Normal Silkworms Research Articles

Fast identification of the BmNPV infected silkworms by portable NIR spectroscopy and chemometrics

A convenient, low-cost, and rapid detection of BmNPV-infected silkworms is of great significance for the safety of the sericulture industry. In this study, a portable NIR system was used to collect the spectra of normal silkworms and the infected silkworms induced by the administration of Bombyx mori nuclear polyhedrosis virus (BmNPV). Different spectral pretreatment methods were applied, then principal component analysis (PCA), linear discriminant analysis (LDA), and partial least squares discriminant analysis (PLSDA) were used for the classification analysis. The results showed that PCA and LDA were unable to achieve the purpose. For the PLSDA calibration, after the pretreatment of SNV combining 2nd derivative, it had a high identification performance, and obtained low classification errors of 0.023, 0.033, and 0.030 for the calibration set, cross-validation set, and test set, respectively, with higher sensitivity and specificity. Therefore, the BmNPV-infected silkworms can be identified by portable NIR spectroscopy, which will effectively reduce losses for the sericulture industry.

Super silkworm cocoons constructed by multi-silkworm larvae: Promising composites with dense structures and excellent mechanical properties

A normal silkworm cocoon (NSC) with a unique nonwoven structure is usually spun by a single silkworm larva. Notably, there is a special Bombyx mori genetic resource that many (three or more) mature larvae tend to collectively spin into one cocoon, which was named “multi-silkworm cocoon” (“MSC”). However, the MSCs display loose structure and poor mechanical properties which limits their further application. In this study, a series of hybrid silkworm cocoons (HMSCs) are obtained by hybridizing “MSC” with a selected commercial silkworm strain successfully. The morphology, microstructures, and mechanical properties of cocoons constructed by one to three silkworm larvae were characterized and compared. The results indicated that about 48.3 % of silkworm larvae could create double and triple cocoons in the F1 generation of the silkworm hybrid, displaying robust fiber networks and dense structures. The mechanical characteristics of the HMSCs, including the tensile, peeling, compression, and needle penetration resistance properties, exceeded those of MSCs, showing significant application potential for high-performance bio-composites. This study provides a practical approach for obtaining silkworm cocoons with controllable structures and mechanical properties to develop and fabricate natural composite and biomimetic materials.

Vitellogenin receptor transports the 30K protein LP1 without cell-penetrating peptide, into the oocytes of the silkworm, Bombyx mori.

Vitellogenin receptors (VgRs) transport vitellogenin (Vg) into oocytes, thereby promoting egg growth and embryonic development. VgRs recognize and transport multiple ligands in oviparous animals, but their role in insects is rarely reported. In this study, we investigated whether Bombyx mori VgR (BmVgR) binds and transports lipoprotein-1 (BmLP1) and lipoprotein-7 (BmLP7) of the 30kDa lipoproteins (30K proteins), which are essential for egg formation and embryonic development in B. mori. Protein sequence analysis showed BmLP7, similar to reported lipoprotein-3 (BmLP3), contains the cell-penetrating peptides and Cysteine position, while BmLP1 has not. Assays using Spodoptera frugiperda ovary cells (sf9) indicated the direct entry of BmLP7 into the cells, whereas BmLP1 failed to enter. However, co-immunoprecipitation (Co-IP) assays indicated that BmVgR could bind BmLP1. Western blotting and immunofluorescence assays further revealed that over-expressed BmVgR could transport BmLP1 into sf9 cells. Co-IP assays showed that SE11C (comprising LBD1+EGF1+OTC domains of BmVgR) or SE22C (comprising LBD2+EGF2+OTC domains of BmVgR) could bind BmLP1. Over-expressed SE11C or SE22C could also transport BmLP1 into sf9 cells. Western blotting revealed that the ability of SE11C to transport BmLP1 might be stronger than that of SE22C. In the vit mutant with BmVgR gene mutation (vit/vit), SDS-PAGE and western blotting showed the content of BmLP1 in the ovary, like BmVg, was lower than that in the normal silkworm. When transgenic with hsp70 promoter over-expressed BmVgR in the vit mutant, we found that the phenotype of the vit mutant was partly rescued after heat treatment. And contents of BmLP1 and BmVg in vit mutant over-expressed BmVgR were higher than in the vit mutant. We conclude that BmVgR and its two repeat domains could bind and transport BmLP1 into the oocytes of the silkworm, besides BmVg. These results will provide a reference for studying the molecular mechanism of VgR transporting ligands in insects.

