Silkworm Disease Research Articles

Characterization and pathogenicity assessment of gut-associated microbes of muga silkworm Antheraea assamensis Helfer (Lepidoptera: Saturniidae)

Antheraea assamensis Helfer (muga silkworm) is an economically important endemic insect species of North Eastern Region of India. The silkworm is often susceptible to infection by pathogenic bacteria, leads to a disease commonly known as flacherie which causes 40% loss per annum to the silk industry. In this study, we have reported isolation, characterization and pathogenicity assessment of gut-associated bacteria of healthy and diseased muga silkworms. Thirty five bacterial isolates were screened from the gut of healthy and diseased silkworms by morphological observation and biochemical tests. 11 and 5 strains from healthy and diseased silkworm respectively were identified by 16S rRNA gene sequencing and analyzed. Pseudomonas aeruginosa (DRK1), Ornithinibacillus bavariensis (DRK2), Achromobacter xylosoxidans (KH3) and Staphylococcus aureus (FLG1) strains were commonly found in healthy as well as diseased larvae whereas, Bacillus thuringiensis (MK1) was found only in diseased larvae. Survivability analysis was performed with the identified strains by injection and oral administration (104CFU/ml). The immune response of the silkworm against the pathogen was also studied by phenoloxidase and lysozyme enzyme activity assay, total and differential hemocyte count and phagocytic activity of hemocytes. It was observed that S. aureus, P. aeruginosa and B. thuringiensis significantly reduced the survivability of silkworm (p<0.001) hence found highly pathogenic. The lethal concentrations (LC50) values of the pathogenic strains were calculated at different time intervals (24, 48, 72 and 96h) within the range from 1.38×102 to 3.63×107CFU/ml. The pathogenic groups demonstrated inhibition of phenoloxidase activity and decreased in total hemocyte count after 48h of infection. However, the lysozyme activity increased significantly in the pathogenic groups compared to the control (p<0.05). Granulocytes and plasmatocytes showed phagocytosis whereas; other types of cells did not show any phagocytic activity. Increasing granulocytes and plasmatocytes counts corroborates the results of phagocytic activity. The present study might be helpful in understanding the disease prognosis and colonization of bacteria causing the disease in muga silkworm.

Silkworm disease diagnosis through molecular approach and their management

Silkworm disease diagnosis through molecular approach and their management

Controle e prevenção da calcinose branca em Bombyx mori L. (Lepidoptera: Bombycidae)

RESUMO: Um dos principais problemas enfrentados pelos sericicultores em todo o mundo são entomopatógenos que causam grandes perdas na produção. Destaca-se a doença calcinose branca, causada pelo fungo Beauveria bassiana (Bals.) Vuill. (Ascomycota: Cordycipitaceae). Doenças fúngicas são facilmente disseminadas, já que os conídios são transportados pelo ar. Além disso, as condições de temperatura e umidade nos barracões são favoráveis ao surgimento e à manutenção do fungo, aumentando sua incidência. Práticas de higiene como a desinfecção do barracão e de utensílios são recomendadas, no entanto a contaminação das lagartas ainda é frequente. Dessa forma, esse artigo reuniu trabalhos voltados para a pesquisa de produtos antimicrobianos alternativos para o uso em sericicultura.

"I make efforts, people make comments": Prof. H. Zanyin Gaw-pioneering the world, the trailblazer and founder of China's virology research.

"I make efforts, people make comments": Prof. H. Zanyin Gaw-pioneering the world, the trailblazer and founder of China's virology research.

Progress in understanding resistance of &amp;lt;italic&amp;gt;Bombyx mori&amp;lt;/italic&amp;gt; to nucleopolyhedrovirus

Bombyx mori nucleopolyhedrovirus (BmNPV) causes an important viral infectious disease in sericulture, and is responsible for 60% of the total cocoon production loss caused by various silkworm diseases. Horizontal resistance of silkworms to BmNPV is well known but we have identified a local line, named NB, whose LD50 is more than 1000 times higher than that of susceptible strains, and that has the highest resistance to BmNPV out of 344 silkworm lines. Through 10 years of crossbreeding and systematic selective breeding using line NB, we have constructed a commercial variety of Bombyx mori that is highly resistant to BmNPV. These genetic studies indicate the existence of BmNPV resistance genes in silkworm, and a pair of dominant genes has been implicated. Recently, we constructed a BmNPV-resistant near-isogenic line (NIL) via interspecific hybridization and backcrossing. Using this model and molecular techniques, such as fluorescent differential display-PCR, suppression subtractive hybridization, DNA microarray and proteomic analyses, we have identified molecules involved in BmNPV resistance. This review summarizes the current research status of BmNPV resistance in silkworms and discusses molecules involved in anti-BmNPV processes.

