Silk Gland Research Articles

Using the multi-omics approach to reveal the silk composition in Plectrocnemia conspersa.

Similar to Lepidoptera, the larvae of Trichoptera are also capable of producing silk. Plectrocnemia conspersa, a predatory species belonging to the suborder Annulipalpia, builds massive silken retreats with preycapturing nets. In this study, we describe the silk glands of P. conspersa and use the multi-omics methods to obtain a complete picture of the fiber composition. A combination of silk gland-specific transcriptome and proteomic analyses of the spun-out fibers yielded 27 significant candidates whose full-length sequences and gene structures were retrieved from the publicly available genome database. About one-third of the candidates were completely novel proteins for which there are no described homologs, including a group of five pseudofibroins, proteins with a composition similar to fibroin heavy chain. The rest were homologs of lepidopteran silk proteins, although some had a larger number of paralogs. On the other hand, P. conspersa fibers lacked some proteins that are regular components in moth silk. In summary, the multi-omics approach provides an opportunity to compare the overall composition of silk with other insect species. A sufficient number of such studies will make it possible to distinguish between the basic components of all silks and the proteins that represent the adaptation of the fibers for specific purposes or environments.

Whole Transcriptome-Based Study to Speculate upon the Silkworm Yellow Blood Inhibitor (I) Gene and Analyze the miRNA-Mediated Gene Regulatory Network

White cocoon is developed and used as a natural fiber, and different silkworm strains have different cocoon colors. Natural-colored cocoons are preferred by people, however, the cocoon color mainly settles on sericin and it basically falls off after reeling. Currently, there are no varieties applied to production due to the formation mechanism of cocoon color is not clear. The formation of cocoon color involves multiple gene regulations. Previous studies have shown that the main genes regulating cocoon traits are the yellow blood (Y) gene, yellow blood inhibitor (I) gene, and yellow cocoon (C) gene. Among them, the products of the Y gene and C gene have been studied, but the I gene is still unclear. In this study, the midgut tissues of the yellow (NB) and the white (306) cocoon silkworm were analyzed by whole transcriptome sequencing. The results showed that there are 1639 DE-circRNAs, 70 DE-miRNAs, and 3225 DE-mRNAs, including 1785 up-regulated genes and 1440 down-regulated genes. GO and KEGG annotation results indicated that DE-mRNAs are mainly involved in intracellular transport, signal transduction, lipid transport, and metabolic processes. