ABSTRACT
Abstract The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.
Resumo O impacto dos antibióticos no crescimento e na produção do casulo foi avaliado, além do isolamento e caracterização das bactérias associadas ao intestino de larvas infectadas do bicho-da-seda. A criação das larvas foi mantida nas condições recomendadas de temperatura e umidade. As larvas do bicho-da-seda com sintomas anormais foram coletadas do grupo controle e dissecadas para coleta do intestino. As bactérias foram isoladas do conteúdo intestinal por espalhamento em placas de ágar e incubadas a 37° C durante 48 horas. A identificação bacteriana e a análise filogenética foram realizadas pelo sequenciamento do gene 16S rRNA. As bactérias isoladas foram submetidas a teste de sensibilidade antimicrobiana (métodos de difusão em disco) com penicilina (10 µg / mL), tetraciclina (30 µg / mL), amoxicilina (25 µg / mL), ampicilina (10 µg / mL) e eritromicina (15 µg / mL). Todas as cepas isoladas apresentaram resultados positivos para o teste da catalase. Isolamos e identificamos cepas bacterianas (n = 06) do intestino de larvas de bicho-da-seda saudáveis e doentes. Com base na sequência do gene 16S rRNA, as bactérias isoladas mostraram estreita relação com Serratia, Bacillus e Pseudomonas spp. Notavelmente, 83,3% das cepas eram resistentes a penicilina, tetraciclina, amoxicilina, ampicilina e eritromicina, mas 16,6% mostraram suscetibilidade aos antibióticos comumente usados mencionados acima. As larvas do bicho-da-seda alimentadas com folhas tratadas com penicilina apresentaram melhora significativa no peso larval, comprimento larval e produção de casulo. Peso larval significativamente maior (6,88g), comprimento larval (5,84cm) e peso do casulo (1,33g) foram registrados para larvas alimentadas com folhas tratadas com penicilina, em comparação com outros antibióticos. Cepas bacterianas isoladas mostraram estreita relação com Serratia spp., Bacillus spp. e Pseudomonas spp.
ABSTRACT
Abstract The impact of antibiotics on growth, cocoon production was assessed in addition to isolation and characterization of bacteria associated with silkworm gut of infected larvae. Larval rearing was maintained at recommended conditions of temperature and humidity. Silkworm larvae showing abnormal symptoms were collected from the control group and dissected for gut collection. Bacteria were isolated from the gut content by spreading on agar plates and incubated at 37 °C for 48 hrs. Bacterial identification and phylogenetic analysis were carried out by 16S rRNA gene sequencing. The isolated bacteria were subjected to antimicrobial susceptibility test (disc diffusion methods) by using Penicillin (10 µg/mL), Tetracycline (30 µg/mL), Amoxicillin (25 µg/mL), Ampicillin (10 µg/mL), and Erythromycin (15 µg/mL). All isolated strains showed positive results for the catalase test. We isolated and identified bacterial strains (n = 06) from the gut of healthy and diseased silkworm larvae. Based on the 16S rRNA gene sequence, isolated bacteria showed close relation with Serratia, Bacillus, and Pseudomonas spp. Notably, 83.3% of strains were resistant to Penicillin, Tetracycline, Amoxicillin, Ampicillin, and Erythromycin but 16.6% showed antibiotic susceptibility to the above-mentioned commonly used antibiotics. Silkworm larvae fed on penicillin-treated leaves showed significant improvement in larval weight, larval length, and cocoon production. Significantly higher larval weight (6.88g), larval length (5.84cm), and cocoon weight (1.33g) were recorded for larvae fed on leaves treated with penicillin as compared to other antibiotics. Isolated bacterial strains showed close relation with Serratia spp., Bacillus spp. and Pseudomonas spp.