Proteomic analysis of silkworm midgut cellular proteins interacting with the 5' end of infectious flacherie virus genomic RNA.

The flacherie disease in the silkworm is caused by the infectious flacherie virus (IFV). IFV relies on its 5' region of genomic RNA to recruit host-related factors to implement viral translation and replication. To identify host proteins bound to the 5'-region of IFV RNA and identify proteins important for its function, mass spectrometry was used to identify proteins from silkworm midgut extracts that were obtained using RNA aptamer-labeled 5' region of IFV RNA. We found 325 protein groups (unique peptide≥2) bound to the 5' region of IFV RNA including translation-related factors (16 ribosomal subunits, 3 eukaryotic initiation factor subunits, 1 elongation factor subunit and 6 potential internal ribosome entry site trans-acting factors), cytoskeleton-related proteins, membrane-related proteins, metabolism enzymes, and other proteins. These results can be used to study the translation and replication related factors of IFV interacting with host silkworm and to control flacherie disease in silkworm.

Identification and Characterization of Bacillus cereus SW7-1 in Bombyx mori (Lepidoptera: Bombycidae).

The bacterial diseases of silkworms cause significant reductions in sericulture and result in huge economic loss. This study aimed to identify and characterize a pathogen from diseased silkworm. SW7-1, a pathogenic bacterial strain, was isolated from the diseased silkworm. The strain was identified on the basis of its bacteriological properties and 16S rRNA gene sequence. The colony was round, slightly convex, opaque, dry, and milky on a nutrient agar medium, the colony also exhibited jagged edges. SW7-1 was Gram-positive, without parasporal crystal, and 0.8–1.2 by 2.6–3.4 µm in length, resembling long rods with rounded ends. The strain was positive to most of the physiological biochemical tests used in this study. The strain could utilize glucose, sucrose, and maltose. The results of its 16S rRNA gene sequence analysis revealed that SW7-1 shared the highest sequence identity (>99%) with Bacillus cereus strain 14. The bacterial strain was highly susceptible to gentamycin, streptomycin, erythromycin, norfloxacin, and ofloxacin and moderately susceptible to tetracycline and rifampicin. It exhibited resistance to other antibiotics. SW7-1 had hemolytic activity and could produce extracellular casease, lipase, and amylase. SW7-1 could reproduce septicemia-like symptoms with high mortality rate when re-fed to healthy silkworm. .The median lethal concentration (LC50) was 5.45 × 104 cfu/ml. Thus, SW7-1 was identified as B. cereus, which is a pathogen for silkworm and human infections are possible.

Characterization of Klebsiella granulomatis pathogenic to silkworm, Bombyx mori L.

Bacterial disease of silkworm causes significant reduction of silk production leading to huge economic loss. This study aims to isolate bacteria from diseased silkworm and to determine its pathogenicity and antibiotic resistance. A strain of Klebsiella granulomatis has been isolated from silkworm haemolymph which was later identified on the basis of biochemical tests and 16S rRNA gene sequencing. The optimum culture condition of K. granulomatis was determined at pH 7.0 and 37 °C temperature. The strain was resistant to most of the antibiotics used in this study except azithromycin, gentamycin and ciprofloxacin. The strain is capable to reproduce flacherrie like symptoms with high mortality rate when re-injected into healthy silkworm. Treatment with low dose of ciprofloxacin was found to be effective to prevent flacherrie induced by the isolated K. granulomatis strain.

PiggyBac transposon-derived targeting shRNA interference against the Bombyx mori nucleopolyhedrovirus (BmNPV).

The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most destructive diseases in silkworm, which has caused the main damage to sericulture industry. In this study, we developed a system of RNAi to prevent the BmNPV infection using the piggyBac transposon-derived targeting short hairpin RNA (shRNA) interference. The shRNAs targeting the genes of i.e.-1, lef-1, lef-2 and lef-3 of BmNPV were designed and used to inhibit the intracellular replication or multiplication of BmNPV in Bm cells. The highest activity was presented in the shRNA targeting the i.e.-1c of BmNPV, of which the inhibition rate reached 94.5 % in vitro. Further a stable Bm cell line of piggyBac transposon-derived targeting shRNA interference against BmNPV was established, which has a highly efficacious suppression on virus proliferation. These results indicated that the recombinant shRNA expression system was a useful tool for resistance to BmNPV in vitro. The approach by recombinant shRNAs opens a door of RNAi technology as a strategy that offering technically simpler, cheaper, and quicker gene knockdown for promising research and biotechnology application on silkworm lethal diseases.