Impact of cytoplasmic polyhedrosis virus on Oak Tasar Silkworm, Antheraea proylei Jolly (Insecta: Lepidoptera: Saturniidae)

Among the diseases causing microbial organisms, cypovirus (CVP) has been found to be very important from the view point of tasar silk production. In this article, impact of CPV infection has been studied in terms of types of haemocytes present in the oak tasar silkworm; changes in the haemocytes as a result of cypovirus (CPV) infection; variation in total haemocyte count (THC) between normal and cypovirus infected larvae; changes that take place in the mid gut due to virus infection; food consumption pattern between the normal and virus infected worms. These aspects are compared between III and IV instar larvae of both normal and virus infected silkworms.

Genetic analysis and transcriptome analysis of the mini mutant of the silkworm, Bombyx mori.

The expression levels of some intrinsic genes, protease activity, and regulation of signaling pathways were distinct during different growth and development stages in the silkworm, Bombyx mori. The silkworm mutant mini was discovered from the normal silkworm strain S8V, and the body-size of themini mutant was smaller than the wild-type from the second-instar and the difference became more significant in the following stages. In this study, genetic analysis of mini mutant showed that mini mutant was controlled by a single recessive gene, manifested as homozygous lethal. Then, the transcriptome analysis of the mini mutant indicated that 2944 differentially expressed genes (DEGs) were identified from the silkworm in the 48 h of the second-instar, of which 1638 genes in the mini mutants were upregulated and 1306 genes were downregulated. These DEGs were mainly distributed in the biological process, cellular component, and molecular function. The functional annotation based on the KEGG database showed that these genes were mainly clustered in metabolic pathways, fatty acid metabolism pathways, ribosome biogenesis in eukaryotes, and so on. Further analysis indicated that some genes involved in the growth and metabolism including enzyme genes, juvenile hormone, and ecdysone exhibited different transcriptional levels. These results provided new experimental evidence regarding the mechanism of the underlying formation of mini mutants.

P25 Gene Knockout Contributes to Human Epidermal Growth Factor Production in Transgenic Silkworms

Transgenic silkworm expression systems have been applied for producing various recombinant proteins. Knocking out or downregulating an endogenous silk protein is considered a viable strategy for improving the ability of transgenic expression systems to produce exogenous proteins. Here, we report the expression of human epidermal growth factor (hEGF) in a P25 gene knockout silkworm. The hEGF gene regulated by the P25 gene promoter was integrated into a silkworm’s genome. Five transgenic positive silkworm lineages were generated with different insertion sites on silkworm chromosomes and the ability to synthesize and secrete proteins into cocoons. Then, a cross-strategy was used to produce transgenic silkworms with a P25 gene knockout background. The results of the protein analysis showed that the loss of an endogenous P25 protein can increase the hEGF production to about 2.2-fold more than normal silkworms. Compared to those of transgenic silkworms with wild type (non-knockout) background, the morphology and secondary structure of cocoon silks were barely changed in transgenic silkworms with a P25 gene knockout background, indicating their similar physical properties of cocoon silks. In conclusion, P25 gene knockout silkworms may become an efficient bioreactor for the production of exogenous proteins and a promising tool for producing various protein-containing silk biomaterials.

Toxicological evaluation of transgenic silkworms.

Safety of transgenic silkworms must be evaluated before their commercial application. We assessed subacute toxicity using a 28-day feeding study in rats. Eighty rats were evenly allocated into four groups, with each group containing 10 male and 10 female rats. Rats of three groups were fed dried transgenic silkworm H19.9A pupae with overexpressed endogenous Bmhsp19.9, transgenic silkworm A4SOR pupae with overexpressed exogenous SOR, or normal silkworm pupae at a dose of 3.0g/kg/day, respectively. The fourth group served as a normal feeding control. The body weight, feed consumption, hematology response variables, serum biochemical parameters, organ weights, gross necropsy, and histopathologic of animals were evaluated. No mortality, adverse effects, or major differences in the evaluated parameters were observed in the groups fed transgenic pupae in comparison with the control, suggesting that transgenic silkworms are toxicologically equivalent to normal silkworms and are safe for consumption in rats.

