation of the silk gland, several silk protein genes are expressed with unique regional specificities. Most of the Hox and some of the homeobox genes are also expressed in the differentiated silk gland with regional specificities. The expression patterns of Hox genes in the silk gland roughly correspond to those in embryogenesis showing “colinearity”. The central Hox class protein Antennapedia (Antp) directly regulates the expression of several middle silk gland–specific silk genes, whereas the Lin-1/Isl-1/Mec3 (LIM)-homeodomain transcriptional factor Arrowhead (Awh) regulates the expression of posterior silk gland–specific genes for silk fiber proteins. We summarize our results and discuss the usefulness of the silk gland of Bombyx mori for analyzing the function of Hox genes. Further analyses of the regulatory mechanisms underlying the region-specific expression of silk genes will provide novel insights into the molecular bases for target-gene selection and regulation by Hox and homeodomain proteins. PMID:29615585

Effects of diet-deprivation and physical stimulation on the feeding behaviour of the larvae of the silkworm, Bombyx mori.

PubMed

Nagata, Shinji; Nagasawa, Hiromichi

2006-08-01

Continuous observations of larvae of the silkworm, Bombyx mori, revealed that feeding occurred at regular intervals throughout larval development. To investigate possible factors influencing meal-timing, the behaviours of diet-deprived Bombyx larvae were also analysed. Diet-deprivation resulted in longer durations of the first meals after diet replacement, but did not affect feeding patterns. Furthermore, long-term diet-deprivation promoted wandering behaviour and a consequent delay in feeding after diet replacement. Under diet-deprivation conditions, meal-starts appeared to be inducible by defecation and physical stimulation. However, stimulation-induced meal-starts were dependent on the time elapsed since the larvae's previous meals. Provided that more than 1h had elapsed since their previous meals, larvae could be induced to feed by defecation and tapping. At less than 1h post-meal, larvae were less likely to begin feeding after defecation or physical stimulation. Activated locomotions such as wandering and feeding were observed in the long-term diet-deprived larvae only after diet blocks were replaced, while long-term diet-deprived larvae did not show activated locomotion during the absence of diet blocks. Collectively, these data suggest that a combination of elevated locomotion activity and the presence of diet may be necessary for the initiation of feeding in diet-deprived larvae.

Cloning and characterization of carboxyl terminus of heat shock cognate 70-interacting protein gene from the silkworm, Bombyx mori.

PubMed

Ohsawa, Takeshi; Fujimoto, Shota; Tsunakawa, Akane; Shibano, Yuka; Kawasaki, Hideki; Iwanaga, Masashi

2016-11-01

Carboxyl terminus of heat shock cognate 70-interacting protein (CHIP) is an evolutionarily conserved E3 ubiquitin ligase across different eukaryotic species and is known to play a key role in protein quality control. CHIP has two distinct functional domains, an N-terminal tetratricopeptide repeat (TPR) and a C-terminal U-box domain, which are required for the ubiquitination of numerous labile client proteins that are chaperoned by heat shock proteins (HSPs) and heat shock cognate proteins (HSCs). During our screen for CHIP-like proteins in the Bombyx mori databases, we found a novel silkworm gene, Bombyx mori CHIP. Phylogenetic analysis showed that BmCHIP belongs to Lepidopteran lineages. Quantitative reverse transcription-PCR analysis indicated that BmCHIP was relatively highly expressed in the gonad and fat body. A pull-down experiment and auto-ubiquitination assay showed that BmCHIP interacted with BmHSC70 and had E3 ligase activity. Additionally, immunohistochemical analysis revealed that BmCHIP was partially co-localized with ubiquitin in BmN4 cells. These data support that BmCHIP plays an important role in the ubiquitin proteasome system as an E3 ubiquitin ligase in B. mori. Copyright © 2016 Elsevier Inc. All rights reserved.

Analysis of differentially expressed genes between fluoride-sensitive and fluoride-endurable individuals in midgut of silkworm, Bombyx mori.

PubMed

Qian, Heying; Li, Gang; He, Qingling; Zhang, Huaguang; Xu, Anying

2016-08-15

Fluoride tolerance is an economically important trait of silkworm. Near-isogenic lines (NILs) of the dominant endurance to fluoride (Def) gene in Bombyx mori has been constructed before. Here, we analyzed the gene expression profiles of midgut of fluoride-sensitive and fluoride-endurable individuals of Def NILs by using high-throughput Illumina sequencing technology and bioinformatics tools, and identified differentially expressed genes between these individuals. A total of 3,612,399 and 3,567,631 clean tags for the libraries of fluoride-endurable and fluoride-sensitive individuals were obtained, which corresponded to 32,933 and 43,976 distinct clean tags, respectively. Analysis of differentially expressed genes indicates that 241 genes are differentially expressed between the two libraries. Among the 241 genes, 30 are up-regulated and 211 are down-regulated in fluoride-endurable individuals. Pathway enrichment analysis demonstrates that genes related to ribosomes, pancreatic secretion, steroid biosynthesis, glutathione metabolism, steroid biosynthesis, and glycerolipid metabolism are down-regulated in fluoride-endurable individuals. qRT-PCR was conducted to confirm the results of the DGE. The present study analyzed differential expression of related genes and tried to find out whether the crucial genes were related to fluoride detoxification which might elucidate fluoride effect and provide a new way in the fluorosis research. Copyright © 2016 Elsevier B.V. All rights reserved.

Serratia marcescens Suppresses Host Cellular Immunity via the Production of an Adhesion-inhibitory Factor against Immunosurveillance Cells*

PubMed Central

Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-01-01

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis. PMID:24398686

Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells.

PubMed

Ishii, Kenichi; Adachi, Tatsuo; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-02-28

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.

Morphological and histomorphological structures of testes and ovaries in early developmental stages of the silkworm, Bombyx mori.

PubMed

Sakai, Hiroki; Kirino, Yohei; Katsuma, Susumu; Aoki, Fugaku; Suzuki, Masataka G

2016-01-01

The gonad develops as a testis in male or an ovary in female. In the silkworm, B. mori , little is known about testis and ovary in the embryonic stages and early larval stages. In this study, we performed morphological and histomorphological observations of ovaries and testes from the late embryonic stage to the 1st instar larval stage. Results obtained with lack of accurate information on sex of examined individuals may be misleading, thus we performed phenotypic observations of gonads by utilizing sex-limited strain that enables us to easily discriminate female embryos from male ones based on those egg colors. In testis, four testicular follicles were clearly observed in the testis at the first instar larval stage, and boundary layers were formed between the testicular follicles. At the late embryonic stage, the testis consisted of four testicular follicles, while the boundary layers were still obscure. In ovary, four ovarioles were easily recognizable in the ovary at the first instar larval stage, and boundary layers were formed between the ovarioles. However, in the late embryonic stage, it was quite difficult to identify four ovarioles. Morphological characteristics were almost similar between testis and ovary in early developmental stages. Our present study demonstrates that the most reliable difference between testis and ovary in early developmental stages is the attaching point of the duct. Formation and development of the duct may be sensitive to the sex-determining signal and display sexual dimorphism in early embryonic stages.

Effect of BBX-B8 overexpression on development, body weight, silk protein synthesis and egg diapause of Bombyx mori.

PubMed

Zheng, Xiaojian; Gong, Yongchang; Kumar, Dhiraj; Chen, Fei; Kuan, Sulan; Liang, Zi; Hu, Xiaolong; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2016-08-01

Bombyxin (BBX) is an insulin-like peptide exists in the silkworm Bombyx mori. Our previous studies on the effects of inhibiting BBX-B8 expression found that BBX-B8 is important for the development of organ, reproduction and trehalose metabolism in the silkworms. In this paper, we investigated the expression profile of the BBX-B8 gene and effect of BBX-B8 overexpression on the development, body weight, silk protein synthesis and egg diapause of B. mori to further understand BBX-B8 functions. BBX-B8 gene expression could be detected in the brains, midguts, anterior silkglands, ovaries, testes, fat bodies, hemolymph, malpighian tubules and embryos by RT-PCR, however it was mainly expressed in the brain. Western blots showed that the change in BBX-B8 expression was not obvious in the brain of 1- to 4-day-old larvae of fifth instar silkworms, but expression increased substantially at 5- to 6-day-old larvae of fifth instar silkworms. Transgenic silkworms overexpressing BBX-B8 were obtained by introducing non-transposon transgenic vector pIZT-B8 containing a BBX-B8 gene driven by Orgyia pseudotsugata nucleopolyhedrovirus IE2 promoter into the genome. Development duration of the transgenic silkworms was delayed by 2.5-3.5 days. Cocoon shell weight of transgenic silkworms was reduced by 4.79 % in females and 7.44 % in males, pupal weight of transgenic silkworms was reduced 6.75 % in females and 13.83 % in males compared to non-transgenic silkworms, and 5.56-14.29 % of transgenic moths laid nondiapausing eggs. All results indicated that BBX-B8 plays an important role in the development, silk protein synthesis and egg diapause of silkworm.

Graphitized Porous Carbon for Rapid Screening of Angiotensin-Converting Enzyme Inhibitory Peptide GAMVVH from Silkworm Pupa Protein and Molecular Insight into Inhibition Mechanism.

PubMed

Tao, Mengliang; Sun, Huaju; Liu, Long; Luo, Xuan; Lin, Guoyou; Li, Renbo; Zhao, Zhenxia; Zhao, Zhongxing

2017-10-04

A novel hydrophobic hexapeptide with high angiotensin-converting enzyme (ACE) inhibitory activity was screened from silkworm pupa protein (SPP) hydrolysate via graphitized porous carbon and reverse-phase high-performance liquid chromatography methods. Graphitized porous carbon derived from dopamine, possessing high surface area and high graphitic carbon, was used to rapidly screen and enrich hydrophobic peptides from SPP hydrolysate. The ACE inhibition pattern and mechanism of the purified peptide were also systematically studied by the classic Lineweaver-Burk model and by molecular docking/dynamic simulation. The novel hydrophobic hexapeptide was identified as Gly-Ala-Met-Val-Val-His (GAMVVH, IC 50 = 19.39 ± 0.21 μM) with good thermal/antidigestive stabilities. Lineweaver-Burk plots revealed that GAMVVH behaved as a competitive ACE inhibitor. It formed hydrogen bonds with S1 and S2 pockets of ACE and established competitive coordination with Zn(II) of ACE. The synergy of hydrogen bonds with active pockets and Zn(II) coordination efficiently changed the three-dimensional structure of ACE and thus inhibited bioactivity of ACE.

An invertebrate hyperglycemic model for the identification of anti-diabetic drugs.

PubMed

Matsumoto, Yasuhiko; Sumiya, Eriko; Sugita, Takuya; Sekimizu, Kazuhisa

2011-03-30

The number of individuals diagnosed with type 2 diabetes mellitus, which is caused by insulin resistance and/or abnormal insulin secretion, is increasing worldwide, creating a strong demand for the development of more effective anti-diabetic drugs. However, animal-based screening for anti-diabetic compounds requires sacrifice of a large number of diabetic animals, which presents issues in terms of animal welfare. Here, we established a method for evaluating the anti-diabetic effects of compounds using an invertebrate animal, the silkworm, Bombyx mori. Sugar levels in silkworm hemolymph increased immediately after feeding silkworms a high glucose-containing diet, resulting in impaired growth. Human insulin and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, decreased the hemolymph sugar levels of the hyperglycemic silkworms and restored growth. Treatment of the isolated fat body with human insulin in an in vitro culture system increased total sugar in the fat body and stimulated Akt phosphorylation. These responses were inhibited by wortmannin, an inhibitor of phosphoinositide 3 kinase. Moreover, AICAR stimulated AMPK phosphorylation in the silkworm fat body. Administration of aminoguanidine, a Maillard reaction inhibitor, repressed the accumulation of Maillard reaction products (advanced glycation end-products; AGEs) in the hyperglycemic silkworms and restored growth, suggesting that the growth defect of hyperglycemic silkworms is caused by AGE accumulation in the hemolymph. Furthermore, we identified galactose as a hypoglycemic compound in jiou, an herbal medicine for diabetes, by monitoring its hypoglycemic activity in hyperglycemic silkworms. These results suggest that the hyperglycemic silkworm model is useful for identifying anti-diabetic drugs that show therapeutic effects in mammals.

An in vivo invertebrate evaluation system for identifying substances that suppress sucrose-induced postprandial hyperglycemia

PubMed Central

Matsumoto, Yasuhiko; Ishii, Masaki; Sekimizu, Kazuhisa

2016-01-01

Sucrose is a major sweetener added to various foods and beverages. Excessive intake of sucrose leads to increases in blood glucose levels, which can result in the development and exacerbation of lifestyle-related diseases such as obesity and diabetes. In this study, we established an in vivo evaluation system using silkworms to explore substances that suppress the increase in blood glucose levels caused by dietary intake of sucrose. Silkworm hemolymph glucose levels rapidly increased after intake of a sucrose-containing diet. Addition of acarbose or voglibose, α-glycosidase inhibitors clinically used for diabetic patients, suppressed the dietary sucrose-induced increase in the silkworm hemolymph glucose levels. Screening performed using the sucrose-induced postprandial hyperglycemic silkworm model allowed us to identify some lactic acid bacteria that inhibit the increase in silkworm hemolymph glucose levels caused by dietary intake of sucrose. The inhibitory effects of the Lactococcus lactis #Ll-1 bacterial strain were significantly greater than those of different strains of lactic acid bacteria. No effect of the Lactococcus lactis #Ll-1 strain was observed in silkworms fed a glucose diet. These results suggest that the sucrose diet-induced postprandial hyperglycemic silkworm is a useful model for evaluating chemicals and lactic acid bacteria that suppress increases in blood glucose levels. PMID:27194587

An in vivo invertebrate evaluation system for identifying substances that suppress sucrose-induced postprandial hyperglycemia.

PubMed

Matsumoto, Yasuhiko; Ishii, Masaki; Sekimizu, Kazuhisa

2016-05-19

Sucrose is a major sweetener added to various foods and beverages. Excessive intake of sucrose leads to increases in blood glucose levels, which can result in the development and exacerbation of lifestyle-related diseases such as obesity and diabetes. In this study, we established an in vivo evaluation system using silkworms to explore substances that suppress the increase in blood glucose levels caused by dietary intake of sucrose. Silkworm hemolymph glucose levels rapidly increased after intake of a sucrose-containing diet. Addition of acarbose or voglibose, α-glycosidase inhibitors clinically used for diabetic patients, suppressed the dietary sucrose-induced increase in the silkworm hemolymph glucose levels. Screening performed using the sucrose-induced postprandial hyperglycemic silkworm model allowed us to identify some lactic acid bacteria that inhibit the increase in silkworm hemolymph glucose levels caused by dietary intake of sucrose. The inhibitory effects of the Lactococcus lactis #Ll-1 bacterial strain were significantly greater than those of different strains of lactic acid bacteria. No effect of the Lactococcus lactis #Ll-1 strain was observed in silkworms fed a glucose diet. These results suggest that the sucrose diet-induced postprandial hyperglycemic silkworm is a useful model for evaluating chemicals and lactic acid bacteria that suppress increases in blood glucose levels.

Silk fibroin produced by transgenic silkworms overexpressing the Arg-Gly-Asp motif accelerates cutaneous wound healing in mice.

PubMed

Baba, Atsunori; Matsushita, Shigeto; Kitayama, Kasumi; Asakura, Tetsuo; Sezutsu, Hideki; Tanimoto, Akihide; Kanekura, Takuro

2018-03-04

We investigated the effect of silk fibroin (SF) on wound healing in mice. SF or an amorphous SF film (ASFF) prepared from silk produced by the wild-type silkworm Bombyx mori (WT-SF, WT-ASFF) or by transgenic worms that overexpress the Arg-Gly-Asp (RGD) sequence (TG-SF, TG-ASFF) was placed on 5-mm diameter full-thickness skin wounds made by biopsy punch on the back of 8-12 week-old BALB/c mice. Each wound was covered with WT-ASFF and urethane film (UF), TG-ASFF plus UF, or UF alone (control). Wound closure, histological thickness, the area of granulation tissue, and neovascularization were analyzed 4, 8, and 12 days later. The effect of SF on cell migration and proliferation was examined in vitro by scratch- and MTT-assay using human dermal fibroblasts. Wound closure was prompted by TG-ASFF, granulation tissue was thicker and larger in ASFF-treated wounds than the control, and neovascularization was promoted significantly by WT-ASFF. Both assays showed that SF induced the migration and proliferation of human dermal fibroblasts. The effects of TG-ASFF and TG-SF on wound closure, granulation formation, and cell proliferation were more profound than that of WT-ASFF and WT-SF. We document that SF accelerates cutaneous wound healing, and this effect is enhanced with TG-SF. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc.