Expression analysis of Bombyx mori bidensovirus structural proteins and assembly of virus-like particles in insect cells.

Bombyx mori bidensovirus (BmBDV) is a new designated species of the new genus Bidensovirus in the new family Bidnaviridae, which contains two single-stranded linear DNAs (VD1 and VD2) and causes the chronic densonucleosis disease of silkworm. Previous researches revealed that VD1-ORF3 encodes the major structural proteins VPs. In this work, through western blot, we found that VPs expressed from 48h post-inoculation and kept increasing until 120h post-inoculation in midgut of Bombyx mori. In order to further investigate the translation of vp gene, the ORFs (vp1 and vp2) of the VP started just up-stream of the first two candidate initiation codons were expressed in Sf9 cells by a baculovirus expression system. The expression products were purified by gradient density centrifugation and analyzed by Western blot and electron microscopy. The results showed that the expressions of vp1 yielded three proteins (VP1, VP1', and VP2), which are the same with the viral VPs expression in midgut of Bombyx mori, and vp2 generated two VPs with the molecular weights of about 51kDa (VP2) and 37kDa. The observation by electron microscopy indicated that these VPs can auto-assemble into virus-like particles that could not be distinguished from virus particles. These findings will provide materials for studying the structure of BmBDV and be helpful in the studies on BmBDV-based disease in silkworms.

Morphological and molecular studies of Vairimorpha necatrix BM, a new strain of the microsporidium V. necatrix (microsporidia, burenellidae) recorded in the silkworm, Bombyx mori

Vairimorpha sp. BM (2012) is a recent isolate of the microsporidia from the silkworm in Shandong, China. The ultrastructure, tissue pathology and molecular characterization of this isolate is described in this study. This pathogenic fungus causes pebrine disease in silkworms which manifests as a systemic infection. Meanwhile, the silkworm eggs produced by the infected moths were examined using a microscope and PCR amplification. Neither spores nor the expected PCR band were observed, suggesting that no vertical transmission occurred in Bombyx mori. In addition, the ultrastructure of the isolate was studied by light microscopy and transmission electron microscopy. Two types of spores were observed: diplokaroytic spores with 13–17 coils of polar tubes and monokaryotic spores with less coils of polar tubes which could form octospores; however, no sporophorous vesicles were observed. Finally, phylogenetic analysis of the small subunit rRNA genes of Vairimorpha species showed that this isolate has a closer relationship to Vairimorpha necatrix than the other species studied. This result also is supported by phylogenetic analysis based on their actin genes, heat shock protein 70 (HSP70) and RNA polymerase II (RPB1). Based on the information gained during this study, we propose that this microsporidian species infecting B. mori should be given the name V. necatrix BM.

Efficacy of sodium hypochlorite in controlling viral and bacterial diseases in muga silkworm, Antheraea assamensis Helfer.

The incidence of the viral and bacterial diseases viz. flacherie and grasserie caused large mortality to the muga silkworm, Antheraea assamensis Helfer thereby affected the cocoon silk production. It was observed that the application of 0.01 % of NaOCl to the silkworms at larval instars, reduced the mortality due to bacterial and viral diseases quite significantly (P < 0.1 & P < 0 .05) as compared to the control without affecting the quality of cocoons during different seasons at the different locations. The values of Effective Rate of Rearing (E.R.R. %) in the treated lots was significantly higher (P < 0.1 & P < 0 .05) than that of control in all the crop seasons at the different locations. The results of the present study deduced that the application of 0.01 % of Sodium hypochlorite during the rearing of muga silkworm, Antheraea assamensis Helfer can effectively control the larval mortality due to bacterial and viral diseases thereby enhance the cocoon production.

Codon Optimization Enhances Protein Expression of Bombyx mori Nucleopolyhedrovirus DNA Polymerase in E. coli

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms, while BmNPV DNA polymerase (BmNPV-pol), encoded by ORF53 gene, plays a central role in viral DNA replication. Efficacy studies of BmNPV-POL are limited because of poor heterologous protein expression in E. coli. Here, we redesigned the BmNPV-pol to preferentially match codon frequencies of E. coli without altering the amino acid sequence. Following de novo synthesis, codon-optimized BmNPV-pol (co-BmNPV-pol) gene was cloned into pET32a and pGEX-4T-2 vector. The expression of co-BmNPV-POL in E. coli was significantly increased when BmNPV-POL was fused with GST protein rather than a His-tag. The co-BmNPV-POL fusion proteins were isolated using GST affinity chromatography and Mono Q iron exchange chromatography. Protein purity and identity were confirmed by western blot and MALDI-TOF analyses. The biological activity of purified proteins was measured on a poly(dA)/oligo(dT) primer/template. The specific polymerasing activity of the recombinant BmNPV-POL was 6,329 units/mg at optimal conditions. Thus, a large amount of purified protein as a soluble form with high activity would provide many benefits for the functional research and application of BmNPV-POL.

