Enzymatic hydrolysis of silkworm pupa and its allergenicity evaluation by animal model with different immunization routes

Silkworm pupa is a nourishing food with high nutritional value,but its consumption has been greatly limited given its allergenicity.Enzyme hydrolytic technique is recognized as an effective method to reduce the allergenicity of protein.In this study,we aimed to investigate the effect of enzymolysis on the allergenicity of silkworm pupa.Crude silkworm pupa protein was extracted through alkali extraction and acid precipitation,which included 5 proteins with the molecular weights ranging from 34 kDa to 76 kDa,and silkworm pupa were then hydrolyzed by alkaline protease.The allergenicity of silkworm pupa protein and its enzymatic hydrolysates was evaluated by establishing BALB/c mice model,and the mice were immunized via intragastric gavage and intraperitoneal injection,respectively.The results indicated that the intraperitoneal inj ection immunization route induced more by detecting with antibodies,histamine and Th2-related cytokines.Moreover,mice treated with silkworm pupa protein peptide displayed no obvious allergic symptoms,indicating that enzyme hydrolytic technique could significantly reduce the allergenicity of silkworm pupa.

Investigation on the Pointed Cocoon of Wild Silkworm Descendant

[Objective] To study the cocoon traits of wild silkworm descendants. [Method] In the selective breeding of domesticated raising wild silkworm and wild silkworm descendants, investigation on the traits of the built cocoons was carried out. [Result] The pointed traits of cocoons displayed obviously in the wild silkworm descendants, its pointed parts were consistent with the rear part of silkworm chrysalis, and the female silkworm has slight difference, indicating the silkworm co- coon pointed trait was hereditary. [Conclusion] This study provided references for the study of silkworm cocoon traits of wild silkworm descendant and guides the selective breeding of silkworm variety.

Induced Expression of Cytochrome P450 CYP305 B1V1 Gene in Different Tissues of Wild Mulberry Silkworm(Bombyx mandarina)

[ Objective] The aim of this study was to investigate effects of various inducers on the expression of cytochrome P450 CYP305 B1 V1 Gene in different tissues of wild mulberry silkworm. [ Method] Referring to the mRNA sequence of CYP305 B1 V1 Gene published in GenBank for wild mulberry silkworm, one pair of primers was designed, and the expression of cytochrome P450 CYP305 B1 V1 Gene in different tissues of wild mulberry silkworm treated by NaF, rutin, cypermethrin and ecdysone was also analyzed by the semi - quantitative RT - PCR. Furthermore, homology comparison and phylogenetic analysis for amino acid sequences of this gene were studied. [ Result] Rutin, cypermethrin and NaF had effects on the expression of P450 CYP305 B1 V1 Gene in different tissues of wild mulberry silkworm, while ecdysone had no significant effect. Homology comparison for amino acids indicated that the amino acid sequence of this gene was the most similar to that of CYP305 B1 gene in Bombyx mori with 100% amino acid identity, and highly similar to those of Tribolium casmneum CYP305A1, Apis mellifera CYP305A1, Drosophi- la melanogaster CYP305A1, Anopheles gambiae CYP305A2and Culex pipiens quinquefasciatus CYP2LI. [ Conclusion] CYP305 B1 V1 Gene of wild mulberry silkworm is likely to mainly take part in the metabolism of exogenous compounds, which is of great significance for revealing the function of cytochrome P450 and the metabolic mechanism of different drugs.

Abnormal Expression of Silkworm Attacin Gene Induced by 3 Kinds of Microorganisms

[Objective] The aim of this research was to study the expression of silkworm attacin gene induced by some different microorganisms.[Method] Three kinds of microorganism,BmNPV,JM109 and Agrobacterium LBA4404,were ingested to silkworm and the expression profile of attacin gene in hemocyte and fat body was detected by semi-quantitative PCR.And subsequently,the PCR products were cloned and sequenced for further analysis.[Result] A specific band,about 800 bp,appeared in fat body of all induced silkworms.As indicated by the results of cloning and sequencing(GenBank accession number:FJ373019),the band was produced because the 2 introns existing in normal expression form were not spliced.Furthermore,when the extended expression sequence was translated into amino acids,the translation stopped earlier by the stop codon TGA at the 5' end of the first intron of the original sequence,leading the loss of attacin C terminus.[Conclusion] There were two splicesomes of attacin gene,which had reference value to study the role of the attacin gene in silkworm immunity.