Potential of transferring transgenic DNA from silkworm to chicken

Safety assessment must be conducted before the commercial release of transgenic silkworms. This study was conducted to assess the potential of transferring transgenic DNA from silkworms to other organisms. One hundred hatched male chickens were evenly assigned into 4 groups (T1–4). Groups T1–3 were fed transgenic silkworms P3+5UI with enhanced green fluorescent protein DNA (EGFP) inserted, A4SOR with superoxide reductase DNA (SOR) inserted, and normal silkworm, respectively. Each chicken was fed one silkworm larva every day for 3 weeks. T4 was the normal feeding control. Twenty chickens were randomly selected from each treatment for sacrifice at 22 days of age. The serum was collected individually for biochemical examination, revealing no difference in the analyzed serum parameters between T4 and T1–3. DNA from the duodenum, jejunum, ileum, liver, kidney, and jejunal digesta was extracted for PCR analysis of EGFP, SOR, silkworm housekeeping gene TIF-4A, and chicken ovalbumin gene. No transgenic DNA or TIF-4A was detected in the digesta and tissues of chickens. The same results were observed in chicken upon increasing the amount and frequency of feeding transgenic silkworms, suggesting that the transgenic DNA from silkworms was degraded in the digestive tract and not transferred into the tissues of chicken. This study revealed that transferr recombinant DNA from transgenic silkworm to another organism is unlikely.

Silkworms with Spider Silklike Fibers Using Synthetic Silkworm Chow Containing Calcium Lignosulfonate, Carbon Nanotubes, and Graphene.

Silkworm silk has become increasingly relevant for material applications. However, the industry as a whole is retracting because of problems with mass production. One of the key problems is the inconsistent properties of the silk. A means by which to improve the silk material properties is through enhanced sericulture techniques. One possible technique is altering the feed of the silkworms to include single-wall carbon nanotubes (SWNTs) or graphene (GR). Recently published results have demonstrated substantial improvement in fiber mechanical properties. However, the effect of the surfactant used to incorporate those materials into the feed on the fiber mechanical properties in comparison to normal silkworm silk has not been studied or reported. Thus, the total effect of feeding the SWNT and GR in the presence of surfactants on silkworms is not understood. Our study focuses on the surfactant [calcium lignosulfonate (LGS)] and demonstrates that it alone results in appreciable improvement of mechanical properties in comparison to nontreated silkworm silk. Furthermore, our study demonstrates that mixing the LGS, SWNT, and GR directly into the artificial diet of silkworms yields improved mechanical properties without decline below the control silk at high doses of SWNT or GR. Combined, we present evidence that mixing surfactants, in this case LGS, directly with the diet of silkworms creates a high-quality fiber product that can exceed 1 GPa in tensile strength. With the addition of nanocarbons, either SWNT or GR, the improvement is even greater and consistently surpasses control fibers. However, feeding LGS alone is a more economical and practical choice to consistently improve the mechanical properties of silkworm fiber.

Proteomic Analysis of Larval Integument in a Dominant Obese Translucent (Obs) Silkworm Mutant.

The dominant obese translucent (Obs) mutant of the silkworm (Bombyx mori) results in a short and stout larval body, translucent phenotype, and abnormal pigmentation in the integument. The Obs mutant also displays deficiency in ecdysis and metamorphosis. In the present study, to gain an understanding of multiple Obs phenotypes, we investigated the phenotypes of Obs and performed a comparative analysis of the larval integument proteomes of Obs and normal silkworms. The phenotypic analysis revealed that the Obs larvae were indeed short and fat, and that chitin and uric acid content were lower but melanin content was higher in the Obs mutant. Proteomic analysis revealed that 244 proteins were significantly differentially expressed between Obs and normal silkworms, some of which were involved in uric acid metabolism and melanin pigmentation. Twenty-six proteins were annotated as cuticular proteins, including RR motif-rich cuticular proteins (CPR), glycine-rich cuticular protein (CPG), hypothetical cuticular protein (CPH), cuticular protein analogous to peritrophins (CPAPs), and the chitin_bind_3 motif proteins, and accounted for over 84% of the abundance of the total significantly differentially expressed proteins. Moreover, 22 of the 26 cuticular proteins were downregulated in the Obs mutant. Comparative proteomic analysis suggested that the multiple phenotypes of the Obs mutant might be related to changes in the expression of proteins that participate in cuticular formation, uric acid metabolism, and melanin pigmentation. These results could lay a basis for further identification of the gene responsible for the Obs mutant. The data have been deposited to ProteomeXchange with identifier PXD010998.