Pebrine Disease of Chinese Silkworm Controlled by Using Atmospheric Cold Plasma Jet

Tussah pebrine disease resulting from Nosema bombycis (NB) spores causes massive production and economic losses in the silk industry in China. This paper reports on highly effective inactivation of NB spores for the control of pebrine disease by using an atmospheric pressure cold plasma jet. Both Giemsa dyeing measurement and tussah breeding experiment show that the atmospheric pressure He plasma jet containing 1% <formula formulatype="inline" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex Notation="TeX">${\rm O}_{2}$</tex></formula> kills almost all the NB spores with a <formula formulatype="inline" xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"> <tex Notation="TeX">$10^{7}$</tex></formula> population within an exposure time of 5 min. Scanning electron microscopy measurements and UV absorbance spectra show that plasma inactivation has withered up the NB spores and resulted in the spores inactivated. Both energy dispersive spectroscopy and optical emission spectroscopy measurements showed that plasma-created reactive particles, such as O and accompanied charged species can play an important role in the inactivation processing.

Expression, purification, and enzymatic characterization of Bombyx mori nucleopolyhedrovirus DNA polymerase

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms. BmNPV DNA polymerase (Bm-DNAPOL), encoded by the ORF53 gene, plays a central role in viral DNA replication. In this work, a His-tagged Bm-DNAPOL fusion protein, constructed using a novel MultiBac expression system, was overexpressed in Sf-9 insect cells, purified to near homogeneity on Ni-NTA agarose beads and further purified by ion-exchange chromatography. About 0.4 mg of enzyme was obtained from about 1 × 10(9) infected Sf-9 cells in suspension culture. Characterization of the highly purified enzyme indicated that Bm-DNAPOL is a monomer with an apparent molecular mass of approximately 110,000 Da. It possessed a specific activity of 15,126.3 U/mg under optimal in vitro reaction conditions and behaved in the manner of a proliferating cell nuclear antigen (PCNA)-independent DNA polymerase on both poly(dA)/oligo(dT) primer/template and singly premiered M13 DNA. BmNPV viral replication may be independent of replication factor C and a PCNA complex, while single-stranded DNA binding protein might play an important role in BmNPV DNA replication. These findings will be significant in studies on BmNPV-based disease in silkworms and for using silkworms as a bioreactor for the production of biomolecules of commercial importance.

Amasya İpekçiliği Üzerine ( 1750-1900)

Amasya is one of the most important cities in the chain of Ottoman silk manufacture. From the standpoint of Ottoman silk production, the early role of the city was in the way of being the entrepot of Iranian silk. In later periods, it is reflected in documents that silkworm-breeding and branches of work and production depending on silk reached very important dimensions. It is observed that from the last quarter of the 18th century onward, tax records regarding silk production are mentioned more than the records concerning the tax of mizan-i harir. It is also observed that at an accelerating rate within the 18th century, records about mulberry fields and wormhouses are intensely mentioned. In line with what has been stated, it is thought that the dependency on Iranian silk decreased in time. Negative effects of the silkworm disease that was effective in the second half of the 19th century were felt in Amasya too. To the degree that it is reflected in the archival material, it is understood that government officials attended perhaps most sensitively to the Amasya silk- production. When examined under the light of the documents and the information, it is thought that there was an Amasya silk production comparable to that of Bursa. Although the city of Bursa played a dominant role in Ottoman silk production, the total production of a few cities such as Amasya was able to nearly balance the Bursa silk production or perhaps provide a production level above that of Bursa.

Gender Influenced Spore Dimorphism in Nosema bombycis Nageli Causing Pebrine Disease in Mulberry Silkworm, Bombyx mori L.

Nosema bombycis is a pathogen causing pebrine disease of the mulberry silkworm, Bombyx mori. The disease spreads mainly through transovarian transmission of environmental spore and secondarily through contaminated food, rearing appliances, etc. by primary spores. Ultra-structure studies using Scanning and Transmission Electron Microscopy of the two spores revealed differences in the primary spore which contained a Short Polar Tube (ST) with a thin wall ( 200 nm). It is observed that the yield of spore with LT is highest in female moths, whereas, it is spores with ST are highest in male moths. Besides ultra-structures, the development pattern of the two types of spores is also different. It is an interesting finding in the present study that, spores of N. bombycis produced two types of spores and multiplied in different gender under the influence of the host’s reproductive role and physiology for transmission of disease. The detailed study on ultra-structure of disporous N. bombycis in both the sexes of B. mori along with their development in the life cycle stages of silkworms with special reference to the inoculum concentration of spore is discussed.