Isolating and Screening of Effective Decomposing Strain of Silkworm Chrysalis Protein and Study on Parts of Its Enzymatic Properties

[Objective] The research aimed to obtain an effectively-decomposing strain of silkworm chrysalis protein and discuss its enzymatic properties.[Method] The effectively-decomposing bacteria of protein was isolated from the decayed silkworm chrysalis by using dilution plate and its enzymatic properties were tested after primary screening and second screening.The enzyme activity was determined and the intermediate and small molecule protein content in silkworm chrysalis was measured after solid-state fermentati...

Breeding of a New Silkworm Variety "Yesanyuan" with Healthiness and Hypersilk

[Objective] The study aimed to breed eminent mulbery wild silb with hypersilk.[Method] Eminent mulberry wild silkworm basic materials Ye A and Ye B were bred by distant complex crossing of wild mulberry silkworm with strong stress resistance and hypersilkgeneous silkworm,then hybridized with Japanese silkworm strain,the new silkworm variety "Yesanyuan" with healthiness and hypersilkgene was bred.[Ressult]The results of laboratory identification and rural productive test in Jiangsu Province showed that it has excellent feeding habit,strong stress resistance,high yield,excellent silk quality and excellent fecundity.The new silkworm variety "Yesanyuan" had been approved by Silkworm Variety Evaluation Commission of Jiangsu Province in 2008,it is suitable to rear in Yangtze River catchment for Spring and Autumn,North sericultural area for all-year.[Conclusion] The study is helpful for the development of silkworm industry.

Synthesis of Biomorphic ZrO2-CeO2 Nanostructures by Silkworm Silk Template

A simple and green technique has been developed to prepare hierarchical biomorphic ZrO2- CeO2, using silkworm silk as the template. Different from traditional immersion technics, the whole synthesis process depends more on the restriction or direction functions of the silkworm silk template. The analytic results showed that ZrO2-CeO2 exhibited a well-crystallized hierarchically interwoven hollow fiber structure with 16-28 μm in diameter. The grain size of the sample calcined at 800 ℃ was about 14 nm. Consequently, the interwoven meshwork at three dimensions is formed due to the direction of biotemplate. The action mechanism is summarily discussed here. It may bring the biomorphic ZrO2-CeO2 nanomaterials with hierarchical interwoven structures to more applications, such as catalysts.

Isolation and Screening of Strains for Silkworm Puparium Chitin Degradation and the Optimization of Chitinase Production by Response Surface Methodology

[Objective] The aim of this study was to optimize the conditions of chitinase-produce strains.[Method] A kind of screened chitinase-produce strain G-254 was habituated cultured,and then the single factor experiment was carried out to explore the effects of different carbon source,nitrogen source and inorganic salt on the activity of produced chitinase;the response surface test was used to determine the optimal conditions for chitinase production.[Result] The optimal conditions for chitinase production were：8% of glucose,5% of beef extract and 0.07% of MgSO4,and the activity of chitinase reached the maximal value（6.86 U）under these conditions.[Conclusion] The study improved the activity of chitinase produced by strain G-254 and provided good foundation for industrial production.

Metabolism of Hydrogen Peroxide in the Course of Embryonic Development in Silkworm

Metabolism of hydrogen peroxide in the course of embryonic development ofsilkworm (variety Guang) was determined by using colorimetric analysis and oxygen electrodemethod. The results indicated that: 1) In the course of fertilization (0-4 h after egg laying), thelevel of H_2O_2 content reached its peak value at 2.5 h of the developmental course and the activity ofsuperoxidase dismutase (SOD) was at higher level while the activity of catalase (CAT) at the lowestcorrespondingly; 2) The H_2O_2 content in embryo, in which the diapause of eggs was being relievedthrough treatment with hydrochloric acid on time in the course of embryonic development, wassignificantly higher than that of diapause eggs except the lower level showed in the embryo whenthe development of it went on for 168~216 h and the activity of SOD reached, lower and higher,tWo peaks in 72 and 168 h after egg-laying, respectively, and was significantly higher in late stagewhile the activity of CAT was shown with a stable level in the period of 72-192 h after egg-laying,and, after then, a rapid rising occurred in the embryo. The level of the CAT activity in embryowas shown significantly lower than that in diapause eggs in early period and higher in late stage ofegg development; 3) In the course of formation of diapause in eggs, the level of H_2O_2contentchanged smoothly and the activity of SOD changed vigorously in early period, and kept stable statelater; and the CAT activity increased gradually; while in the course of relief of diapause under ontime-treatment with hydrochloric acid, the level of H_2O_2 was significantly higher than that indiapause eggs and the SOD activity displayed a new peak value and significantly rose in later stage,while the activity of CAT in relieving embryo from diapause was signincantly lower than that indiapause eggs. Combining the results obtained in other researches with those from ours mentionedabove, we suggest that the metabolism of H_2O_2 might be of importance in the courses of formationand relief of diapause in silkworm eggs. Maybe the esterase A4 timer hypothesis and themicropylar barrier to oxygen hypothesis could be integrated for explanation of the course offormation and relief of diapause in silkworm eggs.