Natural Silkworm Cocoon Composites with High Strength and Stiffness Constructed in Confined Cocooning Space.

In this study, using round paper tubes (PTs) and rectangular cardboard boxes (CBs) as external constraints to control the size of the cocooning space, we fabricated a series of modified silkworm cocoons (PT cocoons and CB cocoons). Their microstructures, morphologies, compositions, and mechanical properties were characterized and compared with normal silkworm cocoons. These two kinds of modified silkworm cocoons exhibit dense and homogeneous layer structures. Tensile test results indicate that above a size limit of cocooning space, their tensile strengths, Young’s moduli, and strain energy densities increase with the decrease in cocooning space. Especially in comparison with the normal cocoons, the tensile strength and Young’s modulus of the PT-14 cocoon increase by 44% and 100%, respectively. Meanwhile, PT cocoons and CB cocoons, except PT-12, also possess better peeling resistance than normal cocoons. Owing to the dense structure and low porosity, the modified cocoons form robust fiber networks that result in high strength and toughness. This study provides a green and efficient method to fabricate mechanically enhanced silkworm cocoons with special shapes and dense layer structures. The method can be easily subjected to further modification processes and has potential applications in the production of high-performance green cocoon composites and biomimetic materials.

Transposable Element Bm1645 is a Source of BmAGO2-associated Small RNAs that affect its expression in Bombyx mori

BackgroundA transposable element (TE) is a DNA fragment that can change its position within a genome. Transposable elements play important roles in maintaining the stability and diversity of organisms by transposition. Recent studies have shown that approximately half of the genes in Bombyx mori are TEs.ResultsWe systematically identified and analyzed the BmAGO2-associated TEs, which exceed 100 in the B. mori genome. Additionally, we also mapped the small RNAs associated with BmAGO2 in B.mori. The transposon Bm1645 is the most abundant TE associated with BmAGO2, and Bm1645-derived small RNAs represent a small RNA pool. We determined the expression patterns of several Bm1645-derived small RNAs by northern blotting, and the results showed there was differential expression of multiple small RNAs in normal and BmNPV-infected BmN cells and silkworms from various developmental stages. We confirmed that four TE-siRNAs could bind to BmAGO2 using EMSA and also validated the recognition sites of these four TE-siRNAs in Bm1645 by dual-luciferase reporter assays. Furthermore, qRT-PCR analysis revealed the overexpression of the four TE-siRNAs could downregulate the expression of Bm1645 in BmN cells, and the transcription of Bm1645 was upregulated by the downregulation of BmAGO2.ConclusionsOur results suggest Bm1645 functions as a source of small RNAs pool and this pool can produce many BmAGO2-associated small RNAs that regulate TE’s expression.

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

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-em) 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-em mutant to 360kb on chromosome 10 using 2287 F2 individuals. Using expression analysis and RNA interference, the best l-em candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~1.9kb region from the 3′ untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a >100kb DNA fragment in the l-em 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-em 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-em mutant. These results indicate that BmEP80 is responsible for the l-em mutation.

Genetic Analysis of the Electrophysiological Response to Salicin, a Bitter Substance, in a Polyphagous Strain of the Silkworm Bombyx mori