Mutation of a cuticular protein, BmorCPR2, alters larval body shape and adaptability in silkworm, Bombyx mori.

PubMed

Qiao, Liang; Xiong, Gao; Wang, Ri-xin; He, Song-zhen; Chen, Jie; Tong, Xiao-ling; Hu, Hai; Li, Chun-lin; Gai, Ting-ting; Xin, Ya-qun; Liu, Xiao-fan; Chen, Bin; Xiang, Zhong-huai; Lu, Cheng; Dai, Fang-yin

2014-04-01

Cuticular proteins (CPs) are crucial components of the insect cuticle. Although numerous genes encoding cuticular proteins have been identified in known insect genomes to date, their functions in maintaining insect body shape and adaptability remain largely unknown. In the current study, positional cloning led to the identification of a gene encoding an RR1-type cuticular protein, BmorCPR2, highly expressed in larval chitin-rich tissues and at the mulberry leaf-eating stages, which is responsible for the silkworm stony mutant. In the Dazao-stony strain, the BmorCPR2 allele is a deletion mutation with significantly lower expression, compared to the wild-type Dazao strain. Dysfunctional BmorCPR2 in the stony mutant lost chitin binding ability, leading to reduced chitin content in larval cuticle, limitation of cuticle extension, abatement of cuticle tensile properties, and aberrant ratio between internodes and intersegmental folds. These variations induce a significant decrease in cuticle capacity to hold the growing internal organs in the larval development process, resulting in whole-body stiffness, tightness, and hardness, bulging intersegmental folds, and serious defects in larval adaptability. To our knowledge, this is the first study to report the corresponding phenotype of stony in insects caused by mutation of RR1-type cuticular protein. Our findings collectively shed light on the specific role of cuticular proteins in maintaining normal larval body shape and will aid in the development of pest control strategies for the management of Lepidoptera.

Uncovering the Molecular Mechanism of Anti-Allergic Activity of Silkworm Pupa-Grown Cordyceps militaris Fruit Body.

PubMed

Wu, Ting-Feng; Chan, Yu-Yi; Shi, Wan-Yin; Jhong, Meng-Ting

2017-01-01

Cordyceps militaris has been widely used as an herbal drug and tonic food in East Asia and has also been recently studied in the West because of its various pharmacological activities such as antitumoral, anti-inflammatory and immunomodulatory effects. In this study, we examined the molecular mechanism underlying the anti-allergic activity of ethanol extract prepared from silkworm pupa-cultivated Cordyceps militaris fruit bodies in activated mast cells. Our results showed that ethanol extract treatment significantly inhibited the release of [Formula: see text]-hexosaminidase (a degranulation marker) and mRNA levels of tumor necrosis factor-[Formula: see text] as well as interleukin-4 in RBL-2H3 cells. The cells were sensitized with 2,4-dinitrophenol specific IgE and then stimulated with human serum albumin conjugated with 2,4-dinitrophenol. Oral administration of 300[Formula: see text]mg/kg ethanol extract significantly ameliorated IgE-induced allergic reaction in mice with passive cutaneous anaphylaxis. Western immunoblotting results demonstrated that ethanol extract incubation significantly inhibited Syk/PI3K/MEKK4/JNK/c-jun biochemical cascade in activated RBL-2H3 cells, which activated the expression of various allergic cytokines. In addition, it suppressed Erk activation and PLC[Formula: see text] evocation, which would respectively evoke the synthesis of lipid mediators and Ca[Formula: see text] mobilization to induce degranulation in stimulated RBL-2H3 cells. A compound, identified as [Formula: see text]-sitostenone, was shown to inhibit [Formula: see text]-hexosaminidase secretion from activated mast cells. Our study demonstrated that ethanol extract contained the ingredients, which could inhibit immediate degranulation and de novo synthesis of allergic lipid mediators and cytokines in activated mast cells.

Antioxidant activities of two sericin proteins extracted from cocoon of silkworm (Bombyx mori) measured by DPPH, chemiluminescence, ORAC and ESR methods.

PubMed

Takechi, Tayori; Wada, Ritsuko; Fukuda, Tsubasa; Harada, Kazuki; Takamura, Hitoshi

2014-05-01

Recent efforts have focused on the use of sericin proteins extracted from cocoons of silkworm as a healthy food source for human consumption. In this study, we focused on the antioxidative properties of sericin proteins. The antioxidative properties were measured in sericin proteins extracted from the shell of the cocoon, designated hereafter as white sericin protein and yellow-green sericin protein, as well as bread without sericin protein and bread to which white sericin powder had been added using four measurement methods: 1,1-Diphenyl-2-picrylhydrazyl (DPPH), chemiluminescence, oxygen radical absorbance capacity (ORAC) and electron spin resonance (ESR). High antioxidative properties of sericin proteins were indicated by all four methods. A comparison of the two types of sericin proteins revealed that yellow-green sericin protein exhibited high antioxidative properties as indicated by the DPPH, chemiluminescence and ORAC methods. By contrast, a higher antioxidative property was determined in white sericin protein by the ESR method. Consequently, our findings confirmed that sericin proteins have antioxidative properties against multiple radicals. In addition, the antioxidative property of bread was enhanced by the addition of sericin powder to the bread. Therefore, findings of this study suggest that sericin proteins may be efficiently used as beneficial food for human health.

Antioxidant activities of two sericin proteins extracted from cocoon of silkworm (Bombyx mori) measured by DPPH, chemiluminescence, ORAC and ESR methods

PubMed Central

TAKECHI, TAYORI; WADA, RITSUKO; FUKUDA, TSUBASA; HARADA, KAZUKI; TAKAMURA, HITOSHI

2014-01-01

Recent efforts have focused on the use of sericin proteins extracted from cocoons of silkworm as a healthy food source for human consumption. In this study, we focused on the antioxidative properties of sericin proteins. The antioxidative properties were measured in sericin proteins extracted from the shell of the cocoon, designated hereafter as white sericin protein and yellow-green sericin protein, as well as bread without sericin protein and bread to which white sericin powder had been added using four measurement methods: 1,1-Diphenyl-2-picrylhydrazyl (DPPH), chemiluminescence, oxygen radical absorbance capacity (ORAC) and electron spin resonance (ESR). High antioxidative properties of sericin proteins were indicated by all four methods. A comparison of the two types of sericin proteins revealed that yellow-green sericin protein exhibited high antioxidative properties as indicated by the DPPH, chemiluminescence and ORAC methods. By contrast, a higher antioxidative property was determined in white sericin protein by the ESR method. Consequently, our findings confirmed that sericin proteins have antioxidative properties against multiple radicals. In addition, the antioxidative property of bread was enhanced by the addition of sericin powder to the bread. Therefore, findings of this study suggest that sericin proteins may be efficiently used as beneficial food for human health. PMID:24748975

20-Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis-related protein in the silkworm, Bombyx mori.

PubMed

Ji, M-M; Liu, A-Q; Sima, Y-H; Xu, S-Q

2016-10-01

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth-instar ecdysis and larval-pupal metamorphosis. By immunoprecipitation with the anti-BmNep1 antibody and liquid chromatography-tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20-hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval-pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx. © 2016 The Royal Entomological Society.

Transcriptome Sequencing and Positive Selected Genes Analysis of Bombyx mandarina

PubMed Central

Wu, Yuqian; Long, Renwen; Liu, Chun; Xia, Qingyou

2015-01-01

The wild silkworm Bombyx mandarina is widely believed to be an ancestor of the domesticated silkworm, Bombyx mori. Silkworms are often used as a model for studying the mechanism of species domestication. Here, we performed transcriptome sequencing of the wild silkworm using an Illumina HiSeq2000 platform. We produced 100,004,078 high-quality reads and assembled them into 50,773 contigs with an N50 length of 1764 bp and a mean length of 941.62 bp. A total of 33,759 unigenes were identified, with 12,805 annotated in the Nr database, 8273 in the Pfam database, and 9093 in the Swiss-Prot database. Expression profile analysis found significant differential expression of 1308 unigenes between the middle silk gland (MSG) and posterior silk gland (PSG). Three sericin genes (sericin 1, sericin 2, and sericin 3) were expressed specifically in the MSG and three fibroin genes (fibroin-H, fibroin-L, and fibroin/P25) were expressed specifically in the PSG. In addition, 32,297 Single-nucleotide polymorphisms (SNPs) and 361 insertion-deletions (INDELs) were detected. Comparison with the domesticated silkworm p50/Dazao identified 5,295 orthologous genes, among which 400 might have experienced or to be experiencing positive selection by Ka/Ks analysis. These data and analyses presented here provide insights into silkworm domestication and an invaluable resource for wild silkworm genomics research. PMID:25806526

Deletion of the Bombyx mori odorant receptor co-receptor (BmOrco) impairs olfactory sensitivity in silkworms.

PubMed

Liu, Qun; Liu, Wei; Zeng, Baosheng; Wang, Guirong; Hao, Dejun; Huang, Yongping

2017-07-01

Olfaction plays an essential role in many important insect behaviors such as feeding and reproduction. To detect olfactory stimuli, an odorant receptor co-receptor (Orco) is required. In this study, we deleted the Orco gene in the Lepidopteran model insect, Bombyx mori, using a binary transgene-based clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system. We initially generated somatic mutations in two targeted sites, from which we obtained homozygous mutants with deletion of a 866 base pair sequence. Because of the flight inability of B. mori, we developed a novel method to examine the adult mating behavior. Considering the specialization in larval feeding, we examined food selection behavior in Orco somatic mutants by the walking trail analysis of silkworm position over time. Single sensillum recordings indicated that the antenna of the homozygous mutant was unable to respond to either of the two sex pheromones, bombykol or bombykal. An adult mating behavior assay revealed that the Orco mutant displayed a significantly impaired mating selection behavior in response to natural pheromone released by a wild-type female moth as well as an 11:1 mixture of bombykol/bombykal. The mutants also exhibited a decreased response to bombykol and, similar to wild-type moths, they displayed no response to bombykal. A larval feeding behavior assay revealed that the Orco mutant displayed defective selection for mulberry leaves and different concentrations of the volatile compound cis-jasmone found in mulberry leaves. Deletion of BmOrco severely disrupts the olfactory system, suggesting that BmOrco is indispensable in the olfactory pathway. The approach used for generating somatic and homozygous mutations also highlights a novel method for mutagenesis. This study on BmOrco function provides insights into the insect olfactory system and also provides a paradigm for agroforestry pest control. Copyright © 2017 Elsevier Ltd. All rights reserved.

Combined Effect of Cameo2 and CBP on the Cellular Uptake of Lutein in the Silkworm, Bombyx mori

PubMed Central

Dong, Xiao-Long; Chai, Chun-Li; Pan, Cai-Xia; Tang, Hui; Chen, Yan-Hong; Dai, Fang-Yin; Pan, Min-Hui; Lu, Cheng

2014-01-01

Formation of yellow-red color cocoons in the silkworm, Bombyx mori, occurs as the result of the selective delivery of carotenoids from the midgut to the silk gland via the hemolymph. This process of pigment transport is thought to be mediated by specific cellular carotenoids carrier proteins. Previous studies indicated that two proteins, Cameo2 and CBP, are associated with the selective transport of lutein from the midgut into the silk gland in Bombyx mori. However, the exact roles of Cameo2 and CBP during the uptake and transport of carotenoids are still unknown. In this study, we investigated the respective contributions of these two proteins to lutein and β-carotene transport in Bombyx mori as well as commercial cell-line. We found that tissues, expressed both Cameo2 and CBP, accumulate lutein. Cells, co-expressed Cameo2 and CBP, absorb 2 fold more lutein (P<0.01) than any other transfected cells, and the rate of cellular uptake of lutein was concentration-dependent and reached saturation. From immunofluorescence staining, confocal microscopy observation and western blot analysis, Cameo2 was localized at the membrane and CBP was expressed in the cytosol. What’s more, bimolecular fluorescence complementation analysis showed that these two proteins directly interacted at cellular level. Therefore, Cameo2 and CBP are necessarily expressed in midguts and silk glands for lutein uptake in Bombyx mori. Cameo2 and CBP, as the membrane protein and the cytosol protein, respectively, have the combined effect to facilitate the cellular uptake of lutein. PMID:24475153

Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae.

PubMed

Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Sasaki-Tabata, Kaori; Tanizaki, Yusuke; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

2011-07-01

A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

Genome-wide comparison of genes involved in the biosynthesis, metabolism, and signaling of juvenile hormone between silkworm and other insects

PubMed Central

Cheng, Daojun; Meng, Meng; Peng, Jian; Qian, Wenliang; Kang, Lixia; Xia, Qingyou

2014-01-01

Juvenile hormone (JH) contributes to the regulation of larval molting and metamorphosis in insects. Herein, we comprehensively identified 55 genes involved in JH biosynthesis, metabolism and signaling in the silkworm (Bombyx mori) as well as 35 in Drosophila melanogaster, 35 in Anopheles gambiae, 36 in Apis mellifera, 47 in Tribolium castaneum, and 44 in Danaus plexippus. Comparative analysis showed that each gene involved in the early steps of the mevalonate (MVA) pathway, in the neuropeptide regulation of JH biosynthesis, or in JH signaling is a single copy in B. mori and other surveyed insects, indicating that these JH-related pathways or steps are likely conserved in all surveyed insects. However, each gene participating in the isoprenoid branch of JH biosynthesis and JH metabolism, together with the FPPS genes for catalyzing the final step of the MVA pathway of JH biosynthesis, exhibited an obvious duplication in Lepidoptera, including B. mori and D. plexippus. Microarray and real-time RT-PCR analysis revealed that different copies of several JH-related genes presented expression changes that correlated with the dynamics of JH titer during larval growth and metamorphosis. Taken together, the findings suggest that duplication-derived copy variation of JH-related genes might be evolutionarily associated with the variation of JH types between Lepidoptera and other insect orders. In conclusion, our results provide useful clues for further functional analysis of JH-related genes in B. mori and other insects. PMID:25071411

Characterization of the chemical structure and innate immune-stimulating activity of an extracellular polysaccharide from Rhizobium sp. strain M2 screened using a silkworm muscle contraction assay.

PubMed

Urai, Makoto; Aizawa, Tomoko; Imamura, Katsutoshi; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2017-11-22

We screened innate immunostimulant-producing bacteria using a silkworm muscle contraction assay, and isolated Rhizobium sp. strain M2 from soil. We purified the innate immunostimulant from strain M2, and characterized the chemical structure by nuclear magnetic resonance spectroscopy and chemical analyses. The innate immunostimulant (M2 EPS) comprised glucose, galactose, pyruvic acid, and succinic acid with a molar ratio of 6.8:1.0:0.9:0.4, and had a succinoglycan-like high molecular-weight heteropolysaccharide structure. To determine the structural motif involved in the innate immunostimulating activity, we modified the M2 EPS structure chemically, and found that the activity was increased by removal of the succinic and pyruvic acid substitutions. Strong acid hydrolysis completely inactivated the M2 EPS. Unmasking of the β-1,3/6-glucan structure of the side-chain by deacylation and depyruvylation may enhance the innate immune-stimulating activity of M2 EPS. These findings suggest that the succinoglycan-like polysaccharide purified from strain M2 has innate immune-stimulating activity, and its glycan structure is necessary for the activity.

Isolation and bioactivities of a non-sericin component from cocoon shell silk sericin of the silkworm Bombyx mori.