Analysis of the Production and Operation Status of Silkworm Eggs Producing Farms in China——Based on Questionnaire Survey to 136 Silkworm Eggs Producing Farms Nationwide

Through statistical analysis on questionnaire survey data from 136 silkworm eggs producing farms in China,it was concluded that most silkworm eggs producing farms had such problems as follows: old production facilities and poor production condition,heavy staff burden,weak scientific and technological support,small operation scale and heavy surplus production capacity; narrow silkworm eggs sale channels and stubborn regional segmentation,insufficient innovation and lack of technological support for diversified expansion. All these problems led to low labor productivity,low asset utilization rate and poor economic benefit in silkworm eggs producing farms in China. Except the provinces( regions) of Guangxi,Guangdong and Yunnan,the silkworm eggs producing farms in other provinces were generally at deficit condition. In the light of ownership,although silkworm eggs producing farms of different ownership systems were in the red,those of the private sector had the minimum deficit. Based on these findings,it is proposed to deepen system reformation of silkworm eggs producing farms,loosen control to price and circulation of silkworm eggs,implement large-scaled production and operation,speed up scientific and technological innovation,expand diversified development channels,and reinforce government support so as to increase production and operation benefits of silkworm eggs producing farms in China.

Toxicity Effects of Water Extract of Euphorbia fischeriana Steud.against Silkworm(Bombyx mori)

[Objective] Taking silkworm （Bombyx mori） as the test material, the research aimed to study the toxicity effects of Euphorbiafischeriana Steud. against the insects in Lepidoptera. [ Methods ] Water extract was prepared with fresh perennial E. fischer/ana Steud. , which was applied in silkworm with different methods including daubing the extract on silkworm back, soaking silkworm in the extract and feeding silkworm with the mulberry leaves daubed by the extract. [ Re- suits] Those silkworms whose backs were daubed with the water extract had no obvious poisoning symptom. The silkworms soaked for 1 and 3 min could be survived for about 7 d; the silkworms soaked for 5 min could be survived for about 2 d; and the silkworms soaked for 15 rain could be survived for 10 h at most. Those silk- worms fed with mulberry leaves daubed by water extract completely died after 2 d. [ Conduslons ] The water extract of E. fischer/ana Steud. had significant contact toxicity and stomach poisoning against silkworm.

Super-strong and Intrinsically Fluorescent Silkworm Silk from Carbon Nanodots Feeding

Fluorescent silk is fundamentally important for the development of future tissue engineering scaffolds.Despite great progress in the preparation of a variety of colored silks,fluorescent silk with enhanced mechanical properties has yet to be explored.In this study,we report on the fabrication of intrinsically super-strong fluorescent silk by feeding Bombyx mori silkworm carbon nanodots(CNDs).The CNDs were incorporated into silk fibroin,hindering the conformation transformation,confining crystallization,and inducing orientation of mesophase.The resultant silk exhibited super-strong mechanical properties with breaking strength of 521.9±82.7 MPa and breaking elongation of 19.2±4.3%,improvements of 55.1%and 53.6%,respectively,in comparison with regular silk.The CNDs-reinforced silk displayed intrinsic blue fluorescence when exposed to 405 nm laser and exhibited no cytotoxic effect on cells,suggesting that multi-functional silks would be potentially useful in bioimaging and other applications.