Resumo O impacto dos antibióticos no crescimento e na produção do casulo foi avaliado, além do isolamento e caracterização das bactérias associadas ao intestino de larvas infectadas do bicho-da-seda. A criação das larvas foi mantida nas condições recomendadas de temperatura e umidade. As larvas do bicho-da-seda com sintomas anormais foram coletadas do grupo controle e dissecadas para coleta do intestino. As bactérias foram isoladas do conteúdo intestinal por espalhamento em placas de ágar e incubadas a 37° C durante 48 horas. A identificação bacteriana e a análise filogenética foram realizadas pelo sequenciamento do gene 16S rRNA. As bactérias isoladas foram submetidas a teste de sensibilidade antimicrobiana (métodos de difusão em disco) com penicilina (10 µg / mL), tetraciclina (30 µg / mL), amoxicilina (25 µg / mL), ampicilina (10 µg / mL) e eritromicina (15 µg / mL). Todas as cepas isoladas apresentaram resultados positivos para o teste da catalase. Isolamos e identificamos cepas bacterianas (n = 06) do intestino de larvas de bicho-da-seda saudáveis e doentes. Com base na sequência do gene 16S rRNA, as bactérias isoladas mostraram estreita relação com Serratia, Bacillus e Pseudomonas spp. Notavelmente, 83,3% das cepas eram resistentes a penicilina, tetraciclina, amoxicilina, ampicilina e eritromicina, mas 16,6% mostraram suscetibilidade aos antibióticos comumente usados mencionados acima. As larvas do bicho-da-seda alimentadas com folhas tratadas com penicilina apresentaram melhora significativa no peso larval, comprimento larval e produção de casulo. Peso larval significativamente maior (6,88g), comprimento larval (5,84cm) e peso do casulo (1,33g) foram registrados para larvas alimentadas com folhas tratadas com penicilina, em comparação com outros antibióticos. Cepas bacterianas isoladas mostraram estreita relação com Serratia spp., Bacillus spp. e Pseudomonas spp.
Subject(s)
Animals , Bombyx , Anti-Bacterial Agents/pharmacology , Phylogeny , Bacteria/genetics , RNA, Ribosomal, 16S/genetics , Microbial Sensitivity Tests , LarvaABSTRACT
Starting with the relationship between mulberry leaves and silkworm droppings as food and metabolites, this study systematically compared the chemical components, screened out differential components, and quantitatively analyzed the main differential components based on ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS) and UPLC-Q-TRAP-MS combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). Moreover, the in vitro enzymatic transformation of the representative differential components was studied. The results showed that(1) 95 components were identified from mulberry leaves and silkworm droppings, among which 27 components only exist in mulberry leaves and 8 components in silkworm droppings. The main differential components were flavonoid glycosides and chlorogenic acids.(2) Nineteen components with significant difference were quantitatively analyzed, and the components with significant differences and high content were neochlorogenic acid, chlorogenic acid, and rutin.(3) The crude protease in the mid-gut of silkworm significantly metabolized neochlorogenic acid and chlorogenic acid, which may be an important reason for the efficacy change in mulberry leaves and silkworm droppings. This study lays a scientific foundation for the development, utilization, and quality control of mulberry leaves and silkworm droppings. It provides references for clarifying the possible material basis and mechanism of the pungent-cool and dispersing nature of mulberry leaves transforming into the pungent-warm and dampness-resolving nature of silkworm droppings, and offers a new idea for the study of nature-effect transformation mechanism of traditional Chinese medicine.
Subject(s)
Animals , Bombyx , Morus/chemistry , Chlorogenic Acid/analysis , Gas Chromatography-Mass Spectrometry , Chromatography, High Pressure Liquid/methods , Plant Leaves/chemistryABSTRACT
Aim: The present study was conducted to investigate the status and diversity of plant species used for rearing of Eri silkworm and their traditional management in the homegardens of Mishing tribe.Methodology: A total of 45 homegardens from three villages were surveyed randomly which were primarily associated with rearing of Eri silkworm. Vegetation was studied using quadrat method. The data on traditional knowledge associated with the management of plants species for rearing of Eri silkworm were collected using semi structured questionnaires. Results: The average size of studied homegardens was 0.35 ha. A total of 243 plant species were recorded from the studied homegardens of which six species were used as food plants for rearing of Eri silkworm and 10 species were used in construction of mountage. Trees related to Eri culture contributed to 37.47% of the total importance value index (IVI) in the studied homegardens. Heteropanax fragrans (Roxb.) Seem and Ricinus communis L. were the primary food plants of Eri silkworm and were found to be dominant in the homegardens. Activities such as land preparation, sowing, pruning, weeding and watering are carried out for the management of Eri silkworm food plant species and were found to vary for different species. Interpretation: There is an urgent need to document the indigenous knowledge and traditional practices associated with the management of host and food plants used in the rearing of Eri silkworm in the present day context as it is a part of their cultural heritage and should be reoriented through integration of modern farming technique.