PubMed

Wang, Hai-Yan; Wang, Yuan-Jing; Zhou, Li-Xia; Zhu, Lin; Zhang, Yu-Qing

2012-02-01

The cocoon shell of the silkworm Bombyx mori consists of silk fibroin fiber (70%) surrounded by a sericin layer made up of sericin (25%) and non-sericin (5%) components. The non-sericin component which consists of carbohydrate, salt, wax, flavonoids and derivatives is often overlooked in applied research into sericin and its hydrolysate. Here, sericin and non-sericin compounds were obtained from the sericin layer of five types of cocoon shell by means of degumming in water followed by extraction and separation in ethanol. These ethanol extracts were found to mainly contain flavonoids and free amino acids possessing scavenging activities of the 2,2-diphenyl -1-picrylhydrazyl (DPPH) free radical and inhibiting activities of tyrosinase, which were much greater than the corresponding activities of the purified sericin proteins. The extracts also strongly inhibited α-glucosidase while the sericins had no such activity. In particular, the inhibitory activities of the ethanol extract of Daizo cocoons were much greater than those of the other cocoons. The IC(50) values of the Daizo cocoons for DPPH free radicals, tyrosinase, and α-glucosidase were 170, 27, and 110 μg mL(-1), respectively. The bioactivities of the non-sericin component were much higher than the activity of sericin alone. In addition, the in vivo test showed preliminarily that the administration of the non-sericin component had effectively resistant activity against streptozocin (STZ) oxidation and that of the purified sericin could also evidently decrease the induction ratio of diabetic mice induced by STZ. Therefore, ethanol extract protocols of the sericin layer of cocoon shells provide a novel stock which, together with sericin protein, has potential uses in functional food, biotechnological and medical applications.

The specificity of immune priming in silkworm, Bombyx mori, is mediated by the phagocytic ability of granular cells.

PubMed

Wu, Gongqing; Li, Mei; Liu, Yi; Ding, Ying; Yi, Yunhong

2015-10-01

In the past decade, the phenomenon of immune priming was documented in many invertebrates in a large number of studies; however, in most of these studies, behavioral evidence was used to identify the immune priming. The underlying mechanism and the degree of specificity of the priming response remain unclear. We studied the mechanism of immune priming in the larvae of the silkworm, Bombyx mori, and analyzed the specificity of the priming response using two closely related Gram-negative pathogenic bacteria (Photorhabdus luminescens TT01 and P. luminescens H06) and one Gram-positive pathogenic bacterium (Bacillus thuringiensis HD-1). Primed with heat-killed bacteria, the B. mori larvae were more likely to survive subsequent homologous exposure (the identical bacteria used in the priming and in the subsequent challenge) than heterologous (different bacteria used in the priming and subsequent exposure) exposure to live bacteria. This result indicated that the B. mori larvae possessed a strong immune priming response and revealed a degree of specificity to TT01, H06 and HD-1 bacteria. The degree of enhanced immune protection was positively correlated with the level of phagocytic ability of the granular cells and the antibacterial activity of the cell-free hemolymph. Moreover, the granular cells of the immune-primed larvae increased the phagocytosis of a previously encountered bacterial strain compared with other bacteria. Thus, the enhanced immune protection of the B. mori larvae after priming was mediated by the phagocytic ability of the granular cells and the antibacterial activity of the hemolymph; the specificity of the priming response was primarily attributed to the phagocytosis of bacteria by the granular cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Traffic Aware Planner for Cockpit-Based Trajectory Optimization

NASA Technical Reports Server (NTRS)

Woods, Sharon E.; Vivona, Robert A.; Henderson, Jeffrey; Wing, David J.; Burke, Kelly A.

2016-01-01

The Traffic Aware Planner (TAP) software application is a cockpit-based advisory tool designed to be hosted on an Electronic Flight Bag and to enable and test the NASA concept of Traffic Aware Strategic Aircrew Requests (TASAR). The TASAR concept provides pilots with optimized route changes (including altitude) that reduce fuel burn and/or flight time, avoid interactions with known traffic, weather and restricted airspace, and may be used by the pilots to request a route and/or altitude change from Air Traffic Control. Developed using an iterative process, TAP's latest improvements include human-machine interface design upgrades and added functionality based on the results of human-in-the-loop simulation experiments and flight trials. Architectural improvements have been implemented to prepare the system for operational-use trials with partner commercial airlines. Future iterations will enhance coordination with airline dispatch and add functionality to improve the acceptability of TAP-generated route-change requests to pilots, dispatchers, and air traffic controllers.

Bombyx mori silk: From mechanical properties to functionalities

NASA Astrophysics Data System (ADS)

Koh, Leng Duei

Bombyx mori silkworms are the main producer of silk worldwide. It has been used as high-end textile fibers and as surgical sutures, and is being further developed for various emerging biomedical applications including drug delivery, tissue engineering, sensing, and imaging. The silk fibroin features a hierarchical architecture consisting of beta-sheet crystallites embedded in a less ordered amorphous matrix, which accounts for its unique combination of lustre appearance, soft-to-touch texture, and impressive mechanical properties. Notably, many applications of silk take advantage of its impressive mechanical properties, which by nature surpass many natural and synthetic materials. Interestingly, both the silkworm silk and spider dragline silk share similar hierarchical architecture but possess great disparity in mechanical properties. Inspired by spider dragline silk with much superior strength and toughness, there is an ever growing interest to enhance the mechanical properties of Bombyx mori silk. Here, we design a green and facile feeding method to modulate the structures of silk fibroin at the nanoscale using citric acid (CA), and achieved greatly enhanced mechanical properties. The silk obtained (i.e., CA silk) emerges to be the intrinsically toughest silkworm silk, with mechanical properties that exceed those of the previously reported natural and enhanced silkworm silk, and compare well with those of naturally produced spider silk (including those from spiders Araneus diadematus, Nephila clavipes, etc.).The underlying interactions of CA with fibroin structures are revealed by both advanced characterizations and simulations. It is found that CA interacts with fibroin, resulted in remarkably shorter crystallites, and thus giving the outstanding strength and toughness of the CA silk. The greatly enhanced mechanical properties are expected to lead to better functionalities and wider applications of the Bombyx mori silkworm silk. Silkworms usually produce white

Lipid transfer particle from the silkworm, Bombyx mori, is a novel member of the apoB/large lipid transfer protein family[S

PubMed Central

Yokoyama, Hiroshi; Yokoyama, Takeru; Yuasa, Masashi; Fujimoto, Hirofumi; Sakudoh, Takashi; Honda, Naoko; Fugo, Hajime; Tsuchida, Kozo

2013-01-01

Lipid transfer particle (LTP) is a high-molecular-weight, very high-density lipoprotein known to catalyze the transfer of lipids between a variety of lipoproteins, including both insects and vertebrates. Studying the biosynthesis and regulation pathways of LTP in detail has not been possible due to a lack of information regarding the apoproteins. Here, we sequenced the cDNA and deduced amino acid sequences for three apoproteins of LTP from the silkworm (Bombyx mori). The three subunit proteins of the LTP are coded by two genes, apoLTP-II/I and apoLTP-III. ApoLTP-I and apoLTP-II are predicted to be generated by posttranslational cleavage of the precursor protein, apoLTP-II/I. Clusters of amphipathic secondary structure within apoLTP-II/I are similar to Homo sapiens apolipoprotein B (apoB) and insect lipophorins. The apoLTP-II/I gene is a novel member of the apoB/large lipid transfer protein gene family. ApoLTP-III has a putative conserved juvenile hormone-binding protein superfamily domain. Expression of apoLTP-II/I and apoLTP-III genes was synchronized and both genes were primarily expressed in the fat body at the stage corresponding to increased lipid transport needs. We are now in a position to study in detail the physiological role of LTP and its biosynthesis and assembly. PMID:23812557

Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm, Bombyx mori.

PubMed

Ji, Ming-Ming; Lee, Jae Man; Mon, Hiroaki; Iiyama, Kazuhiro; Tatsuke, Tsuneyuki; Morokuma, Daisuke; Hino, Masato; Yamashita, Mami; Hirata, Kazuma; Kusakabe, Takahiro

2017-10-01

p62/Sequestosome-1 (p62/SQSTM1, hereafter referred to as p62) is a major adaptor that allows ubiquitinated proteins to be degraded by autophagy, and Atg8 homologs are required for p62-mediated autophagic degradation, but their relationship is still not understood in Lepidopteran insects. Here it is clearly demonstrated that the silkworm homolog of mammalian p62, Bombyx mori p62 (Bmp62), forms p62 bodies depending on its Phox and Bem1p (PB1) and ubiquitin-associated (UBA) domains. These two domains are associated with Bmp62 binding to ubiquitinated proteins to form the p62 bodies, and the UBA domain is essential for the binding, but Bmp62 still self-associates without the PB1 or UBA domain. The p62 bodies in Bombyx cells are enclosed by BmAtg9-containing membranes and degraded via autophagy. It is revealed that the interaction between the Bmp62 AIM motif and BmAtg8 is critical for the autophagic degradation of the p62 bodies. Intriguingly, we further demonstrate that lipidation of BmAtg8 is required for the Bmp62-mediated complete degradation of p62 bodies by autophagy. Our results should be useful in future studies of the autophagic mechanism in Lepidopteran insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Molecular Cloning, Bioinformatic Analysis, and Expression of Bombyx mori Lebocin 5 Gene Related to Beauveria bassiana Infection.

PubMed

Lü, Dingding; Hou, Chengxiang; Qin, Guangxing; Gao, Kun; Chen, Tian; Guo, Xijie

2017-01-01

A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm, Bombyx mori , by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected by Beauveria bassiana , the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defend B. bassiana infection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm.

Expression profiling of Bombyx mori gloverin2 gene and its synergistic antifungal effect with cecropin A against Beauveria bassiana.

PubMed

Lü, Dingding; Geng, Tao; Hou, Chengxiang; Qin, Guangxing; Gao, Kun; Guo, Xijie

2017-02-05

Gloverin2 is a cationic and glycine-rich antimicrobial peptide whose expression can be induced in fat body of silkworm (Bombyx mori) larvae exposed to bacteria. The purpose of this study is to identify the roles of Bombyx mori gloverin2 (Bmgloverin2) during entomopathogenic fungus Beauveria bassiana infection. Fluorescent quantitative real-time PCR analysis indicated that the relative expression level of Bmgloverin2 gene was up-regulated in the silkworm larvae infected by B. bassiana. The cDNA of Bmgloverin2 was cloned from the silkworm by RT-PCR and the DNA segment of the Bmgloverin2 peptide (without signal peptide sequence) was inserted into pCzn1 expression plasmid and expressed in E. coli ArcticExpress (DE3). SDS-PAGE results revealed that soluble recombinant Bmgloverin2 was successfully expressed and purified. Polyclonal antibody against the Bmgloverin2 was successfully produced with the expressed recombinant protein. Western blot analysis indicated that Bmgloverin2 could be detected in the fat body of silkworm larvae infected with B. bassiana, suggesting that the expression of Bmgloverin2 could be induced by B. bassiana infection in silkworm. Antifungal assays indicated that the Bmgloverin2 had a synergistic antifungal activity with B. mori cecropin A (BmCecA) to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that Bmgloverin2 exhibits synergistic effect with BmCecA in antifungal activity against B. bassiana. The study demonstrates that Bmgloverin2 is an antifungal protein which plays an important role in synergistic antifungal activity with other antimicrobial peptide in silkworm. Copyright © 2016 Elsevier B.V. All rights reserved.

Effects of Starvation and Thermal Stress on the Thermal Tolerance of Silkworm, Bombyx mori: Existence of Trade-offs and Cross-Tolerances.

PubMed

Mir, A H; Qamar, A

2017-09-27

Organisms, in nature, are often subjected to multiple stressors, both biotic and abiotic. Temperature and starvation are among the main stressors experienced by organisms in their developmental cycle and the responses to these stressors may share signaling pathways, which affects the way these responses are manifested. Temperature is a major factor governing the performance of ectothermic organisms in ecosystems worldwide and, therefore, the thermal tolerance is a central issue in the thermobiology of these organisms. Here, we investigated the effects of starvation as well as mild heat and cold shocks on the thermal tolerance of the larvae of silkworm, Bombyx mori (Linnaeus). Starvation acted as a meaningful or positive stressor as it improved cold tolerance, measured as chill coma recovery time (CCRT), but, at the same time, it acted as a negative stressor and impaired the heat tolerance, measured as heat knockdown time (HKT). In the case of heat tolerance, starvation negated the positive effects of both mild cold as well as mild heat shocks and thus indicated the existence of trade-off between these stressors. Both mild heat and cold shocks improved the thermal tolerance, but the effects were more prominent when the indices were measured in response to a stressor of same type, i.e., a mild cold shock improved the cold tolerance more than the heat tolerance and vice versa. This improvement in thermal tolerance by both mild heat as well as cold shocks indicated the possibility of cross-tolerance between these stressors.

Evaluation of silkworm excrement and mushroom dreg for the remediation of multiple heavy metal/metalloid contaminated soil using pakchoi.

PubMed

Wang, Ruigang; Guo, Junkang; Xu, Yingming; Ding, Yongzhen; Shen, Yue; Zheng, Xiangqun; Feng, Renwei

2016-02-01

The economical, environmental friendly and efficient materials to remediate the pollution with multiple heavy metals and metalloids are scarce. Silkworm excrement (SE) and mushroom dregs (MD) are two types of agricultural wastes, and they are widely used to improve the soil fertility in many regions of China. A pot experiment with sixteen treatments was set up to assess the possibility of using SE and MD to stabilize heavy metals and metalloids and reduce their uptake in pakchoi cultivated in slightly contaminated soils with arsenic (As), cadmium (Cd), lead (Pb) and zinc (Zn). The results showed that the single addition of SE obviously stimulated the growth of pakchoi, reduced the contents of all tested heavy metals and metalloids in the edible part of pakchoi and availability of Zn and Cd in soil. The single MD treatment showed an inferior ability to enhance the growth and reduce the contents of heavy metals and metalloids in the edible part of pakchoi. The combined utilization of SE and MD appeared not to show better effects than their individual treatment when using them to remediate this contaminated soil. Some potential mechanisms on the stimulation on pakchoi growth and decreasing the accumulation of heavy metals and metalloids in pakchoi subjected to SE were suggested, including: (1) enhancing soil pH to impact the availability of heavy metals and metalloids; (2) improve the fertility of soil; (3) sulfhydryl groups of organic materials in SE play a role in conjugating heavy metals and metalloids to affect their availability in soil; and (4) stimulating the growth of pakchoi so as to show a "dilution effect" of heavy metals and metalloids. Copyright © 2015 Elsevier Inc. All rights reserved.

Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.

PubMed

Obata, Fumiaki; Tanaka, Shiho; Kashio, Soshiro; Tsujimura, Hidenobu; Sato, Ryoichi; Miura, Masayuki

2015-07-08

Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery. Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation. With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

Transgenic Expression of the piRNA-Resistant Masculinizer Gene Induces Female-Specific Lethality and Partial Female-to-Male Sex Reversal in the Silkworm, Bombyx mori.

PubMed

Sakai, Hiroki; Sumitani, Megumi; Chikami, Yasuhiko; Yahata, Kensuke; Uchino, Keiro; Kiuchi, Takashi; Katsuma, Susumu; Aoki, Fugaku; Sezutsu, Hideki; Suzuki, Masataka G

2016-08-01

In Bombyx mori (B. mori), Fem piRNA originates from the W chromosome and is responsible for femaleness. The Fem piRNA-PIWI complex targets and cleaves mRNAs transcribed from the Masc gene. Masc encodes a novel CCCH type zinc-finger protein and is required for male-specific splicing of B. mori doublesex (Bmdsx) transcripts. In the present study, several silkworm strains carrying a transgene, which encodes a Fem piRNA-resistant Masc mRNA (Masc-R), were generated. Forced expression of the Masc-R transgene caused female-specific lethality during the larval stages. One of the Masc-R strains weakly expressed Masc-R in various tissues. Females heterozygous for the transgene expressed male-specific isoform of the Bombyx homolog of insulin-like growth factor II mRNA-binding protein (ImpM) and Bmdsx. All examined females showed a lower inducibility of vitellogenin synthesis and exhibited abnormalities in the ovaries. Testis-like tissues were observed in abnormal ovaries and, notably, the tissues contained considerable numbers of sperm bundles. Homozygous expression of the transgene resulted in formation of the male-specific abdominal segment in adult females and caused partial male differentiation in female genitalia. These results strongly suggest that Masc is an important regulatory gene of maleness in B. mori.