Computational prediction of microRNA genes in silkworm genome

MicroRNAs （miRNAs） constitute a novel, extensive class of small RNAs （-21 nucleotides）, and play important gene-regulation roles during growth and development in various organisms. Here we conducted a homology search to identify homologs of previously validated miRNAs from silkworm genome. We identified 24 potential miRNA genes, and gave each of them a name according to the common criteria. Interestingly, we found that a great number of newly identified miRNAs were conserved in silkworm and Drosophila, and family alignment revealed that miRNA families might possess single nucleotide polymorphisms, miRNA gene clusters and possible functions of complement miRNA pairs are discussed.

1-Deoxynojirimycin Content and Alfa-Glucosidase Inhibitory Activity and Heat Stability of 1-Deoxynojirimycin in Silkworm Powder

Silkworm powder containing 1-deoxynojirimycin (DNJ) has α-glucosidase inhibitory activity and is promising as a complementary and alternative medicine (CAM) agent in Japan. Silkworm powder produced in Korea was extracted with 75% ethanol. The extract was derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl), and DNJ-FMOC content was measured by HPLC. Then, alfa-glucosidase inhibition by silkworm and mulberry powder in pig liver crude enzyme was assayed using 4-nitrophenyl-alfa-D-glucopyranoside as a substrate. Silkworm powder DNJ content (0.39 to 0.58%) was higher than that in mulberry powder (0.08 to 0.12%). The alfa-glucosidase inhibitory activity of silkworm powder was more potent than that of mulberry leaves and green tea. Silkworm powder DNJ was stable upon heating to 121?C for up to 15 min.

Antihypertensive Properties on Spontaneously Hypertensive Rats of Peptide Hydrolysates from Silkworm Pupae Protein

Peptide hydrolysates of silkworm pupae protein with molecular weight of less than 5000 Da were prepared by ultrafiltration. The extracted peptide hydrolysates of silkworm pupae protein had inhibitory action on angiotensin-I-converting enzyme activity in vitro. The hydrolysates were orally administered to spontaneously hypertensive rats (SHR) in one period and long-term (four weeks). The results showed that the systolic blood pressure (SBP) of the treatment groups decreased in a dose-related manner. After one oral administration of silkworm protein hydrolysates with doses of 60, 20 and 5 mg/kg, the SBP of SHR decreased by 21.5, 13.8, and 9.0 mmHg in 1.5 h. After four weeks of the treatment in 80 mg/kg, the SBP decreased by 25 mmHg, with the antihypertensive activity close to 4 mg/kg of captopril;the SBP of the 40 mg/kg dose group also decreased by 17.5 mmHg. The peptide hydrolysate did not affect the SBP in normal, non-hypertensive rats in one period and long-term treatments. The acute toxicity research showed that the peptide hydrolysates were safe and without side effects. This research would be helpful in exploring the silkworm protein peptides as functional components for the antihypertension treatment.

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

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.

Role of juvenile hormone receptor Methoprene-tolerant 1 in silkworm larval brain development and domestication

The insect brain is the central part of the neurosecretory system,which controls morphology,physiology,and behavior during the insect’s lifecycle.Lepidoptera are holometabolous insects,and their brains develop during the larval period and metamorphosis into the adult form.As the only fully domesticated insect,the Lepidoptera silkworm Bombyx mori experienced changes in larval brain morphology and certain behaviors during the domestication process.Hormonal regulation in insects is a key factor in multiple processes.However,how juvenile hormone(JH)signals regulate brain development in Lepidoptera species,especially in the larval stage,remains elusive.We recently identified the JH receptor Methoprene tolerant 1(Met1)as a putative domestication gene.How artificial selection on Met1 impacts brain and behavioral domestication is another important issue addressing Darwin’s theory on domestication.Here,CRISPR/Cas9-mediated knockout of Bombyx Met1 caused developmental retardation in the brain,unlike precocious pupation of the cuticle.At the whole transcriptomelevel,theecdysteroid(20-hydroxyecdysone,20 E)signaling and downstream pathways were overactivated in the mutant cuticle but not in the brain.Pathways related to cell proliferation and specialization processes,such as extracellular matrix(ECM)-receptor interaction and tyrosine metabolism pathways,were suppressed in the brain.Molecular evolutionary analysis and in vitro assay identified an amino acid replacement located in a novel motif under positive selection in B.mori,which decreased transcriptional binding activity.The B.mori MET1 protein showed a changed structure and dynamic features,as well as a weakened co-expression gene network,compared with B.mandarina.Based on comparative transcriptomic analyses,we proposed a pathway downstream of JH signaling(i.e.,tyrosine metabolism pathway)that likely contributed to silkworm larval brain development and domestication and highlighted the importance of the biogenic amine system in larval evolution during silkworm domestication.