Sawa-J is a polyphagous silkworm (Bombyx mori L.) strain that eats various plant leaves that normal silkworms do not. The feeding preference behavior of Sawa-J is controlled by one major recessive gene(s) on the polyphagous (pph) locus, and several minor genes; moreover, its deterrent cells possess low sensitivity to some bitter substances including salicin. To clarify whether taste sensitivity is controlled by the pph locus, we conducted a genetic analysis of the electrophysiological characteristics of the taste response using the polyphagous strain Sawa-J·lem, in which pph is linked to the visible larval marker lemon (lem) on the third chromosome, and the normal strain Daiankyo, in which the wild-type gene of pph (+pph) is marked with Zebra (Ze). Maxillary taste neurons of the two strains had similar dose–response relationships for sucrose, inositol, and strychnine nitrate, but the deterrent cell of Sawa-J·lem showed a remarkably low sensitivity to salicin. The F1 generation of the two strains had characteristics similar to the Daiankyo strain, consistent with the idea that pph is recessive. In the BF1 progeny between F1 females and Sawa-J·lem males where no crossing-over occurs, the lem and Ze phenotypes corresponded to different electrophysiological reactions to 25 mM salicin, indicating that the gene responsible for taste sensitivity to salicin is located on the same chromosome as the lem and Ze genes. The normal and weak reactions to 25 mM salicin were segregated in crossover-type larvae of the BF1 progeny produced by a reciprocal cross, and the recombination frequency agreed well with the theoretical ratio for the loci of lem, pph, and Ze on the standard linkage map. These results indicate that taste sensitivity to salicin is controlled by the gene(s) on the pph locus.

Effects of BmKIT3R Gene Transfer on Pupal Development of Bombyx mori Linnaeus Using a Gal4/UAS Binary Transgenic System

The pupal stage of the silkworm Bombyx mori Linnaeus lasts for approximately two weeks. However, prolongation of pupal duration would reduce the labor required to process and dry fresh cocoons. This study investigated the effects of BmKIT(3)(R) gene (from the Chinese scorpion Buthus martensii Karsch) transfer on the pupal development of B. mori using a Gal4/UAS binary transgenic system. Gal4 driven by a pupa-specific promoter BmWCP4 (from a B. mori wing-cuticle protein gene) or PDP (from a B. mori cocoonase gene), and BmKIT(3)(R) driven by a UAS cis-acting element were used to construct novel piggyBac-derived plasmids containing a neomycin-resistance gene (neo) controlled by the Bombyx mori nucleopolyhedrovirus (BmNPV) ie-1 (immediate-early gene) promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes, and gene transfer was verified by polymerase chain reaction and dot-blot hybridization. The larval development of the hybrid progeny of Gal4- and UAS-transgenic silkworms was similar to that of normal silkworms, but some pupae failed to metamorphose into moths, and the development of surviving pupae was arrested as a result of BmKIT(3)(R) expression. Moreover, Gal4 driven by the BmWCP4 promoter delayed pupal development more effectively than that driven by the PDP promoter in the Gal4/UAS binary transgenic system. Pupal durations of hybrid transgenic silkworm progeny with BmWCP4 and PDP promoters were approximately 5, 2, and 4 days longer, respectively, compared to corresponding normal silkworms, BmWCP4/Gal4, and UAS/BmKIT(3)(R) transgenic silkworms, respectively. These results suggest new avenues of research for prolonging the pupal duration of silkworms.

Molecular Cloning and Characterization of a Putative cDNA Encoding Endoglucanase IV from Trichoderma Viride and its Expression in Bombyx Mori

The development of cellulase production technology has greatly contributed to the successful use of cellulosic materials as renewable carbon sources. In this study, a putative endoglucanase IV (EG IV) complementary DNA was cloned from the mycelium of a strain of the filamentous fungus Trichoderma viride using a PCR-based exon-splicing method and expressed in both a silkworm BmN cell line and in silkworm larvae. Western blot analysis detected a band of 42 kDa in BmN cells after infection with a recombinant mBacmid/BmNPV/EG IV baculovirus. Sequence alignment analysis of the T. viride EG IV gene showed two domains that were highly conserved with glycosyl hydrolases and a funga-type cellulose-binding domain. Analysis of variance showed that silkworms infected with recombinant baculoviruses exhibited significantly higher enzyme activity that was 48.84% higher than silkworms infected with blank baculoviruses and 46.61% higher than normal silkworms. The expressed bioactive EG IV was also stable at the pH range from 5.0 to 10.0. The availability of large quantities of bioactive EG IV in silkworm provided a possibility to produce cellulase transgenic silkworm, which express bioactive cellulase specially in its digestive tract and improve its metabolism efficiency of mulberry leaves. Its application in the sericulture industry may be very promising.