Airspace Technology Demonstration 3 (ATD-3): Applied Traffic Flow Management Project Overview

NASA Technical Reports Server (NTRS)

Gong, Chester

2016-01-01

ATD-3 Project Overview for 3rd Joint Workshop for KAIA-KARI - NASA ATM Research Collaboration. This presentation gives a high level description of the ATD-3 project and related technologies. These technologies include Multi-Flight Common Routes (MFCR), Traffic Aware Strategic Aircrew Requests (TASAR) and Dynamic Routes for Arrivals in Weather (DRAW).

Silk and silkworm pupa peptides suppress adipogenesis in preadipocytes and fat accumulation in rats fed a high-fat diet.

PubMed

Lee, Sun Hee; Park, Dongsun; Yang, Goeun; Bae, Dae-Kwon; Yang, Yun-Hui; Kim, Tae Kyun; Kim, Dajeong; Kyung, Jangbeen; Yeon, Sungho; Koo, Kyo Chul; Lee, Jeong-Yong; Hwang, Seock-Yeon; Joo, Seong Soo; Kim, Yun-Bae

2012-12-01

The objective was to confirm the anti-obesity activity of a silk peptide (SP) and a silkworm pupa peptide (SPP) in rats fed a high-fat diet (HFD) and to elucidate their action mechanism(s) in a preadipocyte culture system. In an in vitro mechanistic study, the differentiation and maturation of 3T3-L1 preadipocytes were stimulated with insulin (5 μg/mL), and effects of SP and SPP on the adipogenesis of mature adipocytes were assessed. In an in vivo anti-obesity study, male C57BL/6 mice were fed an HFD containing SP or SPP (0.3, 1.0, or 3.0%) for 8 weeks, and blood and tissue parameters of obesity were analyzed. Hormonal stimulation of preadipocytes led to a 50-70% increase in adipogenesis. Polymerase chain reaction and Western blot analyses revealed increases in adipogenesis-specific genes (leptin and Acrp30) and proteins (peroxisome proliferator-activated receptor-γ and Acrp30). The hormone-induced adipogenesis and activated gene expression was substantially inhibited by treatment with SP and SPP (1-50 μg/mL). The HFD markedly increased body weight gain by increasing the weight of epididymal and mesenteric fat. Body and fat weights were significantly reduced by SP and SPP, in which decreases in the area of abdominal adipose tissue and the size of epididymal adipocytes were confirmed by magnetic resonance imaging and microscopic examination, respectively. Long-term HFD caused hepatic lipid accumulation and increased blood triglycerides and cholesterol, in addition to their regulatory factors Acrp30 and leptin. However, SP and SPP recovered the concentrations of Acrp30 and leptin, and attenuated steatosis. SP and SPP inhibit the differentiation of preadipocytes and adipogenesis by modulating signal transduction pathways and improve HFD-induced obesity by reducing lipid accumulation and the size of adipocytes.

Bombyx neuropeptide G protein-coupled receptor A7 is the third cognate receptor for short neuropeptide F from silkworm.

PubMed

Ma, Qiang; Cao, Zheng; Yu, Yena; Yan, Lili; Zhang, Wenjuan; Shi, Ying; Zhou, Naiming; Huang, Haishan

2017-12-15

The short neuropeptide F (sNPF) neuropeptides, closely related to vertebrate neuropeptide Y (NPY), have been suggested to exert pleiotropic effects on many physiological processes in insects. In the silkworm ( Bombyx mori ) two orphan G protein-coupled receptors, Bombyx neuropeptide G protein-coupled receptor (BNGR) A10 and A11, have been identified as cognate receptors for sNPFs, but other sNPF receptors and their signaling mechanisms in B. mori remain unknown. Here, we cloned the full-length cDNA of the orphan receptor BNGR-A7 from the brain of B. mori larvae and identified it as a receptor for Bombyx sNPFs. Further characterization of signaling and internalization indicated that BNGR-A7, -A10, and -A11 are activated by direct interaction with synthetic Bombyx sNPF-1 and -3 peptides. This activation inhibited forskolin or adipokinetic hormone-induced adenylyl cyclase activity and intracellular Ca 2+ mobilization via a G i/o -dependent pathway. Upon activation by sNPFs, BNGR-A7, -A10, and -A11 evoked ERK1/2 phosphorylation and underwent internalization. On the basis of these findings, we designated the receptors BNGR-A7, -A10, and -A11 as Bommo -sNPFR-1, -2, and -3, respectively. Moreover, the results obtained with quantitative RT-PCR analysis revealed that the three Bombyx sNPF receptor subtypes exhibit differential spatial and temporal expression patterns, suggesting possible roles of sNPF signaling in the regulation of a wide range of biological processes. Our findings provide the first in-depth information on sNPF signaling for further elucidation of the roles of the Bombyx sNPF/sNPFR system in the regulation of physiological activities. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

U.S.-Thailand Relations: Analysis of U.S. Military Support to the Kingdom of Thailand

DTIC Science & Technology

2008-01-01

Chen, 1. 7 David K. Wyatt, Thailand: A Short History ( Chiang Mai , Thailand: Silkworm Books, 2003),278. 8 Wyatt, 277. 9 U.S. Department of State...westhawk.blogspot.com/2008/02/marine-corps-expeditionary-plan-is.html Wyatt, David K. Thailand: A Short History. Chiang Mai , Thailand: Silkworm Books

Rheology and dynamic light scattering of silk fibroin solution extracted from the middle division of Bombyx mori silkworm.

PubMed

Ochi, Akie; Hossain, Khandker S; Magoshi, Jun; Nemoto, Norio

2002-01-01

Dynamic light scattering (DLS) and rheological measurements were performed on aqueous silk fibroin solutions extracted from the middle division of Bombyx mori silkworm over a wide range of polymer concentration C from 0.08 to 27.5 wt %. DLS results obtained in the dilute region of C less than 1 wt % are consistent with a model that an elementary unit is a large protein complex consisting of silk fibroin and P25 with a 6:1 molar ratio. Rheological measurements in the dilute C region reveal that those units (or clusters) with the hydrodynamic radius of about 100 nm form a network extending over the whole sample volume with small pseudoplateau modulus mainly by ionic bonding between COO(-) ions of the fibroin molecules and divalent metallic ions such as Ca(2+) or Mg(2+) ions present in the sample and also that, after a yield stress is reached, steady plastic flow is induced with viscosity much lower than the zero-shear viscosity estimated from creep and creep recovery measurements by 4-6 orders of magnitude. Angular frequency omega dependencies of the storage and the loss shear moduli, G'(omega) and G' '(omega), measured in the linear viscoelastic region, indicate that all solutions possess the pseudoplateau modulus in the low omega region and samples become highly viscoleastic for C greater, similar 4.2 wt %. Above C = 11.2 wt % another plateau appears at the high omega end accompanied by a distinct maximum of G' ' in the intermediate omega region. The relaxation motion with tau = 0.5 s corresponding to the maximum of G' ' is one of characteristic properties of the fibroin solutions in the high C region. Thermorheological behaviors of the solution with C = 27.5 wt % show that the network structure formed in the MM part of the silk gland is susceptible to temperature and a more stable homogeneous network is realized by raising the temperature up to T = 65 degrees C.

Surface ultrastructural studies on the germination, penetration and conidial development of Aspergillus flavus Link:Fries infecting silkworm, Bombyx mori Linn.

PubMed

Kumar, Vineet; Singh, G P; Babu, A M

2004-01-01

Aspergillosis is a common disease of the silkworm Bombyx mori Linn., caused by an insect mycopathogen Aspergillus flavus Link:Fries. The present study reveals the germination, penetration and conidial development of A. flavus on the larval integument of B. mori under SEM. Four different strains (NB18, KA, NB4D2 and NB7) of B. mori was surface inoculated with ca. of 1 x 10(6) conidia/ml. Each conidium germinated on the cuticle approximately 6 h after inoculation, forming a humpy or suctorial appressoria within 24 h. The hyphae which entered into haemocoel 2 day post-inoculation, grew and multiplied extensively, forming a mycelial complex, causing death of the host larva in about 4-5 days. This occurred with minimal breakdown of the internal tissues. Death of the host was followed by ramification of the fungus through the mesodermal and epidermal tissues, leading to larval mummification about 5-6 days after inoculation. Extensive fungal growths on the entire larval body followed, consisting of aerial hyphae, which developed branched conidiophores. The aerial hyphae with abundant conidiophores formed a confluent yellowish green fungal mat over the entire larval body in 6-7 days of post-inoculation. The tip of each emerging conidiophores gradually dilated and developed to become a bulbous head known as the vesicle. A large number of conidiogenous cells were produced over the entire surface of vesicle, which later developed into finger-like projections termed as sterigmata or phialides. The phialides matured within 2 days after the aerial hyphae emerged as evidenced by chains of conidia at their tips. The conidia were globose with externally roughened walls. The life cycle of the fungus on B. mori was completed in six to seven days.

YorkieCA overexpression in the posterior silk gland improves silk yield in Bombyx mori.

PubMed

Zhang, Panli; Liu, Shumin; Song, Hong-Sheng; Zhang, Guozheng; Jia, Qiangqiang; Li, Sheng

2017-07-01

The traditional hybrid breeding techniques can no longer meet the increasing demands for silk production by the silkworm, Bombyx mori, and further improvement of the silk yield will depend on modern molecular breeding techniques. Here, we report improved silk yield in transgenic silkworms overexpressing the oncogene Yorkie CA specifically in the posterior silk gland (PSG). The Yorkie CA cDNA was ligated downstream of the hr3 enhancer and the fibroin L-chain (Fil) promoter, then inserted into a piggyBac vector for transgene. Overexpression of Yorkie CA in the PSG significantly increased the weight of the PSG, and also increased the weight of the cocoon, larval body, and pupal body to decreasing degrees. Overexpression of Yorkie CA up-regulated the Yorkie target genes resulting in increased cell size, endomitosis, the number of protein synthesis organelles, the expression of fibroin genes in the PSG, and eventually silk yield. Additionally, as we reported previously using the binary GAL4/UAS system, transgenic silkworms overexpressing Ras1 CA with the hr3 enhancer and the Fil promoter also showed improved silk yield. Unfortunately, the hybrid progeny of Yorkie CA -overexpressing silkworms and Ras1 CA -overexpressing silkworms did not show overlapping improved silk yield due to the failure to increase expression of both Yorkie and Ras1. Copyright © 2017. Published by Elsevier Ltd.

Alternative splicing and trans-splicing events revealed by analysis of the Bombyx mori transcriptome

PubMed Central

Shao, Wei; Zhao, Qiong-Yi; Wang, Xiu-Ye; Xu, Xin-Yan; Tang, Qing; Li, Muwang; Li, Xuan; Xu, Yong-Zhen

2012-01-01

Alternative splicing and trans-splicing events have not been systematically studied in the silkworm Bombyx mori. Here, the silkworm transcriptome was analyzed by RNA-seq. We identified 320 novel genes, modified 1140 gene models, and found thousands of alternative splicing and 58 trans-splicing events. Studies of three SR proteins show that both their alternative splicing patterns and mRNA products are conserved from insect to human, and one isoform of Srsf6 with a retained intron is expressed sex-specifically in silkworm gonads. Trans-splicing of mod(mdg4) in silkworm was experimentally confirmed. We identified integrations from a common 5′-gene with 46 newly identified alternative 3′-exons that are located on both DNA strands over a 500-kb region. Other trans-splicing events in B. mori were predicted by bioinformatic analysis, in which 12 events were confirmed by RT-PCR, six events were further validated by chimeric SNPs, and two events were confirmed by allele-specific RT-PCR in F1 hybrids from distinct silkworm lines of JS and L10, indicating that trans-splicing is more widespread in insects than previously thought. Analysis of the B. mori transcriptome by RNA-seq provides valuable information of regulatory alternative splicing events. The conservation of splicing events across species and newly identified trans-splicing events suggest that B. mori is a good model for future studies. PMID:22627775

Fertilized eggs obtained from transplantation of frozen ovaries and parthenogenesis in combination with artificial insemination of frozen semen of the silkworm, Bombyx mori.

PubMed

Mochida, Yuji; Takemura, Yoko; Kanda, Toshio; Horie, Yasuhiro

2003-04-01

A reliable method is reported for the long-term preservation of ovaries and spermatozoa of the silkworm (Bombyx mori). Three studies are presented. In the first, ovaries were removed from larvae at either 3rd, 4th, or 5th instar, cryopreserved, and stored in liquid nitrogen. Thawed ovaries were transplanted to surgically castrated female larvae at the same or a different developmental stage. The highest percentage of recipient females producing eggs resulted into either 3rd or 4th instar larvae (respectively, 22.1 and 8.7%). Similarly, the highest levels of other measurements of successful cryopreservation and transplanted ovary, and number of eggs laid, occurred with the same combination of donor and recipient developmental stages. Other combinations of ovary/recipient developmental stages yielded lower results. In the second experiment, semen was collected from male moths, cryopreserved, and then thawed semen was diluted with trypsin solution and artificially inseminated into females obtained from the best conditions of first experiment. A small percentage of inseminated moths laid eggs (8-10.3%) compared to that of controls (100%). In addition, the fertility of eggs from experimental moths was lower than that of control females (respectively, 40.3-88% and 97.5%). In the third experiment, eggs were surgically removed from ovarian tubules of moth following transplantation of thawed ovaries and subjected to parthenogenetic activation and artificial hatching. As expected, all resulting moths were female and, following natural mating or artificial insemination with thawed semen, yielded normal offspring at high rates.

Genetic engineered color silk: fabrication of a photonics material through a bioassisted technology.

PubMed

Shimizu, Katsuhiko

2018-05-15

Silk produced by the silkworm Bombyx mori is an attractive material because of its luster, smooth and soft texture, conspicuous mechanical strength, good biocompatibility, slow biodegradation, and carbon neutral synthesis. Silkworms have been domesticated and bred for production of better quality and quantity of silk, resulting in the development of sericulture and the textile industry. Silk is generally white, so dyeing is required to obtain colored fiber. However, the dyeing process involves harsh conditions and generates a large volume of waste water, which have environmentally and economically negative impacts. Although some strains produce cocoons that contain pigments derived from the mulberry leaves that they eat, the pigments are distributed in the sericin layer and are lost during gumming. In trials for production of colored silk by feeding silkworms on diets containing dyes, only limited species of dye molecules were incorporated into the silk threads. A method for the generation of transgenic silkworm was established in conjunction with the discovery of green fluorescent protein (GFP), and silkworms carrying the GFP gene spun silk threads that formed cocoons that glowed bright green and still retained the original properties of silk. A wide range of color variation of silk threads has been obtained by replacing the GFP gene with the genes of other fluorescent proteins chosen from the fluorescent protein palette. The genetically modified silk with photonic properties can be processed to form various products including linear threads, 2D fabrics, and 3D materials. The transgenic colored silk could be economically advantageous due to addition of a new value to silk and reduction of cost for water waste, and environmentally preferable for saving water. Here, I review the literature regarding the production methods of fluorescent silk from transgenic silkworms and present examples of genetically modified color silk.

A Hypothetical Model of Crossing Bombyx mori Nucleopolyhedrovirus through Its Host Midgut Physical Barrier

PubMed Central

Cheng, Yang; Wang, Xue-Yang; Hu, Hao; Killiny, Nabil; Xu, Jia-Ping

2014-01-01

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV. PMID:25502928

Genome-wide identification, characterization of sugar transporter genes in the silkworm Bombyx mori and role in Bombyx mori nucleopolyhedrovirus (BmNPV) infection.