ABSTRACT
The purpose of this article is to study the degradation of chemical compositions after the silkworm excrement being expelled from the silkworm, and to determine its main metabolic compositions and their changing relationships. This research is based on UPLC-Q-TOF-MS technology. Based on the systematic analysis of the main chemical compositions contained in silkworm excrement, the principal compositions analysis(PCA) and partial least squares discriminant analysis(OPLS-DA) on commercial silkworm excrement and fresh silkworm excrement were analyzed for differences. The S-plot chart of OPLS-DA was used to select and identify the chemical compositions that contributed significantly to the difference. At the same time, the relative peak areas of the different compositions were extracted by Masslynx to obtain the relative content of different compositions in fresh silkworm excrement. The results showed that there was a significant difference in the chemical compositions between fresh silkworm excrement and commercial silkworm excrement. The difference compositions were mainly flavonoid glycosides and Diels-Alder type composition, and two types of compounds are degradated during the storage of silkworm sand. In this study, the chemical compositions of fresh silkworm excrement were systematically identified and analyzed for the first time by mass spectrometry, and it was found that some chemical compositions of silkworm excrement were degradated with time during storage.
Subject(s)
Animals , Bombyx , Chromatography, High Pressure Liquid , Discriminant Analysis , Drugs, Chinese Herbal , Mass SpectrometryABSTRACT
The aim of the present study was to assess genetics variations within and among strains with different geographical origin, belonging to Bulgarian germplasm bank of mulberry silkworm (Bombyx moriL.)and to establish their relationships using isoenzyme markers. Polyacrilamide gel electrophoresis (PAGE) was used to study the isoenzymes of nonspecific esterases (EST), malate dehydrogenase (MDH) and acid phosphatase (ACP) from haemolymph, phosphoglucomutase (PGM) and hexokinase (HK) from silk glands and alkaline phosphatase (ALP) from midgut of mulberry silkworm (Bombyx moriL.). Variability was evident in all of these enzyme systems among twelve strains from Bulgarian germplasm resources of B. mori.Total of nine lociwere detected. All of them (100%) were polymorphic. Null alleles in four lociwere determined. Intra-and inter-strain polymorphism was obtained. The degree of polymorphism ranged from 0% to 77.80%. Low levels of observed heterozygosity in comparison with the expected one have been calculated in almost all of strainsas well as deviations from Hardy-Weinberg equilibrium in almost all analyzed loci, resultingof excess of homozygotes. The value of FSTwas 0.4903. The dendrogram constructed with the values of genetic distance manifests that Romanian strain Cislau Tokay is formed one main clade while the rest strains studied (from Bulgaria, Japan, China, Vietnam, Spain, Syria and Egypt) are formed the other clade withsubclades. The genetic data of the tested strains from different geographical regions, would be used for identifying suitable parents for breeding programs with a view to yield improvement in this species with economic significance.
Subject(s)
Animals , Seed Bank , Electrophoresis, Polyacrylamide Gel , Lepidoptera/genetics , GeneticsABSTRACT
Antheraea assamensis Helfer, popularly known as Muga silkworm, the golden silk producer of northeast India is economically important and unique among the Saturniid silkworms. In this study, the genetic diversity and phylogeny of semidomesticated and wild morphs of Muga silkwormcollected from different geographical locations of northeast India were investigated based on the sequences of five mitochondrial loci, i.e. 12S rRNA, 16S rRNA, CoxI, Cytb and CR. All the five mitochondrial loci showed a strong bias towards higher ‘A’ and ‘T’ contents. Transitional substitutions were found to be more than the transversional substitutions. The rate of nucleotide substitution and average genetic divergence were found to be highest in CR sequences and lowest in 12S rRNA gene sequences among the morphs of Muga silkworm. The morphs collected from same geographical area had identical 12S rRNA, 16S rRNA, CoxI and Cytb gene sequences. Moreover, the 12S rRNA and 16S rRNA gene sequences of somesemi-domesticated and wild morphs collected from different geographical locations were also found to be similar. In the phylogenetic trees generated based on themitochondrial loci, mixing of semi-domesticated and wild morphs was observed as they shared the same group. The information generated in this study will help in formulating strategies to conserve the natural biodiversity present among these unique silkworms in northeast India. In addition, this will be useful in identifying diverse morphs of Muga silkworm, which will help in effective breeding programmes to improve its productivity.