Evolution of nanoparticle-induced distortion on viral polyhedra

Morphological changes in the polyhedra of the Bombyx mori L. nuclear polyhedrosis virus (BmNPV), a baculovirus causing the deadly grasserie disease in silkworms, brought about by mixing with lipophilically capped amorphous silica nanoparticles (LASN, average size 10 ± 2 nm) were studied with scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectroscopy. SEM shows that the regular octagonal polyhedra facets are replaced by a larger number of newly formed irregular ones. The average number of facets reveals a nonlinear growth pattern with nanoparticle (NP) concentration, where an initial linear region ends in a plateau. IR bands corresponding to vibration modes of the capping show (a) a saturation of the area under the band with NP concentration, indicating a correlation with attachment to viral polyhedra and (b) a narrowing of the band per NP from the linear to the plateau portions of the distortion curve, suggesting non-equilibrium and equilibrium situations, respectively.

Prevalence of flacherie disease and pathogenicity of isolated pathogensin silkworm,Bombyx mori(L.) under different environmental conditions

Flacherie is one of the diseases in silkworm caused by different bacteria and viruses. Flacherie diseased silkworm larvae were collected from different sericultural areas of Karnataka during different seasons and the pathogens were isolated. The data revealed that prevalence of flacherie was high in summer (5.0 – 20.0%) followed by rainy (2.5 – 15.0%) and winter (0.0 – 7.5%) seasons. It was also observed that the flacherie larvae were infected individually with bacteria (7.5 – 20.0%) or viruses (0.0 – 17.5%) and combined infections of both (5.0 – 32.5%). Streptococcus faecalis Andrewes and Horder, Staphylococcus aureus Rosenbach, Serratia marcescens Bizio and Bacillus thuringiensis Berliner were found pathogenic to silkworm. BmIFV and BmDNV1 were detected in the midgut larval samples and BmNPV was noticed in very few haemolymph samples. LC50 value was calculated for the pathogenic bacteria viz., S. faecalis, S. aureus and S. marcescens under three different environmental conditions (E1, E2 and E3) and the LC50 value against these bacteria was 5.5 × 107, 3.3 × 108 and 6.1 × 108 cells/ml, respectively at E3. LC50 could not be made for the above bacteria at E1 and E2 environment as there was less than 50% mortality even in high concentration. In the case of B. thuringiensis, the mortality was due to toxicity and the LC50 value was 3.2 × 106, 2.8 × 106 and 2.4 × 106 cells/ml at E1, E2 and E3, respectively. LC50 was also calculated against all the viruses (BmIFV, BmDNV1 and BmNPV) and it was 10−4.8 dilutions, 10−2.9 dilutions and 2.0 × 105 polyhedra/ml (E1), 10−4.9 dilutions, 10−3.1 dilutions and 1.1 × 105 polyhedra/ml (E2) and 10−5.1 dilutions, 10−3.6 dilutions and 5.0 × 104 polyhedra/ml (E3), respectively.

Detailed investigation of the sequential pathological changes in silkworm larvae infected with Bombyx densovirus type 1

Bombyx mori densovirus type 1 (BmDNV-1) is a pathogen causing flacherie disease in silkworms. BmDNV-1 multiplies only in the nuclei of the columnar cells of larval midgut epithelium. Although several immunohistochemical studies using anti-BmDNV-1 antibody have been reported to date, sequential pathological changes in BmDNV-1-infected larvae have not been completely elucidated. In this paper, sequential investigations were performed on the pathological features of BmDNV-1-infected larvae and BmDNV-1 propagation. Oral infection experiments using newly ecdysed 4th instar larvae revealed that the larvae began to die 9days post infection (dpi), and the remaining died 10dpi. Histological observations revealed phenotypic alterations in the midgut cells from 4dpi, and complete disruption of the midgut structure at 9dpi. Quantitative RT-PCR of two BmDNV-1 genes indicated that BmDNV-1 began to propagate from 4dpi, and gradually increased until the larvae died. These expression patterns revealed marked correlation with the histological changes observed in the virus-infected midgut cells. Moreover, bioassays using larvae at various developmental stages clearly indicated that the pathogenicity of this virus is not dependent on the larval stage or the molting process.