PubMed

Govindaraj, Lekha; Gupta, Tania; Esvaran, Vijaya Gowri; Awasthi, Arvind Kumar; Ponnuvel, Kangayam M

2016-04-01

Sugar transporters play an essential role in controlling carbohydrate transport and are responsible for mediating the movement of sugars into cells. These genes exist as large multigene families within the insect genome. In insects, sugar transporters not only have a role in sugar transport, but may also act as receptors for virus entry. Genome-wide annotation of silkworm Bombyx mori (B. mori) revealed 100 putative sugar transporter (BmST) genes exists as a large multigene family and were classified into 11 sub families, through phylogenetic analysis. Chromosomes 27, 26 and 20 were found to possess the highest number of BmST paralogous genes, harboring 22, 7 and 6 genes, respectively. These genes occurred in clusters exhibiting the phenomenon of tandem gene duplication. The ovary, silk gland, hemocytes, midgut and malphigian tubules were the different tissues/cells enriched with BmST gene expression. The BmST gene BGIBMGA001498 had maximum EST transcripts of 134 and expressed exclusively in the malphigian tubule. The expression of EST transcripts of the BmST clustered genes on chromosome 27 was distributed in various tissues like testis, ovary, silk gland, malphigian tubule, maxillary galea, prothoracic gland, epidermis, fat body and midgut. Three sugar transporter genes (BmST) were constitutively expressed in the susceptible race and were down regulated upon BmNPV infection at 12h post infection (hpi). The expression pattern of these three genes was validated through real-time PCR in the midgut tissues at different time intervals from 0 to 30hpi. In the susceptible B. mori race, expression of sugar transporter genes was constitutively expressed making the host succumb to viral infection. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of the biosynthesis and signaling pathway of ecdysterone on silkworm (Bombyx mori) following exposure to titanium dioxide nanoparticles.

PubMed

Li, Fanchi; Gu, Zhiya; Wang, Binbin; Xie, Yi; Ma, Lie; Xu, Kaizun; Ni, Min; Zhang, Hua; Shen, Weide; Li, Bing

2014-08-01

Silkworm (Bombyx mori), a model Lepidoptera insect, is economically important. Its growth and development are regulated by endogenous hormones. During the process of transition from larvae to pupae, 20-hydroxyecdysone (20E) plays an important role. The recent surge in consumer products and applications using metallic nanoparticles has increased the possibility of human or ecosystem exposure due to their unintentional release into the environment. We investigated the effects of exposure to titanium dioxide nanoparticles (TiO2 NPs) on the action of 20E in B. mori. Titanium dioxide nanoparticle treatment shortened the molting duration by 8 hr and prolonged the molting peak period by 10 %. Solexa sequencing profiled the changes in gene expression in the brain of fifth-instar B. mori in response to TiO2NPS exposure for 72 hr, to address the effects on hormone metabolism and regulation. Thirty one genes were differentially expressed. The transcriptional levels of pi3k and P70S6K, which are involved in the target of the rapamycin (TOR) signaling pathway, were up-regulated. Transcriptional levels of four cytochrome P450 genes, which are involved in 20E biosynthesis, at different developmental stages (48, 96, 144, and 192 hr) at 5th instars of all displayed trends of increasing expression. Simultaneously, the ecdysterone receptors, also displayed increasing trends. The 20E titers at four developmental stages during the 5th instar were 1.26, 1.23, 1.72, and 2.16 fold higher, respectively, than the control group. These results indicate that feeding B. mori with TiO2 NPs stimulates 20E biosynthesis, shortens the developmental progression, and reduces the duration of molting. Thus, application of TiO2 NPs is of high significance for saving the labor force in sericulture, and our research provides a reference for the ecological problems in the field of Lepidoptera exposured to titanium dioxide nanoparticles.

Effects of Titanium Dioxide Nanoparticles on the Synthesis of Fibroin in Silkworm (Bombyx mori).

PubMed

Ni, Min; Li, FanChi; Tian, JiangHai; Hu, JingSheng; Zhang, Hua; Xu, KaiZun; Wang, BinBin; Li, YangYang; Shen, WeiDe; Li, Bing

2015-08-01

Silkworm (Bombyx mori) is an economically important insect, and its silk production capacity largely depends on its ability to synthesize fibroin. While breeding of B. mori varieties has been a key strategy to improve silk production, little improvement of B. mori silk production has been achieved to date. As a result, the development of sericulture economy has not progressed well, pointing to the need of new ways for improvement of B. mori silk production. Titanium dioxide nanoparticles (TiO2 NPs), a food additive widely used for livestock, have been shown to promote animal growth and increase the protein synthesis in animals. However, no studies on effect of TiO2 NPs on fibroin synthesis in B. mori have been available. In this study, the differential expression profiles of genes and proteins in the silk gland of B. mori fed without or with TiO2 NPs (5 μg ml(-1)) were analyzed and compared using digital gene expression (DGE), reverse transcription quantitative polymerase chain reaction (RT-qPCR), semi-qPCR, and Western blot analysis. The effects of TiO2 NPs feeding on the activity of proteases in the midgut and the synthesis and transportation of amino acids in hemolymph were also investigated. DGE analyses showed that among a total of 4,741 genes detected, 306 genes were differentially expressed after the TiO2 NPs feeding, of which 137 genes were upregulated whereas 169 genes were downregulated. 106 genes were shown to be involved in fibroin synthesis, of which 97 genes, including those encoding cuticular protein glycine-rich 10, serine protease inhibitor 28, aspartate aminotransferase, lysyl-tRNA synthetase, and splicing factor arginine/serine-rich 6, and silk gland factor-1 (SGF-1), were upregulated with the maximum induction of 8.52-folds, whereas nine genes, including those encoding aspartylglucosaminidase, the cathepsin L in Tribolium castaneum, and similar to SPRY domain-containing SOCS box protein 3, were downregulated with the maximum reduction of 8

Abnormal swelling of the peritrophic membrane in Eri silkworm gut caused by MLX56 family defense proteins with chitin-binding and extensin domains.

PubMed

Konno, Kotaro; Shimura, Sachiko; Ueno, Chihiro; Arakawa, Toru; Nakamura, Masatoshi

2018-03-01

MLX56 family defense proteins, MLX56 and its close homolog LA-b, are chitin-binding defense proteins found in mulberry latex that show strong growth-inhibitions against caterpillars when fed at concentrations as low as 0.01%. MLX56 family proteins contain a unique structure with an extensin domain surrounded by two hevein-like chitin-binding domains, but their defensive modes of action remain unclear. Here, we analyzed the effects of MLX56 family proteins on the peritrophic membrane (PM), a thin and soft membrane consisting of chitin that lines the midgut lumen of insects. We observed an abnormally thick (>1/5 the diameter of midgut) hard gel-like membrane consisted of chitin and MLX56 family proteins, MLX56 and LA-b, in the midgut of the Eri silkworms, Samia ricini, fed a diet containing MLX56 family proteins, MLX56 and LA-b. When polyoxin AL, a chitin-synthesis-inhibitor, was added to the diet containing MLX56 family proteins, the toxicity of MLX56 family proteins disappeared and PM became thinner and fragmented. These results suggest that MLX56 family proteins, through their chitin-binding domains, bind to the chitin framework of PM, then through their extensin-domain (gum arabic-like structure), which functions as swelling agent, expands PM into an abnormally thick membrane that inhibits the growth of insects. This study shows that MLX56 family proteins are plant defense lectins with a totally unique mode of action, and reveals the functions of extensin domains and arabinogalactan proteins as swelling (gel-forming) agents of plants. Copyright © 2018 Elsevier Ltd. All rights reserved.

The molecular mechanism of the termination of insect diapause, part 1: A timer protein, TIME-EA4, in the diapause eggs of the silkworm Bombyx mori is a metallo-glycoprotein.

PubMed

Isobe, Minoru; Kai, Hidenori; Kurahashi, Takuya; Suwan, Sathorn; Pitchayawasin-Thapphasaraphong, Suthasinee; Franz, Thomas; Tani, Naoki; Higashi, Kenichiro; Nishida, Hideo

2006-10-01

TIME-EA4 is an ATPase that measures time intervals, functioning as a diapause duration clock in diapause eggs of the silkworm, Bombyx mori. Characterization of the primary and higher structures of the TIME-EA4 would be desirable to clarify the mechanism by which the protein measures the time intervals. In our current studies, the whole sequence of TIME-EA4 has been established as that of a metallo-glycoprotein by combinational means involving peptide sequence analysis, nano-HPLC-ESI-Q-TOF-MS and MS/MS, and cDNA dictation. The amino acid sequence of TIME-EA4 showed 46-55 % homology with the reported proteins of the Cu,Zn-SOD (superoxide dismutase) family; in particular, the SOD active site (core domain) includes metal-binding amino acid ligands and a disulfide bond, and these structures are completely identical in Bombyx SOD, bovine SOD, and TIME-EA4 proteins. We found, however, that TIME-EA4 contains one more copper ion than other SODs, as was proven under neutral nondenaturing conditions. ESI mass spectrometry revealed that the timer function was not in the SOD core domain. In addition, TIME-EA4 has an attached sugar chain, which is indispensable to its functioning as a timer protein.

Effect of octopamine on the activity of juvenile-hormone esterase in the silkworm Bombyx mori and the red flour beetle Tribolium freemani.

PubMed

Hirashima, A; Suetsugu, E; Hirokado, S; Kuwano, E; Taniguchi, E; Eto, M

1999-12-01

This study focuses on the effect of octopamine (OA) on metamorphosis of the silkworm Bombyx mori and the red flour beetle Tribolium freemani Hinton. Titers of OA and juvenile-hormone esterase (JHE) were measured at various larval and pupal stadia of both insects. Effects of OA, OA agonists, and antagonists on metamorphosis and JHE activity were also examined. At day 2, peaks of OA and JHE activity were observed in third instars, and at day 3, a sharp peak of OA was observed, followed by a large peak of JHE activity at day 4 in last instars of B. mori. However, no peaks of OA and JHE activity were observed in fourth instars. A high titer of OA appeared at days 2-4, followed by a peak of JHE activity at day 7 and the second OA peak at day 9 after the start of assay of T. freemani. At pupation, a small peak of OA and the highest activity of JHE were observed. The effects of OA on JHE activity were examined in vitro, because the relationship could be responsible for triggering pupation in B. mori and T. freemani larvae. Exogeneous OA (0.1-10 mM) stimulated the JHE activity of final instars (day 2) of B. mori in vitro. Similarly, the presence of OA (10 mM) activated the JHE activity of newly ecdysed T. freemani pupae in vitro. OA antagonists chlorpromazine and gramine delayed the start of spinning and reduced the JHE activity of B. mori, when applied in diet at 10-100 ppm. Some OA agonists stimulated the pupation and JHE activity of T. freemani larvae reared under crowded conditions, when topically applied. Thus, OA may contribute to activation of the events preparatory to a pupal molt, i.e., the secretion of OA increases JHE activity followed by stimulation of pupation. Copyright 1999 Academic Press.

Purification of functional baculovirus particles from silkworm larval hemolymph and their use as nanoparticles for the detection of human prorenin receptor (PRR) binding

PubMed Central

2011-01-01

Background Baculovirus, which has a width of 40 nm and a length of 250-300 nm, can display functional peptides, receptors and antigens on its surface by their fusion with a baculovirus envelop protein, GP64. In addition, some transmembrane proteins can be displayed without GP64 fusion, using the native transmembrane domains of the baculovirus. We used this functionality to display human prorenin receptor fused with GFPuv (GFPuv-hPRR) on the surface of silkworm Bombyx mori nucleopolyhedrovirus (BmNPV) and then tested whether these baculovirus particles could be used to detect protein-protein interactions. Results BmNPV displaying GFPuv-hPRR (BmNPV-GFPuv-hPRR) was purified from hemolymph by using Sephacryl S-1000 column chromatography in the presence of 0.01% Triton X-100. Its recovery was 86% and the final baculovirus particles number was 4.98 × 108 pfu. Based on the results of enzyme-linked immunosorbent assay (ELISA), 3.1% of the total proteins in BmNPV-GFPuv-hPRR were GFPuv-hPRR. This value was similar to that calculated from the result of western blot by a densitometry (2.7%). To determine whether BmNPV-GFPuv-hPRR particles were bound to human prorenin, ELISA results were compared with those from ELISAs using protease negative BmNPV displaying β1,3-N-acetylglucosaminyltransferase 2 fused with the gene encoding GFPuv (GGT2) (BmNPV-CP--GGT2) particles, which do not display hPRR on their surfaces. Conclusion The display of on the surface of the BmNPV particles will be useful for the detection of protein-protein interactions and the screening of inhibitors and drugs in their roles as nanobioparticles. PMID:21635720

Synthesis of prolyl 4-hydroxylase alpha subunit and type IV collagen in hemocytic granular cells of silkworm, Bombyx mori: Involvement of type IV collagen in self-defense reaction and metamorphosis.

PubMed

Adachi, Takahiro; Tomita, Masahiro; Yoshizato, Katsutoshi

2005-04-01

The present study shows that hemocytic granular cells synthesize and secrete type IV collagen (ColIV) in the silkworm Bombyx mori (B. mori) and suggests that these cells play roles in the formation of basement membrane, the encapsulation of foreign bodies, and the metamorphic remodeling of the gut. The full- and partial-length cDNA of B. mori prolyl 4-hydroxylase alpha subunit (BmP4Halpha) and B. mori ColIV (BmColIV) were cloned, respectively. In situ hybridization and immunocytochemistry on larval tissues and cells identified hemocytic granular cells as the cells that express mRNAs and proteins of both BmP4Halpha and BmColIV. Immunohistochemistry and immunocytochemistry demonstrated that BmColIV was present in the basement membrane and in the secretory granules of granular cells, respectively. Granular cells in culture secreted BmColIV without accompanying the degranulation and discharged it from the granules when the cells were degranulated. Nylon threads were inserted into the hemocoel of larvae. Granular cells concentrated around the nylon threads and encapsulated them as a self-defense reaction. BmColIV was found to be a component of the capsules. Furthermore, the present study showed that actively BmColIV-expressing granular cells accumulated around the midgut epithelium and formed BmColIV-rich thick basal lamina-like structures there in larval to pupal metamorphosis.

Antifreeze Production and Cold-Tolerance in Overwintering Purple Martin Fleas, Ceratophyllus idius Jordan and Rothschild.

DTIC Science & Technology

1986-08-01

Bombyx mori , isotope [ 14C] studies showed that about 1/3 of the free glycerol pool came from lipids (Yaginuma and Yamashita 1980). However, the same... Bombyx silkworm. Nature, Lond. 180: 606-607. Chino, H. 1958. Carbohydrate metabolism in the diapause egg of the silkworm, Bombyx mori -II. Conversion of...1980. The origin of free glycerol accumulated in diapause eggs of Bombyx mori . Physiol. Entomol. 5: 93-97. Zachariassen, K.E. 1977. Effects of

The angiotensin converting enzyme (ACE) inhibitor, captopril disrupts the motility activation of sperm from the silkworm, Bombyx mori.

PubMed

Nagaoka, Sumiharu; Kawasaki, Saori; Kawasaki, Hideki; Kamei, Kaeko

2017-11-01

peptide) that was expressed in Escherichia coli cells exhibited captopril-sensitive carboxypeptidase activities. Our findings show that the BmAcre2 gene encodes a secreted ACE protein included in the Bombyx seminal plasma. In particular, the silkworm ACE protein in the seminal fluid might be involved in the signaling pathway that leads to the activation and regulation of sperm motility. Copyright © 2017 Elsevier Ltd. All rights reserved.

Histamine poisoning from insect consumption: an outbreak investigation from Thailand.

PubMed

Chomchai, Summon; Chomchai, Chulathida

2018-02-01

Insect consumption is a common practice in the Asian culture and all over the world. We are reporting an outbreak investigation of histamine poisoning from ingestion of fried insects. On 24 July 2014, a group of students at a seminar presented to Angthong Provincial Hospital, Thailand, with pruritic rash after ingesting snacks consisting of fried insects from a vendor. We initiated an outbreak investigation with retrospective cohort design and collected samples of remaining foods for analyses. Attack rates, relative risks and their confidence intervals (CI) were calculated. Out of 227 students, 28 developed illnesses that were consistent with our case definition which included, flushing, pruritus, urticarial rashes, headache, nausea, vomiting, diarrhea, dyspnea and bronchospasm. Two children were hospitalized for progressive bronchospasm overnight without serious complications. The types of food ingested included a lunch that was provided at the seminar for all students and snacks that 41 students bought from the only vendor in the vicinity. The snacks included fried grasshoppers, silkworm pupae, common green frogs, bamboo borers, crickets and meat balls. The attack rates were highest (82.6 and 85.0%) among students who ingested fried grasshoppers and silkworm pupae and lowest (4.4 and 5.3%) among those who did not ingest them, with relative risk of 18.7 (95% CI 9.6-36.4) for grasshoppers and 16.0 (95% CI 8.8-29.3) for silkworm pupae. Histamine concentrations in the fried grasshoppers and silkworm pupae were 9.73 and 7.66 mg/100g, respectively. Through epidemiological analysis and laboratory confirmation, we have illustrated that histamine poisoning can occur from ingestion of fried insects. We postulate that histidine, which is present in high concentration in grasshoppers and silkworm pupae, is decarboxylated by bacteria to histamine, a heat stable toxin. The ingestion of histamine is responsible for the clinical pictures being reported.