ABSTRACT
Identifying and comparing the chemical constituents of wild silkworm cocoon and silkworm cocoon is of great significance for understanding the domestication of silkworm. In this study, we used high temperature and high pressure and methanol-water system to extract cocoon chemical constituents. We used UHPLC-MS to identify and compare cocoon chemical constituents of wild silkworm and domestic silkworm Dazao and Haoyue strains. The cocoon metabolic fingerprints of wild silkworm and domestic silkworm Dazao and Haoyue strains were obtained by using the UHPLC-MS in the positive ion mode and negative ion mode. By annotation, we found that cocoon chemical compounds with high abundances contained amino acids, flavonoids, alkaloids, terpenes, organic acids, and lignans. PLS-DA showed that the cocoon components were significantly different among the wild silkworm and two domestic silkworm strains Dazao and Haoyue. Proline, leucine/isoleucine and phenylalanine showed significantly higher abundances in the cocoon of domestic silkworm Dazao strain than in those of wild silkworm and domestic silkworm Haoyue strain. The flavonoid secondary metabolites are abundant in the Dazao cocoon, including quercetin, isoquercetin, quercetin 3-O-sophoroside, quercetin-3-O-α-L-rhamnoside, quercetin-3-O- rutinoside, and kaempferol. The other secondary metabolites, alkaloids, terpenes and lignans, showed higher abundances in the wild silkworm cocoon than in the domestic silkworm cocoon, including neurine, candicine, pilocarpidine, artemisiifolin, eupassopin, and eudesobovatol. By exposing cocoons to UV light and observing the green fluorescence of flavonoids, we found that Dazao cocoon had the most flavonoids, and Haoyue cocoon had least flavonoids and wild silkworm cocoon had mediate flavonoids. Alkaloids and organic acids are good anti-insect and antimicrobial agents, which have high abundance in the wild silkworm cocoon and could enhance the defense ability of wild silkworm cocoon. Flavonoids are abundant in the cocoon of domestic silkworm Dazao strain, which the main factors are leading to the yellow-green cocoon of Dazao.
Subject(s)
Animals , Bombyx , Chromatography, High Pressure Liquid , Flavonoids , Mass SpectrometryABSTRACT
The biogenic monoamine 5-hydroxytryptamine (5-HT) is an ancient intracellular signaling molecule widely distributed in all animals with nervous systems, and has been implicated in principal behaviors. Tryptophan hydroxylase (TRH) induces a highly specific catalytic reaction that converts L-tryptophan (tryptophan) to 5-hydroxy-L-tryptophan (5-HTP) that is subsequently used as a substrate by aromatic L-amino acid decarboxylase (DDC) to form 5-HT. Five-HT is an ancient intracellular signaling molecule that is widely distributed in the animal kingdom and has been implicated in regulating the behaviors of animals with nervous systems. However, the role of TRH in Lepidoptera is not well understood. In this study, we cloned 1 667 bp cDNAs of Bombyx mori TRH (BmTRH), which contains a 1 632 bp open reading frame (ORF). Homology analysis revealed that BmTRH shared high amino acid identity with Homo sapiens TPH and Drosophila TRH (DmTRH). The high homology (70%) of BmTRH with DmTRH suggested that BmTRH could have a function similar to DmTRH. Gene expression analysis revealed that BmTRH was mainly expressed in head and central nervous (CNS). Moreover, immunohistochemistry and Western blotting analyses showed that BmTRH was detected only in larval nervous tissues. Taken together, our results indicate that BmTRH could likely function in the regulation of neural activities in B. mori. The transcripts of B. mori decarboxylase (BmDDC) and B. mori phenylalanine hydroxylase (BmPAH) whose proteins had TRH activity, were also expressed in the CNS tissues, indicating that unlike in Drosophila, two distinct mechanisms likely regulate 5-HT synthesis in silkworm.
Subject(s)
Animals , Amino Acid Sequence , Bombyx , Cloning, Molecular , DNA, Complementary , Insect Proteins , Phenylalanine Hydroxylase , Tryptophan HydroxylaseABSTRACT
Genes belonging to the elongases of very long chain fatty acid (ELOVL) family affect many physiological functions in organism. In this paper, Bmelo424 gene, a member of the ELOVL family in silkworm, was cloned and its ORF was 558 bp. Its protein sequence was predicted to have four transmembrane domains, six serine phosphorylation sites, eight threonine phosphorylation sites and four tyrosine phosphorylation sites, and its subcellular localization was in the endoplasmic reticulum. Secondary structure analysis showed that the percentage of alpha-helix and beta-strand was 26.7% and 20% respectively. The results of fluorescence quantitative PCR showed that Bmelo424 gene was expressed in all tissues of silkworm, especially with the highest expression in head. By heterologous expression of Bmelo424 gene in Saccharomyces cerevisiae, the effect of Bmelo424 gene on fatty acid elongation was studied. GC-MS results indicated that the fatty acid content of C16:1n-7 in S. cerevisiae with pYES2-Bmelo424 recombinant plasmid increased significantly, whereas the content of C16:0, C18:0 and C18:1n-9 decreased. The results of temperature stress revealed that Bmelo424 gene could improve the low temperature adaptability of S. cerevisiae, but its high temperature adaptability decreased. This provides a reference for exploring the function of Bmelo424 gene in silkworm.
Subject(s)
Animals , Acetyltransferases , Amino Acid Sequence , Bombyx , Cloning, Molecular , Fatty Acids , Saccharomyces cerevisiaeABSTRACT
ABSTRACT Bombyx mori nucleopolyhedrovirus (BmNPV) disease is one of the most serious silkworm diseases, and it has caused great economic losses to the sericulture industry. So far, the disease has not been controlled effectively by therapeutic agents. Breeding resistant silkworm varieties breeding may be an effective way to improve resistance to BmNPV and reduce economic losses. A precise resistance-detection method will help to accelerate the breeding process. For this purpose, here we described the individual inoculation method (IIM). Details of the IIM include pathogen BmNPV preparation, mulberry leaf size, pathogen volume, rearing conditions, course of infection, and breeding conditions. Finally, a resistance comparison experiment was performed using the IIM and the traditional group inoculation method (GIM). The incidence of BmNPV infection and the within-group variance results showed that the IIM was more precise and reliable than the GIM.
ABSTRACT
PURPOSE: Obesity, a worldwide epidemic, is associated with insulin resistance, hyperlipidemia, hypertension, cardiovascular disease, and certain cancers. Many strategies, including natural alternative anti-obesity agents, are used widely to prevent obesity. This study examined the effects of silkworm hemolymph on the weight control of C57BL/6N mice fed with a high-fat diet. METHODS: The mice were divided into five groups: normal group (N), high-fat diet group (HFC), high-fat diet and silkworm hemolymph (at dose of 1 mL/kg BW (HFS-1), 5 mL/kg BW (HFS-5) and 10 mL/kg (HFS-10) for 12 weeks. RESULTS: After 12 weeks treatment, the administration of silkworm hemolymph decreased the final body weight significantly along with a decrease in the weights of epididymal fat and total fat. The plasma LDL-cholesterol concentration was significantly lower in the HFS-1, HFS-5, and HFS-10 groups than in the HFC group. In addition, the leptin level of the HFS groups was significantly lower than those of the HFC group without a change in the plasma insulin concentration. CONCLUSION: These findings suggest that the silkworm hemolymph may have the potential to prevent obesity.
Subject(s)
Animals , Mice , Anti-Obesity Agents , Body Weight , Bombyx , Cardiovascular Diseases , Diet, High-Fat , Hemolymph , Hyperlipidemias , Hypertension , Insulin , Insulin Resistance , Leptin , Obesity , Plasma , Weights and MeasuresABSTRACT
The dual luciferase reporter gene system provides sensitive readout, while it relies on a constitutively-expressed control gene for readout normalization. However, most standard control reporter genes are not constitutively expressed under all conditions. Here, we report an effective method to construct a control reporter plasmid for the dual luciferase reporter gene system that would be suitable for hormone research in silkworm cell lines. First, we modified BmVgP78M, a stably-expressed constitutive promoter in silkworm cells by mutating its hormone-related element. Then, we constructed the pRL-VgP78M control reporter plasmid by replacing the SV40 promoter and chimeric intron sequences in pRL-SV40 with the BmVgP78M sequence. Finally, we confirmed that the pRL-VgP78M control reporter plasmid could be stably expressed in silkworm cell lines via cell transfection experiments, and it was unresponsive to the induction of ecdysone, juvenile hormone, or their transcription factors. We thus obtained a control reporter plasmid pRL-VgP78M that could be expressed stably and moderately in silkworm cells. It can be readily used as the control reporter plasmid of the dual luciferase reporter gene system for hormone research in silkworm cell lines. It will also provide a reference for construction of control reporter plasmids of dual luciferase reporter gene systems that are adaptable to cell lines isolated from other species.