Effects of egt gene transfer on the development of Bombyx mori

This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein ( hsp) 23.7 promoter, with a neomycin-resistance gene ( neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene ( gfp) under the control of the B. mori actin 3 ( A3) promoter was constructed. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths.

Synnemata Production Using Silkworm Variety, Female Yangwonjam by Isaria tenuipes

This study was conducted to confirm the possible use of female Yangwonjam as a host for synnemata production of Isaria tenuipes in eight local areas in Korea. Silkworm pupation rate, infection rate and synnemata characteristics of I. tenuipes were examined. Normal silkworms had a higher pupation rate than silkworms inoculated with I. tenuipes. The pupae survival percentage of normal silkworm in cocoons was 92.5~97.6%, whereas it ranged from 91.1~95.6% in silkworms sprayed with I. tenuipes. Female Yangwonjam showed the highest survival percentage at 97.6% among the silkworm varieties tested. I. tenuipes infection rate of larvae of 5th instar newly-exuviated silkworms was 89.2~90.7% in the spring rearing season and 98.2~99.3% in the autumn rearing season. Synnemata production of I. tenuipes was excellent in female Yangwonjam with an incidence rate of 98.0% followed by male Yangwonjam (94.1%) and Baegokjam (93.3%) in the spring rearing season. Synnemata living weight ranged from 1.44~0.94 g in the spring rearing season. The female Yangwonjam had the heaviest synnemata weight (1.44 g) in the spring rearing season. The synnemata of I. tenuipes produced on pupae were white or milky-white in color, and were similar in shape and color to wild synnemata collected in Korea.

Heterologous Expression Characteristics of Trichoderma viride Endoglucanase V in the Silkworm, Bombyx mori L.

Efficient degradation of cellulose needs a synergistic reaction of the cellulolytic enzymes, which include exoglucanases, endoglucanases, and β-1,4-glucosidase. In this study, we used an improved Bac-to-Bac/BmNPV baculovirus expression system, which lacks the virus-encoded chitinase cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV), to express the endoglucanase V (EG V) gene from Trichoderma viride in silkworm BmN cells and silkworm larvae, and analyzed the characteristics of the recombinant enzyme in silkworm larvae. The result showed that an around 36-kDa protein was visualized in BmN cells at 48 h after the second-generation recombinant mBacmid/BmNPV/EG V baculovirus infection. The crude enzyme extract from the recombinant baculoviruses-infected silkworms exhibited a significant maximum activity at the environmental condition of pH 5.0 and a temperature of 50 °C, and increased 39.86% and 37.76% compared with that from blank mBacmid/BmNPV baculovirus-infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 10.0 and at temperature range from 40 to 60 °C. The availability of large quantities of EG V that the silkworm provides might greatly facilitate the future research and the potential application in industries.

Synnemata Production by Isaria tenuipes using Colored Cocoon Silkworm, Golden Silk

This study was conducted to test the use of colored cocoon silkworm, Golden Silk, as a host for the fruiting body production of Cordyceps mushroom. Golden Silk variety had a shorter larval period than Yangwonjam at <TEX>$25.3^{\circ}C$</TEX> and 78.5% RH: The average larval period of the Golden Silk was 24 days and 7 hr, and that of the Yangwonjam was 24 days and 15 hr. In the infection test of I. tenuipes on silkworm, the larval period of normal silkworms was longer than the silkworms inoculated with I. tenuipes in both Golden Silk and Yangwonjam. The pupae survival percentage of Golden Silk was 94.4%, whereas it was 91.9% in Yangwonjam. Golden Silk had a higher pupation rate than Yangwonjam. The pupation rate of normal silkworms was about 5% higher than that of silkworms inoculated with I. tenuipes. Infection rate of I. tenuipes into larvae of 5th instar newly exuviated silkworm was 93.8% in Golden Silk, and 96.9% in Yangwonjam. Synnemata production of I. tenuipes was better in Yangwonjam with an incidence rate of 99.8% than Golden Silk with 98.6%. The average synnemata weight of Golden Silk was 1.12 g and that of Yangwonjam 1.29 g. Golden Silk had a lower synnemata weight than Yangwonjam. But no significant difference was observed in synnemata weight varieties. The synnemata of I. tenuipes produced on pupae were white or milky-white in color, and similar in shape and color to wild synnemata collected in Korea.