Proteomic analysis of Bombyx mori molting fluid: Insights into the molting process.

PubMed

Liu, Hua-Wei; Wang, Luo-Ling; Tang, Xin; Dong, Zhao-Ming; Guo, Peng-Chao; Zhao, Dong-Chao; Xia, Qing-You; Zhao, Ping

2018-02-20

Molting is an essential biological process occurring multiple times throughout the life cycle of most Ecdysozoa. Molting fluids accumulate and function in the exuvial space during the molting process. In this study, we used liquid chromatography-tandem mass spectrometry to investigate the molting fluids to analyze the molecular mechanisms of molting in the silkworm, Bombyx mori. In total, 375 proteins were identified in molting fluids from the silkworm at 14-16h before pupation and eclosion, including 12 chitin metabolism-related enzymes, 35 serine proteases, 15 peptidases, and 38 protease inhibitors. Gene ontology analysis indicated that "catalytic" constitutes the most enriched function in the molting fluid. Gene expression patterns and bioinformatic analyses suggested that numerous enzymes are involved in the degradation of cuticle proteins and chitin. Protein-protein interaction network and activity analyses showed that protease inhibitors are involved in the regulation of multiple pathways in molting fluid. Additionally, many immune-related proteins may be involved in the immune defense during molting. These results provide a comprehensive proteomic insight into proteolytic enzymes and protease inhibitors in molting fluid, and will likely improve the current understanding of physiological processes in insect molting. Insect molting constitutes a dynamic physiological process. To better understand this process, we used LC-MS/MS to investigate the proteome of silkworm molting fluids and identified key proteins involved in silkworm molting. The biological processes of the old cuticle degradation pathway and immune defense response were analyzed in the proteome of silkworm molting fluid. We report that protease inhibitors serve as key factors in the regulation of the molting process. The proteomic results provide new insight into biological molting processes in insects. Copyright © 2017 Elsevier B.V. All rights reserved.

Silk-based biomaterials.

PubMed

Altman, Gregory H; Diaz, Frank; Jakuba, Caroline; Calabro, Tara; Horan, Rebecca L; Chen, Jingsong; Lu, Helen; Richmond, John; Kaplan, David L

2003-02-01

Silk from the silkworm, Bombyx mori, has been used as biomedical suture material for centuries. The unique mechanical properties of these fibers provided important clinical repair options for many applications. During the past 20 years, some biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue-like proteins) was the likely cause. More recent studies with well-defined silkworm silk fibers and films suggest that the core silk fibroin fibers exhibit comparable biocompatibility in vitro and in vivo with other commonly used biomaterials such as polylactic acid and collagen. Furthermore, the unique mechanical properties of the silk fibers, the diversity of side chain chemistries for 'decoration' with growth and adhesion factors, and the ability to genetically tailor the protein provide additional rationale for the exploration of this family of fibrous proteins for biomaterial applications. For example, in designing scaffolds for tissue engineering these properties are particularly relevant and recent results with bone and ligament formation in vitro support the potential role for this biomaterial in future applications. To date, studies with silks to address biomaterial and matrix scaffold needs have focused on silkworm silk. With the diversity of silk-like fibrous proteins from spiders and insects, a range of native or bioengineered variants can be expected for application to a diverse set of clinical needs.

Azadirachtin induced apoptosis in the prothoracic gland in Bombyx mori and a pronounced Ca2+ release effect in Sf9 cells

PubMed Central

Zhang, Jing; Liu, Hongmei; Sun, Zhipeng; Xie, Jianjun; Zhong, Guohua; Yi, Xin

2017-01-01

Azadirachtin is a bio-rational insecticide used as an antifeedant and growth disruption agent against many insect species. However, recent studies have shown that there is a potential risk of this compound harming some beneficial insects. In such cases its application does not normally lead to death, but it may result in altered developmental regulation. Therefore, it is essential to obtain toxicological data to understand the mechanism of such sub-lethal effects, especially where they relate to important beneficial insects. Here, we found that azadirachtin could regulate growth and cocooning in silkworms, which may be associated with induced apoptotic effect on the prothoracic gland. However, azadirachtin treatment could not induce apoptosis in the prothoracic gland in vitro, in contrast to the effect of 20-hydroxyecdysone in vitro, which suggesting that the apoptosis might not be direct effect of azadirachtin. Then we examined the activity of Ca2+-Mg2+-ATPase and found that azadirachtin could trigger a significant increase in intracellular Ca2+ release in the Sf9 cell line, which suggested that the calcium signaling pathway might be involved in the process of apoptosis in prothoracic gland and growth regulation in vivo silkworms. Although more evidence is needed to fully understand the mechanism of azadirachtin in perturbing the growth of silkworms, this study provides some toxicological information and highlights the potential risks of azadirachtin in relation to silkworms. PMID:29230101

Azadirachtin induced apoptosis in the prothoracic gland in Bombyx mori and a pronounced Ca2+ release effect in Sf9 cells.

PubMed

Zhang, Jing; Liu, Hongmei; Sun, Zhipeng; Xie, Jianjun; Zhong, Guohua; Yi, Xin

2017-01-01

Azadirachtin is a bio-rational insecticide used as an antifeedant and growth disruption agent against many insect species. However, recent studies have shown that there is a potential risk of this compound harming some beneficial insects. In such cases its application does not normally lead to death, but it may result in altered developmental regulation. Therefore, it is essential to obtain toxicological data to understand the mechanism of such sub-lethal effects, especially where they relate to important beneficial insects. Here, we found that azadirachtin could regulate growth and cocooning in silkworms, which may be associated with induced apoptotic effect on the prothoracic gland. However, azadirachtin treatment could not induce apoptosis in the prothoracic gland in vitro , in contrast to the effect of 20-hydroxyecdysone in vitro, which suggesting that the apoptosis might not be direct effect of azadirachtin. Then we examined the activity of Ca 2+ -Mg 2+ -ATPase and found that azadirachtin could trigger a significant increase in intracellular Ca 2+ release in the Sf9 cell line, which suggested that the calcium signaling pathway might be involved in the process of apoptosis in prothoracic gland and growth regulation in vivo silkworms. Although more evidence is needed to fully understand the mechanism of azadirachtin in perturbing the growth of silkworms, this study provides some toxicological information and highlights the potential risks of azadirachtin in relation to silkworms.

Characterization of Bombyx mori mitochondrial transcription factor A, a conserved regulator of mitochondrial DNA.

PubMed

Sumitani, Megumi; Kondo, Mari; Kasashima, Katsumi; Endo, Hitoshi; Nakamura, Kaoru; Misawa, Toshihiko; Tanaka, Hiromitsu; Sezutsu, Hideki

2017-04-15

In the present study, we initially cloned and characterized a mitochondrial transcription factor A (Tfam) homologue in the silkworm, Bombyx mori. Bombyx mori TFAM (BmTFAM) localized to mitochondria in cultured silkworm and human cells, and co-localized with mtDNA nucleoids in human HeLa cells. In an immunoprecipitation analysis, BmTFAM was found to associate with human mtDNA in mitochondria, indicating its feature as a non-specific DNA-binding protein. In spite of the low identity between BmTFAM and human TFAM (26.5%), the expression of BmTFAM rescued mtDNA copy number reductions and enlarged mtDNA nucleoids in HeLa cells, which were induced by human Tfam knockdown. Thus, BmTFAM compensates for the function of human TFAM in HeLa cells, demonstrating that the mitochondrial function of TFAM is highly conserved between silkworms and humans. BmTfam mRNA was strongly expressed in early embryos. Through double-stranded RNA (dsRNA)-based RNA interference (RNAi) in silkworm embryos, we found that the knockdown of BmTFAM reduced the amount of mtDNA and induced growth retardation at the larval stage. Collectively, these results demonstrate that BmTFAM is a highly conserved mtDNA regulator and may be a good candidate for investigating and modulating mtDNA metabolism in this model organism. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparative proteomic analysis of Bombyx mori hemolymph and fat body after calorie restriction.

PubMed

Chen, Huiqing; Li, Yijia; Chen, Keping; Yao, Qin; Li, Guohui; Wang, Lin

2010-01-01

Calorie restriction (CR) is known to extend life span from yeast to mammals. To gain an insight into the effects of CR on growth and development of the silkworm Bombyx mori at protein level, we employed comparative proteomic approach to investigate proteomic differences of hemolymph and fat body of the silkworm larvae subjected to CR. Thirty-nine differentially expressed proteins were identified by MALDI TOF/TOF MS. Among them, 19 were from the hemolymph and 20 from the fat body. The hemolymph of the CR group contained two down-regulated and 17 up-regulated proteins, whereas the fat body contained 15 down-regulated and five up-regulated ones. These proteins belonged to those functioning in immune system, in signal transduction and apoptosis, in regulation of growth and development, and in energy metabolism. Our results suggest that CR can alter the expression of proteins related to the above four aspects, implying that these proteins may regulate life span of the silkworm through CR.

GluCl a target of indole alkaloid okaramines: a 25 year enigma solved

NASA Astrophysics Data System (ADS)

Furutani, Shogo; Nakatani, Yuri; Miura, Yuka; Ihara, Makoto; Kai, Kenji; Hayashi, Hideo; Matsuda, Kazuhiko

2014-08-01

In 1989, indole alkaloid okaramines isolated from the fermentation products of Penicillium simplicissimum were shown to be insecticidal, yet the mechanism of their toxicity to insects remains unknown. We therefore examined the action of okaramine B on silkworm larval neurons using patch-clamp electrophysiology. Okaramine B induced inward currents which reversed close to the chloride equilibrium potential and were blocked by fipronil. Thus it was tested on the silkworm RDL (resistant-to-dieldrin) γ-aminobutyric-acid-gated chloride channel (GABACl) and a silkworm L-glutamate-gated chloride channel (GluCl) expressed in Xenopus laevis oocytes. Okaramine B activated GluCl, but not RDL. GluCl activation by okaramines correlated with their insecticidal activity, offering a solution to a long-standing enigma concerning their insecticidal actions. Also, unlike ivermectin, okaramine B was inactive at 10 μM on human α1β2γ2 GABACl and α1β glycine-gated chloride channels and provides a new lead for the development of safe insect control chemicals.

Carbon dioxide dynamics in an artificial ecosystem

NASA Astrophysics Data System (ADS)

Hu, Enzhu; Hu, Dawei; Tong, Ling; Li, Ming; Fu, Yuming; He, Wenting; Liu, Hong

An experimental artificial ecosystem was established as a tool to understand the behavior of closed ecosystem and to develop the technology for a future bioregenerative life support system for lunar or planetary exploration. Total effective volume of the system is 0.7 m3 . It consists of a higher plant chamber, an animal chamber and a photo-bioreactor which cultivated lettuce (Lactuca sativa L.), silkworm (Bombyx Mori L.) and microalgae (Chlorella), respectively. For uniform and sustained observations, lettuce and silkworms was cultivated using sequential cultivation method, and microalgae using continuous culture. Four researchers took turns breathing the system air through a tube for brief periods every few hours. A mathematic model, simulating the carbon dioxide dynamics was developed. The main biological parameters concerning photosynthesis of lettuce and microalgae, respiration of silkworms and human were validated by the experimental data. The model described the respiratory relationship between autotrophic and heterotrophic compartments. A control strategy was proposed as a tool for the atmosphere management of the artificial ecosystem.

Serratia marcescens Induces Apoptotic Cell Death in Host Immune Cells via a Lipopolysaccharide- and Flagella-dependent Mechanism*

PubMed Central

Ishii, Kenichi; Adachi, Tatsuo; Imamura, Katsutoshi; Takano, Shinya; Usui, Kimihito; Suzuki, Kazushi; Hamamoto, Hiroshi; Watanabe, Takeshi; Sekimizu, Kazuhisa

2012-01-01

Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH2-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity. PMID:22859304

Green cocoons in silkworm Bombyx mori resulting from the quercetin 5-O-glucosyltransferase of UGT86, is an evolved response to dietary toxins.

PubMed

Xu, Xu; Wang, Meng; Wang, Ying; Sima, Yanghu; Zhang, Dayan; Li, Juan; Yin, Weiming; Xu, Shiqing

2013-05-01

The glycosylation of UDP-glucosyltransferases (UGTs) is of great importance in the control and elimination of both endogenous and exogenous toxins. Bm-UGT10286 (UGT86) is the sole provider of UGT activity against the 5-O position of quercetin and directly influences the formation of green pigment in the Bombyx cocoon. To evaluate whether cocoon coloration evolved for mimetic purposes, we concentrated on the expression pattern of Ugt86 and the activities of the enzyme substrates. The expression of Ugt86 was not only detected in the cocoon absorbing and accumulating tissues such as the digestive tube and silk glands, but also in quantity in the detoxification tissues of the malpighian tubes and fat body, as well as in the gonads. As in the green cocoon strains, Ugt86 was clearly expressed in the yellow and white cocoon strains. In vitro, the fusion protein of UGT86 showed quercetin metabolic activity. Nevertheless, Ugt86 expression of 5th instar larvae was not up-regulated in the silk gland by exogenous quercetin. However, it was significantly up-regulated in the digestive tube and gonads (P < 0.05). A similar result was observed in experiments where larvae were exposed to rutin, an insect resistance inducer and growth inhibitor typically found in plants, and to 20-hydroxylecdysone (20E), an insect endocrine and plant source hormone. On the contrary, up-regulated Ugt86 expression was almost nil in larvae exposed to juvenile hormone III (P > 0.05). The results of HPLC revealed that a new substance was formed by mixing 20E with the recombinant UGT86 protein in vitro, indicating that the effect of Ugt86 on 20E was similar to that on exogenous quercetin derived from plant food, and that the effect probably initiated the detoxification reaction against rutin. The conclusion is that the reaction of Ugt86 on the silkworm cocoon pigment quercetin is not the result of active mimetic ecogenesis, but derives from the detoxification of UGTs.

BNGR-A25L and -A27 are two functional G protein-coupled receptors for CAPA periviscerokinin neuropeptides in the silkworm Bombyx mori.

PubMed

Shen, Zhangfei; Chen, Yu; Hong, Lingjuan; Cui, Zhenteng; Yang, Huipeng; He, Xiaobai; Shi, Ying; Shi, Liangen; Han, Feng; Zhou, Naiming

2017-10-06

CAPA peptides, such as periviscerokinin (PVK), are insect neuropeptides involved in many signaling pathways controlling, for example, metabolism, behavior, and reproduction. They are present in a large number of insects and, together with their cognate receptors, are important for research into approaches for improving insect control. However, the CAPA receptors in the silkworm ( Bombyx mori ) insect model are unknown. Here, we cloned cDNAs of two putative CAPA peptide receptor genes, BNGR-A27 and -A25, from the brain of B. mori larvae. We found that the predicted BNGR-A27 ORF encodes 450 amino acids and that one BNGR-A25 splice variant encodes a full-length isoform (BNGR-A25L) of 418 amino acid residues and another a short isoform (BNGR-A25S) of 341 amino acids with a truncated C-terminal tail. Functional assays indicated that both BNGR-A25L and -A27 are activated by the PVK neuropeptides Bom -CAPA-PVK-1 and -PVK-2, leading to a significant increase in cAMP-response element-controlled luciferase activity and Ca 2+ mobilization in a G q inhibitor-sensitive manner. In contrast, BNGR-A25S was not significantly activated in response to the PVK peptides. Moreover, Bom -CAPA-PVK-1 directly bound to BNGR-A25L and -A27, but not BNGR-A25S. Of note, CAPA-PVK-mediated ERK1/2 phosphorylation and receptor internalization confirmed that BNGR-A25L and -A27 are two canonical receptors for Bombyx CAPA-PVKs. However, BNGR-A25S alone is a nonfunctional receptor but serves as a dominant-negative protein for BNGR-A25L. These results provide evidence that BNGR-A25L and -A27 are two functional G q -coupled receptors for Bombyx CAPA-PVKs, enabling the further elucidation of the endocrinological roles of Bom -CAPA-PVKs and their receptors in insect biology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Multibiological life support system experiments with humans partially involved

NASA Astrophysics Data System (ADS)

Liu, Hong; Tong, Ling; Li, Ming; Hu, Dawei; Fu, Yuming; He, Wenting; Hu, Enzhu

To establish bioregenerative life support system in lunar or mars bases in the future, manned stimulation experiments including several kinds of creatures are needed to be conducted first. Gas exchange relation, element transfer and transformation principles, etc. between human beings and the multibiological system composed of plants, animals, Chlorella vulgaris and so on must be investigated in order to place different organisms with appropriate numbers and proportions. This research cultivated lettuce (Lactuca sativa L.) and silkworm (Bombyx Mori L.) in the Closed Integrative Cultivating System (CICS) of the Integrative Experimental Sys-tem (IES) with Chlorella vulgaris cultivated in the Plate Photo Bioreactor (PPB) of the IES. Gas exchange between testers and the IES were conducted periodically. The automotive control system of the PPB changed the illumination intensity of the photo bioreactor according to the CO2 concentration in the IES to make CO2 /O2 in the system maintain at stable levels by regu-lating the photosynthesis of alga. The conveyor-type cultivation method which was harvesting the biggest batch of lettuce and silkworms through the mass exchange chamber of IES every four days and transferring the smallest batch of lettuce and silkworms into the system; carrying certain amount of alga liquid out of the bioreactor every day with nutrient liquid replenished into the system was implemented in the experiments. In terms of gas circulation, CO2 /O2 concentration changes in the system with trace gas contaminants (CH4 , NH3 and C2 H4 ) were measured. As to the mass transfer and transformation, element (C, H, O, N) contents, height, crown width and biomasses of lettuce in different developing stages, silkworms' bioconversion rates, alga's biomass changes, the amount and community change trends of the microorganism in different positions of the system, the quality of condensates gained under different running conditions and so on were studied. Results showed

iTRAQ-based quantitative proteomics analysis of molecular mechanisms associated with Bombyx mori (Lepidoptera) larval midgut response to BmNPV in susceptible and near-isogenic strains.

PubMed

Yu, Haizhong; Wang, Xueyang; Xu, Jiaping; Ma, Yan; Zhang, Shangzhi; Yu, Dong; Fei, Dongqiong; Muhammad, Azharuddin

2017-08-08

Bombyx mori nucleopolyhedrovirus (BmNPV) has been identified as a major pathogen responsible for severe economic loss. Most silkworm strains are susceptible to BmNPV, with only a few highly resistant strains thus far identified. Here we investigated the molecular basis of silkworm resistance to BmNPV using susceptible (the recurrent parent P50) and resistant (near-isogenic line BC9) strains and a combination of iTRAQ-based quantitative proteomics, reverse-transcription quantitative PCR and Western blotting. By comparing the proteomes of infected and non-infected P50 and BC9 silkworms, we identified 793 differentially expressed proteins (DEPs). By gene ontology and KEGG enrichment analyses, we found that these DEPs are preferentially involved in metabolism, catalytic activity, amino sugar and nucleotide sugar metabolism and carbon metabolism. 114 (14.38%) DEPs were associated with the cytoskeleton, immune response, apoptosis, ubiquitination, translation, ion transport, endocytosis and endopeptidase activity. After removing the genetic background and individual immune stress response proteins, we identified 84 DEPs were found that are potentially involved in resistance to BmNPV. Further studies showed that a serine protease was down-regulated in P50 and up-regulated in BC9 after BmNPV infection. Taken together, these results provide insights into the molecular mechanism of silkworm response to BmNPV. Bombyx mori nucleopolyhedrovirus (BmNPV) is highly pathogenic, causing serious losses in sericulture every year. However, the molecular mechanisms of BmNPV infection and host defence remain unclear. Here we combined quantitative proteomic, bioinformatics, RT-qPCR and Western blotting analyses and found that BmNPV invasion causes complex protein alterations in the larval midgut, and that these changes are related to cytoskeleton, immune response, apoptosis, ubiquitination, translation, ion transport, endocytosis and endopeptidase activity. Five important differentially

Bm-muted, orthologous to mouse muted and encoding a subunit of the BLOC-1 complex, is responsible for the otm translucent mutation of the silkworm Bombyx mori.

PubMed

Zhang, Haokun; Kiuchi, Takashi; Wang, Lingyan; Kawamoto, Munetaka; Suzuki, Yutaka; Sugano, Sumio; Banno, Yutaka; Katsuma, Susumu; Shimada, Toru

2017-09-20

"Tanaka's mottled translucent" (otm) is a mutation of the silkworm Bombyx mori that exhibits translucent skin during larval stages. We performed positional cloning of the gene responsible for otm and mapped it to a 364-kb region on chromosome 5 that contains 22 hypothetical protein-coding genes. We performed RNA-seq analysis of the epidermis and fat body of otm larvae and determined that the gene BGIBMGA002619 may be responsible for the otm mutation. BGIBMGA002619 encodes the biosynthesis of lysosome-related organelles complex 1 (BLOC-1) subunit 5, whose ortholog is responsible for the Muted mutant in mouse. Accordingly, we named this gene Bm-muted. We discovered that the expression of Bm-muted in the epidermis and fat body of otm mutants was dramatically suppressed compared with the wild type. We determined the nucleotide sequences of the full-length cDNA and genomic region corresponding to Bm-muted and found that a 538-bp long DNA sequence similar to B. mori transposon Organdy was inserted into the 3' end of the first intron of Bm-muted in two otm strains. The Bm-muted cDNA of otm mutants lacked exon 2, and accordingly generated a premature stop codon in exon 3. In addition, short interfering RNA (siRNA)-mediated knockdown of this gene caused localized partial translucency of larval skin. These data indicate that the mutation in Bm-muted caused the otm-mutant phenotype. We propose that the insertion of Organdy caused a splicing disorder in Bm-muted in the otm mutant, resulting in a null mutation of Bm-muted. This mutation is likely to cause deficiencies in urate granule formation in epidermal cells that result in translucent larval skin. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of specific activators of intestinal amino acid transport in Bombyx mori larval growth and nutrition.

PubMed

Leonardi, M G; Casartelli, M; Fiandra, L; Parenti, P; Giordana, B

2001-12-01

Nutrient absorption and its modulation are critical for animal growth. In this paper, we demonstrate that leucine methyl ester (Leu-OMe) can greatly increase the activity of the transport system responsible for the absorption of most essential amino acids in the larval midgut of the silkworm Bombyx mori. We investigated leucine uptake activation by Leu-OMe in brush border membrane vesicles and in the apical membrane of epithelial cells in the midgut incubated in vitro. Moreover, the addition of this strong activator of amino acid absorption to diet significantly affected larval growth. Silkworms fed on artificial diet supplemented with Leu-OMe reached maximum body weight 12-18 h before control larvae, and produced cocoon shells up to 20% heavier than those of controls. The activation of amino acid absorption plays an essential role in larval development so that larval growth and cocoon production similar to controls reared on an artificial diet with 25% of dry mulberry leaf powder were observed in silkworms fed on an artificial diet with only 5% of mulberry powder. Arch. Copyright 2001 Wiley-Liss, Inc.

Use of Morus alba-Bombyx mori as a useful template to assess Pb entrance in the food chain from wastewater.

PubMed

Ashfaq, Muhammad; Khan, Muhammad Imran; Hanif, Muhammad Asif

2009-08-01

This study focused on a 75-d microplot experiment to access lead (Pb) entrance into the terrestrial ecosystem using Morus alba (mulberry) and Bombyx mori (silkworm) as pollution indicator sources. The Pb(II) mobility in different systems was in the following order: synthetic effluents > soil > plant > larva > larva-fecal. The Pb(II) mobility from an inorganic source to different life forms was found to be highly dependent on soil pH and the concentration of Pb in synthetic effluents. At lower concentrations, Pb was less lethal to B. mori larvae. Fifth instars of B. mori were found to be more tolerant to toxicity caused by Pb compared with lower instars. M. alba was not a hyperaccumulator plant because Pb(II) concentration was <1,000 mg/kg at all soil pHs and Pb(II) concentrations in effluents. The maximum amount of Pb(II) detected in soil, mulberry leaves, silkworm larvae, and silkworm feces was 326.5 +/- 0.04, 42.78 +/- 0.02, 61.24 +/- 0.02, and 22.3 +/- 0.02 mg/kg, respectively.

Bombyx mori Serpin6 regulates prophenoloxidase activity and the expression of antimicrobial proteins.

PubMed

Li, Bing; Yu, Hai-Zhong; Ye, Chong-Jun; Ma, Yan; Li, Xing; Fan, Tao; Chen, Fu-Sheng; Xu, Jia-Ping

2017-04-30

Serpins are a family of serine protease inhibitors that are found widely in insects. They play an important role in insect physiological responses, such as innate immunity and development. In this study, we obtained the Bombyx mori serpin6 (BmSerpin6) sequence from National Center for Biotechnology Information (NCBI) and the silkworm genome database (SilkDB). Reverse transcription PCR (RT-PCR) results showed that BmSerpin6 was expressed highly in hemocytes, the midgut, and the fat body. After challenging with Micrococcus luteus (Mi) and Serratia marcescens (Sm), the BmSerpin6 expression level was induced significantly. Transcript levels of gloverin2 and prophenoloxidase (PPO) activity were reduced significantly in the fat body and hemocytes after injecting the recombinant BmSerpin6 protein into silkworm larvae. A BmSerpin6 recombinant plasmid (BmSerpin6-pAC 5.1) was constructed successfully and transfected into Drosophila S2 cells, which resulted in significantly reduced expression of the drosomycin protein. These results indicated that BmSerpin6 might regulate silkworm immune responses. Copyright © 2017 Elsevier B.V. All rights reserved.

Silk sericin/polyacrylamide in situ forming hydrogels for dermal reconstruction.

PubMed

Kundu, Banani; Kundu, Subhas C

2012-10-01

In situ forming tissue sealants are advantageous due to ease in application, complete coverage of defect site and assured comfort levels to patients. The interconnected three-dimensional hydrophilic networks perfectly manage typical dermal wounds by suitably scaffolding skin fibroblast, diffusing the nutrients, therapeutics and exudates while still maintaining an adequately moist environment. We evaluate the cell homing ability of semi-interpenetrating non-mulberry tropical tasar silk sericin/polyacrylamide hydrophilic network with a keen understanding of its network characteristics and correlation of protein concentration with the performance as cell scaffold. Interconnectivity of porous networks observed through scanning electron micrograph revealed pore sizes ranging from 23 to 52 μm. The enhanced β-sheet content with the increasing sericin concentration in far red spectroscopy study supported their corresponding improved compressive strength. These semi-interpenetrating networks were found to possess a maximum fluid uptake of 112% of its weight, hence preventing the accumulation of exudates at the wound area. The present systems appear to possess characteristics like rapid gelation (~5min) at 37 °C, 98% porosity enabling the migration of fibroblasts during healing (observed through confocal and scanning electron micrographs), cell adhesion together with the absence of any cyto-toxic effect suggesting its potential as in situ tissue sealants. The compressive strength up to 61 kPa ensured ease in handling even when wet. The results prove the suitability to use non-mulberry tasar cocoon silk sericin/polyacrylamide semi-interpenetrating network as a reconstructive dermal sealant. Copyright © 2012 Elsevier Ltd. All rights reserved.

Bombyx mori cecropin A has a high antifungal activity to entomopathogenic fungus Beauveria bassiana.

PubMed

Lu, Dingding; Geng, Tao; Hou, Chengxiang; Huang, Yuxia; Qin, Guangxing; Guo, Xijie

2016-05-25

A cDNA encoding cecropin A (CecA) was cloned from the larvae of silkworm, Bombyx mori, using RT-PCR. It encodes a protein of 63 amino acids, containing a 22 amino acid signal peptide and a 37 amino acid mat peptide of functional domain. The CecA secondary structure contains two typical amphiphilic α-helices. Real-time qPCR analysis revealed that CecA was expressed in all the tissues tested, including cuticle, fat body, hemocytes, Malpighian tubule, midgut and silk gland in the silkworm larvae with the highest expression in the fat body and hemocytes. The gene expression of B. mori CecA was rapidly induced by Beauveria bassiana challenge and reached maximum levels at 36h after inoculation in third instar larvae. In the fifth instar larvae infected with B. bassiana, the relative expression level of CecA was upregulated in fat body and hemocytes, but not in cuticle, Malpighian tubule, midgut and silk gland. The cDNA segment of the CecA was inserted into the expression plasmid pET-30a(+) to construct a recombinant expression plasmid. Western blot results revealed that his-tagged fusion protein was successfully expressed and purified. Then the mat peptide of CecA was chemically synthesized with C-terminus amidation for in vivo antifungal assay and purity achieved 93.7%. Mass spectrometry and SDS-PAGE showed its molecular weight to be 4046.95Da. Antifungal assays indicated that the B. mori CecA had a high antifungal activity to entomopathogenic fungus B. bassiana both in vitro and in vivo in the silkworm larvae. This is the first report that the CecA is effective to inhibit B. bassiana inside the body of silkworm. Copyright © 2016 Elsevier B.V. All rights reserved.

Overview of research on Bombyx mori microRNA

PubMed Central

Wang, Xin; Tang, Shun-ming; Shen, Xing-jia

2014-01-01

Abstract MicroRNAs (miRNAs) constitute some of the most significant regulatory factors involved at the post-transcriptional level after gene expression, contributing to the modulation of a large number of physiological processes such as development, metabolism, and disease occurrence. This review comprehensively and retrospectively explores the literature investigating silkworm, Bombyx mori L. (Lepidoptera: Bombicidae), miRNAs published to date, including discovery, identification, expression profiling analysis, target gene prediction, and the functional analysis of both miRNAs and their targets. It may provide experimental considerations and approaches for future study of miRNAs and benefit elucidation of the mechanisms of miRNAs involved in silkworm developmental processes and intracellular activities of other unknown non-coding RNAs. PMID:25368077

Detection of Nosema bombycis by FTA cards and loop-mediated isothermal amplification (LAMP).

PubMed

Yan, Wei; Shen, Zhongyuan; Tang, Xudong; Xu, Li; Li, Qianlong; Yue, Yajie; Xiao, Shengyan; Fu, Xuliang

2014-10-01

We successfully established a detection method which exhibited a markedly higher sensitivity than previously developed detection methods for Nosema bombycis by combining glass beads, FTA card, and LAMP. Spores of N. bombycis were first broken by acid-washed glass beads; the DNA was subsequently extracted and purified with the FTA card, and LAMP was performed using primers (LSU296) designed based on the sequence of the LSU rRNA of N. bombycis. The minimum detection concentration was 10 spores/mL. When this method was used to detect pebrine disease in silkworm egg, the detection rate for 500 silkworm eggs, in which only one egg was infected with N. bombycis, was 100 % under our optimized conditions. If the number of eggs in the sample increased to 800 or 1,000, the sample was divided into two equal portions, and the eggs were smashed with glass beads after the addition of 1 mL of TE buffer. The liquid in two tubes was later mixed and applied to the FTA card, and the detection rates were 100 %. Furthermore, the LAMP method established in our study could detect N. bombycis infection in silkworm 24 h earlier than microscopy.