ABSTRACT
Alphabaculovirus is a genus of the entomopathogenic virus, whose species Bombyx mori nucleopolyhedrovirus (BmNPV) infects the silkworm, Bombyx mori, which is an important insect in the sericulture industry. A geographic isolate of BmNPV was identified in the state of Paraná, Brazil. It was infecting B. mori larvae and various organs and target tissues were identified, however, there was no information about the infection of Malpighian tubules (MT). The MT comprises the excretory system of B. mori and acts in the elimination of toxic substances and in hydroelectrolytic homeostasis. Thus, the present study examined the susceptibility and cytopathology of B. mori MT to BmNPV. To this end, hybrid fifth instar larvae were inoculated with a virus suspension at different days post-inoculation (dpi). MT segments were collected and divided into the ampullae, proximal, medial and distal regions. These were processed for light microscopy and transmission electron analysis. The MT regions revealed differences in susceptibility to BmNPV and the ampullae in its transition area was infected from the sixth dpi; the other regions did not reveal any evidence of infection. The transition area of the ampullae has not been previously described in Lepidoptera and its cytopathology revealed a hypertrophic nucleus with viroplasm, followed by the formation and development of viral polyhedra, which are common characteristics of infections by Alphabaculovirus. Thus, infection of the ampullae of the MT of B. mori by BmNPV, together with other known targets, compromises the metabolic balance of the insect, which results in consequences for silk production and damage to the sericulture sector.
Alphabaculovirus é um gênero de vírus entomopatogênico, cuja espécie Bombyx mori nucleopolyhedrovirus (BmNPV) infecta o bicho-da-seda, Bombyx mori, inseto importante na indústria sericícola. Um isolado geográfico do BmNPV foi identificado no estado do Paraná, Brasil, infectando lagartas de B. mori e vários órgãos e tecidos alvos foram identificados; entretanto, não há informações sobre a infecção do túbulo de Malpighi (TM). O TM compõe o sistema excretor de B. mori, atuando na eliminação de substâncias tóxicas e na homeostase hidroeletrolítica. Assim, o presente estudo, analisou a susceptibilidade e a citopatologia do TM de B. mori ao BmNPV. Para tanto, lagartas híbridas de 5° instar foram inoculadas com uma suspensão viral e em diferentes dias pós-inoculação (dpi), segmentos do TM foram coletados e subdivididos nas regiões da ampola, proximal, média e distal; sendo processados para análises em microscopias de luz e eletrônica de transmissão. As regiões do TM revelaram diferenças na susceptibilidade ao BmNPV e a ampola, na sua área de transição, foi infectada a partir do 6° dpi, já as demais regiões não revelaram quaisquer indícios de infecção. A área de transição da ampola, ainda não havia sido descrita em lepidópteros e sua citopatologia revelou núcleo hipertrófico com viroplasma, seguido da formação e desenvolvimento dos poliedros virais, características comuns das infecções pelo Alphabaculovirus. Assim, a infecção da ampola do TM de B. mori ao BmNPV, somada a de outros alvos conhecidos, compromete o equilíbrio metabólico do inseto, com consequências na produção de seda e prejuízos ao setor sericícola.
Subject(s)
Bombyx , Baculoviridae , Nucleocapsid , Lepidoptera , Malpighian TubulesABSTRACT
Objective:To obtain recombinant Profilin of silkworm,identify its immunogenicity,predict its B cell epitopes and construct the evolutionary trees. Methods: The nucleotide sequence of Profilin was acquired from NCBI,synthesized it and cloned it into pET-28 vector. Then,the recombinant plasimids were transformed to E. coli BL21. After induced by IPTG,recombinant protein was purified by Affinity chromatography. Furtherly,its allergenicity was identified by Western blot,the potential B cell epitopes was analyzed through DNAStar and build the evolutionary trees by MEGA5. 05. Results: The recombinant protein of Profilin was successfully expressed and purified by affinity chromatography. Besides,the protein contains a high IgE-binding activity with IgE existing in serum of patients allergic to silkworm. Conclusion: The recombinant proflilin has IgE-binding activity, and it is meaningful for fundamental research and specific diagnosis studies of allergic diseases caused by silkworm.