ANALYSISES OF GENE EFFECTS ON SOME COCOON CHARACTERS IN SILKWORM MORIL

1.Estimates of genetic variance and covariance for individual characters showed that cocoon weight was controlled by both gene direct effect and maternal effect;Cocoon shell weight was controlled by gene direct effect mainly;Cocoon shell ratio was mainly due to maternal effect and se x-linked effect.2.Estimates of genetic covariance components of each pair of ch aracters indicated that there were high significant positive relationships among most of genetic effects except maternal additive effect in cocoon weight and cocoon shell weight.There was a high positive relationship idirect effect between cocoon weight and cocoon shell ratio,but in cytop lasm effect and maternal effect were high negative relationships between them.Also there were high positive relationships in direct effect and maternal dominance between cocoon shell weight and cocoon shell ratio.3.Predictions of gene effects pointed out that sex-chromosome effects （Σi8=1Lii） on cocoon weight and cocoon shell weight both were lar ger positive values,that means cocoon weight and cocoon shell weight of females are generally higher than those of males in Silkworm. Standardized donmiance directions of direct effects on cocoon weight and cocoon shell weight were high positive values,but standardized domiance directieon of maternal effects on both of them were high negative values,it showed the two characters had larger heterosis in F1 generation and depressed in F2 generation.Sex-chromosome effect （Σi8=1Lii） on cocoon she11 ratio was high negative value,explaining that females usually have lower cocoon shell ratio than males.Standardized domiance directions of direct effect and maternal effect on cocoon shell ratio were not significant,predicting that the heterosis of cocoon shell ratio in F1 generation is not obvious comparing to cocoon weight and cocoon shell weight.

Analysis of Two-Dimensional Gel Electrophoresis Images of Protein from Posterior Silk Gland of Silkworm (Bombyx mori) on Day 1 and Day 4 in the 5th Instar Stage

The posterior silk gland （PSG） of silkworm is an important organ where fibroin is synthesized and secreted exclusively. Because fibroin constitutes 75-80% of the silk filament, the mechanism governing fibroin secretion, quality and yield of cocoon can be elucidated by the study on the PSG. Using two-dimensional gel electrophoresis （2-DE） and image analysis system, the changes in the protein composition in the PSG cell were investigated on the day 1 （D1） and day 4 （D4） in the 5th instar stage from five different strains of silkworm （Bombyx mori）. While differences at protein level between days and strains were far less than those observed at the gene level using EST analysis. The change trends in protein composition from D1 to D4 were diverse among the different strains. The results suggest that the secretion of fibroin is regulated by multiple proteins. The site of regulation and the proteins responsible for the regulation vary with the strain, which leads to differences between strains in the capacity of fibroin secretion in the PSG cell.

Transformation Efficiency of Sulfur for a Mulberry Leaf-Silkworm Cocoon System in the Lower-Middle Reaches of the Yangtze River, China

Cocoon samples were collected from fifty-two mulberry gardens with high, intermediate, and low silkworm cocoon productivities in the lower-middle reaches of the Yangtze River in the six China’s provinces of Jiangsu, Jiangxi, Anhui, Fujian, Hunan, and Hubei to determine the transformation efficiency of S from mulberry leaves to silkworm cocoons, and to evaluate the sulfur cycle (uptake and output) in the mulberry leaf-silkworm cocoon system with typical mulberry gardens in the lower-middle reaches of the Yangtze River in China. The transformation efficiency of sulfur (TES) from mulberry leaves into silkworm cocoons in the high-productivity mulberry gardens was significantly lower (P < 0.05) than that in the low-productivity gardens. For the high-productivity mulberry gardens the TES from mulberry leaves into the cocoon shells was significantly higher (P < 0.05) than that for low-yield mulberry gardens. Producing 1 kg dry cocoon in mulberry gardens required uptake of about 20 g S, however 1 kg of dry cocoon only removed about 4 g S. Therefore, recycling of these organic wastes with silkworm cultivation was important for sulfur balances.