Identifying potential maternal genes of Bombyx mori using digital gene expression profiling

PubMed Central

Xu, Pingzhen

2018-01-01

Maternal genes present in mature oocytes play a crucial role in the early development of silkworm. Although maternal genes have been widely studied in many other species, there has been limited research in Bombyx mori. High-throughput next generation sequencing provides a practical method for gene discovery on a genome-wide level. Herein, a transcriptome study was used to identify maternal-related genes from silkworm eggs. Unfertilized eggs from five different stages of early development were used to detect the changing situation of gene expression. The expressed genes showed different patterns over time. Seventy-six maternal genes were annotated according to homology analysis with Drosophila melanogaster. More than half of the differentially expressed maternal genes fell into four expression patterns, while the expression patterns showed a downward trend over time. The functional annotation of these material genes was mainly related to transcription factor activity, growth factor activity, nucleic acid binding, RNA binding, ATP binding, and ion binding. Additionally, twenty-two gene clusters including maternal genes were identified from 18 scaffolds. Altogether, we plotted a profile for the maternal genes of Bombyx mori using a digital gene expression profiling method. This will provide the basis for maternal-specific signature research and improve the understanding of the early development of silkworm. PMID:29462160

Genetic Characterization of Bombyx mori (Lepidoptera: Bombycidae) Breeding and Hybrid Lines With Different Geographic Origins

PubMed Central

Furdui, Emilia M.; Mărghitaş, Liviu A.; Dezmirean, Daniel S.; Paşca, Ioan; Pop, Iulia F.; Erler, Silvio; Schlüns, Ellen A.

2014-01-01

Abstract The domesticated silkworm Bombyx mori L. comprises a large number of geographical breeds and hybrid lines. Knowing the genetic structure of those may provide information to improve the conservation of commercial lines by estimating inbreeding over generations and the consequences of excessive use of those lineages. Here, we analyzed the genetic diversity of seven breeds and eight hybrid lines from Eastern Europe and Asia using highly polymorphic microsatellites markers to determine its genetical impact on their use in global breeding programs. No consistent pattern of deviation from Hardy–Weinberg equilibrium was found for most breed and hybrids; and the absence of a linkage disequilibrium also suggests that the strains are in equilibrium. A principal coordinate analysis revealed a clear separation of two silkworm breeds from the rest: one (IBV) originated from India and the other one (RG 90 ) from Romania/Japan. The tendency of the other breeds from different geographic origins to cluster together in a general mix might be due to similar selection pressures (climate and anthropogenic factors) in different geographic locations. Phylogenetic analyses grouped the different silkworm breeds but not the hybrids according to their geographic origin and confirmed the pattern found in the principal coordinate analysis. PMID:25502023

Crystal Structure of Silkworm Bombyx mori JHBP in Complex With 2-Methyl-2,4-Pentanediol: Plasticity of JH-Binding Pocket and Ligand-Induced Conformational Change of the Second Cavity in JHBP

PubMed Central

Fujimoto, Zui; Suzuki, Rintaro; Shiotsuki, Takahiro; Tsuchiya, Wataru; Tase, Akira; Momma, Mitsuru; Yamazaki, Toshimasa

2013-01-01

Juvenile hormones (JHs) control a diversity of crucial life events in insects. In Lepidoptera which major agricultural pests belong to, JH signaling is critically controlled by a species-specific high-affinity, low molecular weight JH-binding protein (JHBP) in hemolymph, which transports JH from the site of its synthesis to target tissues. Hence, JHBP is expected to be an excellent target for the development of novel specific insect growth regulators (IGRs) and insecticides. A better understanding of the structural biology of JHBP should pave the way for the structure-based drug design of such compounds. Here, we report the crystal structure of the silkworm Bombyx mori JHBP in complex with two molecules of 2-methyl-2,4-pentanediol (MPD), one molecule (MPD1) bound in the JH-binding pocket while the other (MPD2) in a second cavity. Detailed comparison with the apo-JHBP and JHBP-JH II complex structures previously reported by us led to a number of intriguing findings. First, the JH-binding pocket changes its size in a ligand-dependent manner due to flexibility of the gate α1 helix. Second, MPD1 mimics interactions of the epoxide moiety of JH previously observed in the JHBP-JH complex, and MPD can compete with JH in binding to the JH-binding pocket. We also confirmed that methoprene, which has an MPD-like structure, inhibits the complex formation between JHBP and JH while the unepoxydated JH III (methyl farnesoate) does not. These findings may open the door to the development of novel IGRs targeted against JHBP. Third, binding of MPD to the second cavity of JHBP induces significant conformational changes accompanied with a cavity expansion. This finding, together with MPD2-JHBP interaction mechanism identified in the JHBP-MPD complex, should provide important guidance in the search for the natural ligand of the second cavity. PMID:23437107

Electricity from the silk cocoon membrane.

PubMed

Tulachan, Brindan; Meena, Sunil Kumar; Rai, Ratan Kumar; Mallick, Chandrakant; Kusurkar, Tejas Sanjeev; Teotia, Arun Kumar; Sethy, Niroj Kumar; Bhargava, Kalpana; Bhattacharya, Shantanu; Kumar, Ashok; Sharma, Raj Kishore; Sinha, Neeraj; Singh, Sushil Kumar; Das, Mainak

2014-06-25

Silk cocoon membrane (SCM) is an insect engineered structure. We studied the electrical properties of mulberry (Bombyx mori) and non-mulberry (Tussar, Antheraea mylitta) SCM. When dry, SCM behaves like an insulator. On absorbing moisture, it generates electrical current, which is modulated by temperature. The current flowing across the SCM is possibly ionic and protonic in nature. We exploited the electrical properties of SCM to develop simple energy harvesting devices, which could operate low power electronic systems. Based on our findings, we propose that the temperature and humidity dependent electrical properties of the SCM could find applications in battery technology, bio-sensor, humidity sensor, steam engines and waste heat management.

Electricity from the Silk Cocoon Membrane

NASA Astrophysics Data System (ADS)

Tulachan, Brindan; Meena, Sunil Kumar; Rai, Ratan Kumar; Mallick, Chandrakant; Kusurkar, Tejas Sanjeev; Teotia, Arun Kumar; Sethy, Niroj Kumar; Bhargava, Kalpana; Bhattacharya, Shantanu; Kumar, Ashok; Sharma, Raj Kishore; Sinha, Neeraj; Singh, Sushil Kumar; Das, Mainak

2014-06-01

Silk cocoon membrane (SCM) is an insect engineered structure. We studied the electrical properties of mulberry (Bombyx mori) and non-mulberry (Tussar, Antheraea mylitta) SCM. When dry, SCM behaves like an insulator. On absorbing moisture, it generates electrical current, which is modulated by temperature. The current flowing across the SCM is possibly ionic and protonic in nature. We exploited the electrical properties of SCM to develop simple energy harvesting devices, which could operate low power electronic systems. Based on our findings, we propose that the temperature and humidity dependent electrical properties of the SCM could find applications in battery technology, bio-sensor, humidity sensor, steam engines and waste heat management.

Comparative study on the mechanical property of silk thread from cocoons of Bombyx mori L.

PubMed

Iizuka, E; Hachimori, A; Abe, K; Sunohara, M; Hiraide, Y; Ueyama, A; Kamo, K; Fujiwara, T; Nakamura, F; Uno, T

1983-01-01

Specimens of bave (undegummed silk thread) were collected from cocoons of various origins of parent silkworm races, such as Japanese, Chinese, European, Korean and tropical origins, and from as many races as possible. An apparatus was set up to measure the dynamic elastic modulus of these specimens. In all the categories of the races tested, the elastic modulus was linearly related to the size of bave, regardless of the portion of cocoon layer from which the specimens were taken. This correlation was concluded to be universal to the silk thread of Bombyx mori L. species; however, values of the regression coefficient and of the elastic modulus were susceptible to the origin of silkworm races, depending on whether they were native or improved.

Genetic characterization of Bombyx mori (Lepidoptera: Bombycidae) breeding and hybrid lines with different geographic origins.

PubMed

Furdui, Emilia M; Mărghitaş, Liviu A; Dezmirean, Daniel S; Paşca, Ioan; Pop, Iulia F; Erler, Silvio; Schlüns, Ellen A

2014-01-01

The domesticated silkworm Bombyx mori L. comprises a large number of geographical breeds and hybrid lines. Knowing the genetic structure of those may provide information to improve the conservation of commercial lines by estimating inbreeding over generations and the consequences of excessive use of those lineages. Here, we analyzed the genetic diversity of seven breeds and eight hybrid lines from Eastern Europe and Asia using highly polymorphic microsatellites markers to determine its genetical impact on their use in global breeding programs. No consistent pattern of deviation from Hardy-Weinberg equilibrium was found for most breed and hybrids; and the absence of a linkage disequilibrium also suggests that the strains are in equilibrium. A principal coordinate analysis revealed a clear separation of two silkworm breeds from the rest: one (IBV) originated from India and the other one (RG90) from Romania/Japan. The tendency of the other breeds from different geographic origins to cluster together in a general mix might be due to similar selection pressures (climate and anthropogenic factors) in different geographic locations. Phylogenetic analyses grouped the different silkworm breeds but not the hybrids according to their geographic origin and confirmed the pattern found in the principal coordinate analysis. © The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.

Purification and Initial Functions of Sex-Specific Storage Protein 2 in Bombyx mori.

PubMed

Chen, Jianqing; Shu, Tejun; Chen, Jian; Ye, Man; Lv, Zhengbing; Nie, Zuoming; Gai, Qijing; Yu, Wei; Zhang, Yaozhou

2015-08-01

In this study, we identified a heat-resistant protein from the chrysalis stage of the silkworm which we named sex-specific storage protein 2 (SSP2). This protein was stable even at 80 °C, and has an amino acid sequence that is 90.65 % homologous to SP2. We utilized the heat-resistant characteristics of SSP2 to purify the protein and maintain its biological activity. In addition, using flow cytometry and the MTT assay, we found that SSP2 had anti-apoptotic effects on BmN cells, and that SSP2 could also inhibit cell apoptosis induced by chemical factors. These results suggest that SSP2 has a cell-protective function, and provides a basis for future work on the function of storage proteins in silkworm.

Electricity from the Silk Cocoon Membrane

PubMed Central

Tulachan, Brindan; Meena, Sunil Kumar; Rai, Ratan Kumar; Mallick, Chandrakant; Kusurkar, Tejas Sanjeev; Teotia, Arun Kumar; Sethy, Niroj Kumar; Bhargava, Kalpana; Bhattacharya, Shantanu; Kumar, Ashok; Sharma, Raj Kishore; Sinha, Neeraj; Singh, Sushil Kumar; Das, Mainak

2014-01-01

Silk cocoon membrane (SCM) is an insect engineered structure. We studied the electrical properties of mulberry (Bombyx mori) and non-mulberry (Tussar, Antheraea mylitta) SCM. When dry, SCM behaves like an insulator. On absorbing moisture, it generates electrical current, which is modulated by temperature. The current flowing across the SCM is possibly ionic and protonic in nature. We exploited the electrical properties of SCM to develop simple energy harvesting devices, which could operate low power electronic systems. Based on our findings, we propose that the temperature and humidity dependent electrical properties of the SCM could find applications in battery technology, bio-sensor, humidity sensor, steam engines and waste heat management. PMID:24961354

Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila

USDA-ARS?s Scientific Manuscript database

The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle...

A Cockpit-Based Application for Traffic Aware Trajectory Optimization

NASA Technical Reports Server (NTRS)

Woods, Sharon E.; Vivona, Robert A.; Roscoe, David A.; LeFebvre, Brendan C.; Wing, David J.; Ballin, Mark G.

2013-01-01

The Traffic Aware Planner (TAP) is a cockpit-based advisory tool designed to be hosted on a Class 2 Electronic Flight Bag and developed to enable the concept of Traffic Aware Strategic Aircrew Requests (TASAR). This near-term concept provides pilots with optimized route changes that reduce fuel burn or flight time, avoids interactions with known traffic, weather and restricted airspace, and may be used by the pilots to request a trajectory change from air traffic control. TAP's internal architecture and algorithms are derived from the Autonomous Operations Planner, a flight-deck automation system developed by NASA to support research into aircraft self-separation. This paper reviews the architecture, functionality and operation of TAP.

Entomophagy and space agriculture

NASA Astrophysics Data System (ADS)

Katayama, N.; Ishikawa, Y.; Takaoki, M.; Yamashita, M.; Nakayama, S.; Kiguchi, K.; Kok, R.; Wada, H.; Mitsuhashi, J.; Space Agriculture Task Force, J.

Supplying food for human occupants remains one of the primary issues in engineering space habitation Evidently for long-term occupation on a distant planet it is necessary to start agriculture on site Historically humans have consumed a variety of animals and it is required to fill our nutritional need when they live in space Among many candidate group and species of animal to breed in space agriculture insects are of great interest since they have a number of advantages over mammals and other vertebrates or invertebrates About 70-75 of animal species is insects and they play an important role in materials recycle loop of terrestrial biosphere at their various niche For space agriculture we propose several insect species such as the silkworm Bombyx mori the drugstore beetle Stegobium paniceum and the termite Macrotermes subhyalinus Among many advantages these insects do not compete with human in terms of food resources but convert inedible biomass or waste into an edible food source for human The silkworm has been domesticated since 5 000 years ago in China Silk moth has lost capability of flying after its domestication history This feature is advantageous in control of their breeding Silkworm larvae eat specifically mulberry leaves and metamorphose in their cocoon Silk fiber obtained from cocoon can be used to manufacture textile Farming system of the drugstore beetle has been well established Both the drugstore beetle and the termite are capable to convert cellulose or other inedible biomass

Synthetic Engineering of Spider Silk Fiber as Implantable Optical Waveguides for Low-Loss Light Guiding.

PubMed

Qiao, Xin; Qian, Zhigang; Li, Junjie; Sun, Hongji; Han, Yao; Xia, Xiaoxia; Zhou, Jin; Wang, Chunlan; Wang, Yan; Wang, Changyong

2017-05-03

A variety of devices used for biomedical engineering have been fabricated using protein polymer because of their excellent properties, such as strength, toughness, biocompatibility, and biodegradability. In this study, we fabricated an optical waveguide using genetically engineered spider silk protein. This method has two significant advantages: (1) recombinant spider silk optical waveguide exhibits excellent optical and biological properties and (2) biosynthesis of spider silk protein can overcome the limitation to the research on spider silk optical waveguide due to the low yield of natural spider silk. In detail, two kinds of protein-based optical waveguides made from recombinant spider silk protein and regenerative silkworm silk protein were successfully prepared. Results suggested that the recombinant spider silk optical waveguide showed a smoother surface and a higher refractive index when compared with regenerative silkworm silk protein. The optical loss of recombinant spider silk optical waveguide was 0.8 ± 0.1 dB/cm in air and 1.9 ± 0.3 dB/cm in mouse muscles, which were significantly lower than those of regenerative silkworm silk optical waveguide. Moreover, recombinant spider silk optical waveguide can meet the demand to guide and efficiently deliver light through biological tissue. In addition, recombinant spider silk optical waveguide showed low toxicity to cells in vitro and low-level inflammatory reaction with surrounding tissue in vivo. Therefore, recombinant spider silk optical waveguide is a promising implantable device to guide and deliver light with low loss.

Identification of Enterococcus mundtii as a pathogenic agent involved in the "flacherie" disease in Bombyx mori L. larvae reared on artificial diet.

PubMed

Cappellozza, Silvia; Saviane, Alessio; Tettamanti, Gianluca; Squadrin, Marta; Vendramin, Elena; Paolucci, Paolo; Franzetti, Eleonora; Squartini, Andrea

2011-03-01

Enterococcus mundtii was shown to be directly correlated with flacherie disease of the silkworm larvae reared on artificial diet supplemented with chloramphenicol. Its identification was carried out by means of light and electron microscopy and nucleotide sequencing of 16S gene. The bacterium is capable of rapidly multiplying in the silkworm gut and of invading other body tissues, as demonstrated by deliberate infection of germfree larvae and by subsequent TEM observations. E. mundtii can endure alkaline pH of the silkworm gut and it has been proved to adapt in vitro to commonly applied doses of chloramphenicol, whose use can further contribute to reduce competition by other bacteria in Bombyx mori alimentary canal. The modality of transmission of the infection to the larvae was among the objectives of the present research. Since contamination of the progeny by mother moths can be avoided through routine egg shell disinfection, a trans-ovarian vertical transmission can be ruled out. On the other hand the bacterium was for the first time identified on mulberry leaves, and therefore artificial diet based on leaf powder could be a source of infection. We showed that while microwaved diet could contain live E. mundtii cells, the autoclaved diet is safe in this respect. Being E. mundtii also part of the human-associated microbiota, and since B. mori is totally domestic species, a possible role of man in its epidemiology can be postulated. Copyright © 2010 Elsevier Inc. All rights reserved.