ABSTRACT
AIM To prepare the silkworm pupa polysaccharide capsules.METHODS The crude silkworm pupa polysaccharide was extracted by alkali-assisted ultrasonic wave.After deproteinization and decolorization,it was dialysed,and then isolated and purified by SephadexG-150 column.Single factor test was applied to optimizing three influencing factors including ethanol concentration,soild-liquid ratio and drying temperature.The polysaccharide powder's angle of repose,bulk density and critical relative humidity were determined,after which the water content,load difference and disintegration time of obtained capsules were investigated.RESULTS The optimal conditions were determined to be 90％ for ethanol concentration,1 ∶ 1 for soild-liquid ratio,and 60 ℃ for drying temperature.For polysaccharide powder,the average angle of repose was 37.74℃,the average bulk density was 0.51 g/mL,and the critical relative humidity was 63％.With 6.87％ for water content,0.132 3 g for average load,and 12.12 min for average disintegration time,the capsules were qualified according to standard requirements of Chinese Pharmacopoeia.CONCLUSION The good formability of silkworm pupa polysaccharide capsules demonstrates that this simple,stable and controllable technology can provide an experimental basis for the development of related drugs.
ABSTRACT
Molting fluid, a liquid between the old epidermis and new epidermis, plays an important role in the process of ecdysis and metamorphosis for insect. In order to explore the function of molting fluid, we used two-dimensional electrophoresis to study the molting fluid during the prepupal stage and pre-eclosion stage. More than 200 protein spots were found in the molting fluid of the 2 stages, which distributed in the 4-9 of pI and 10-180 kDa of molecular weight. We selected 42 spots to be analyzed by the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF/TOF) from the molting fluid of pre-eclosion stage, of which 34 proteins were identified successfully, including apolipoprotein, protease and protease inhibitors, chitin-binding protein and protein involved in immunity. Some of the proteins demonstrated differential expression between the two stages of metamorphosis. In order to validate the result from proteomics analysis, we studied expression of the apolipoprotein D by Q-PCR from the developmental stages. The results showed that the gene encoding apolipoprotein D had the high expression from the 1st day to the 4th day of the pupa stage, which indicated they could be involved in eclosion due to the abundant accumulation in the late pupa. Our results offered more clues for understanding the mechanism of ecdysis and metamorphosis in insect and could give reference for further study of molting fluid.
ABSTRACT
This paper investigated the diversity of the silkworm excrement bacterial communities in different ages before and after drying, aiming to clarify the differences of bacterial communities in composition and bacterial abundance and the influences of drying treatment, and provide scientific basis for the efficacy of scientific connotation and utilization of silkworm excrement. High-throughput sequencing technique was used to measure the sequence of 16S rDNA-V4 variable region of bacteria in silkworm excrement. QIIME, Mothur and PICRUSt software programs were employed to sort and calculate the number of sequences and operational taxonomic units (OTUs) for each sample. Thereafter, the abundance, distribution, alpha diversity index of species, beta diversity and bacterial communities diversity among different sample groups and predicted the bacterial gene functions were analyzed. In this study, the numbers of effective sequences for six samples were 259 250; the rarefaction curves showed a sufficient sequencing depth, and the number of OTUs was close to saturation. The bacteria in silkworm excrement belonged to the following five phylums: Proteobacteria (89.3%), Actinobacteria (5.0%), Firmicutes (4.4%), Bacteroidetes (1.1%) and Cyanobacteria (0.2%). The dominant specie was Cyanobacteria of the total bacteria identified, respectively. The abundances and diversities of the silkworm excrement bacterial communities have been reduced after drying treatment, especially the silkworm excrement of the fifth instar. PICRUSt analysis was performed to show that abundance of the functional genes such as membrane transport, carbohydrate metabolism, amino acid metabolism, lipid metabolism, nucleotide metabolism, cellular processes and signaling were relatively high. The result showed that the drying treatment could decreased the species and numbers of pathogenic bacteria in silkworm excrement obviously and improve the quality of medicinal materials. Compared with the lower ages, silkworm excrement of fifth instar seems like to be more suitable for use in medicine. Illumina MiSeq high-throughput sequencing system provides a more accurate and scientific data resource for the study of bacteria in silkworm excrement.
ABSTRACT
This work aimed to evaluate the effects of the products Biogermex®, Agro-mos®, Soil-Set® and Planta Clean® on the development of white muscardine in Bombyx mori. Products have been tested at two stages for effects on biology of caterpillars and on silk production (1), and then they were evaluated to know their potential in the prevention/control of white muscardine (2). Products were sprayed on mulberry leaves (preventive treatment) and also on the integument of silkworms (curative treatment). At the first stage, the products were applied on four consecutive days, in one of the daily feeds. Planta Clean® - curative treatment - and Soil-Set® - preventive treatment - caused mortality in 63.3% and 30% of silk worms, respectively. The fungus Beauveria bassiana was inoculated at the concentration 5 × 107 conidia/mL on the leaves and insects. The products were applied three hours after application on the fungus, in a single application, and also for four days in the other groups. A group of silkworms sprayed only with the fungus served as control. Biogermex® - curative treatment applied once and for four times - was the most effective, reducing mortality by 25 and 26.7%, respectively, as compared with the control group. None of the evaluated products acts preventively against white muscardine.
Este trabalho objetivou avaliar os efeitos de produtos Biogermex®, Agro-mos®, Soil-Set® e Planta Clean® no desenvolvimento da calcinose branca em Bombyx mori. Os produtos foram testados em duas etapas: efeitos sobre a biologia e produção de seda pela lagarta (1) e, em seguida, em função do seu potencial na prevenção/no controle da calcinose branca (2). Os produtos foram pulverizados sobre as folhas de amoreira (tratamento preventivo) e sobre o tegumento das lagartas (tratamento curativo). Na primeira etapa, os produtos foram aplicados durante quatro dias consecutivos, em uma das alimentações do dia. Planta Clean® - tratamento curativo - e Soil-Set® - tratamento preventivo - causaram mortalidade em 63,3 e 30% das lagartas, respectivamente. O fungo Beauveria bassiana foi inoculado na concentração 5 × 107 conídios/mL sobre folhas e insetos. Os produtos foram aplicados três horas após o fungo, em uma aplicação, e também por quatro dias em outro grupo. Um grupo de lagartas pulverizadas apenas com fungo serviu como controle. Biogermex® - tratamento curativo aplicado uma e quatro vezes - foi o mais eficiente, reduzindo a mortalidade em 25 e 26,7% respectivamente, em relação ao grupo controle. Nenhum dos produtos avaliados atuou de forma preventiva contra muscardina branca.
Subject(s)
Animals , Beauveria , Bombyx , Calcinosis , Pesticides , InsectaABSTRACT
This work aimed to evaluate the effects of the products Biogermex®, Agro-mos®, Soil-Set® and Planta Clean® on the development of white muscardine in Bombyx mori. Products have been tested at two stages for effects on biology of caterpillars and on silk production (1), and then they were evaluated to know their potential in the prevention/control of white muscardine (2). Products were sprayed on mulberry leaves (preventive treatment) and also on the integument of silkworms (curative treatment). At the first stage, the products were applied on four consecutive days, in one of the daily feeds. Planta Clean® - curative treatment - and Soil-Set® - preventive treatment - caused mortality in 63.3% and 30% of silk worms, respectively. The fungus Beauveria bassiana was inoculated at the concentration 5 × 107 conidia/mL on the leaves and insects. The products were applied three hours after application on the fungus, in a single application, and also for four days in the other groups. A group of silkworms sprayed only with the fungus served as control. Biogermex® - curative treatment applied once and for four times - was the most effective, reducing mortality by 25 and 26.7%, respectively, as compared with the control group. None of the evaluated products acts preventively against white muscardine.(AU)
Este trabalho objetivou avaliar os efeitos de produtos Biogermex®, Agro-mos®, Soil-Set® e Planta Clean® no desenvolvimento da calcinose branca em Bombyx mori. Os produtos foram testados em duas etapas: efeitos sobre a biologia e produção de seda pela lagarta (1) e, em seguida, em função do seu potencial na prevenção/no controle da calcinose branca (2). Os produtos foram pulverizados sobre as folhas de amoreira (tratamento preventivo) e sobre o tegumento das lagartas (tratamento curativo). Na primeira etapa, os produtos foram aplicados durante quatro dias consecutivos, em uma das alimentações do dia. Planta Clean® - tratamento curativo - e Soil-Set® - tratamento preventivo - causaram mortalidade em 63,3 e 30% das lagartas, respectivamente. O fungo Beauveria bassiana foi inoculado na concentração 5 × 107 conídios/mL sobre folhas e insetos. Os produtos foram aplicados três horas após o fungo, em uma aplicação, e também por quatro dias em outro grupo. Um grupo de lagartas pulverizadas apenas com fungo serviu como controle. Biogermex® - tratamento curativo aplicado uma e quatro vezes - foi o mais eficiente, reduzindo a mortalidade em 25 e 26,7% respectivamente, em relação ao grupo controle. Nenhum dos produtos avaliados atuou de forma preventiva contra muscardina branca.(AU)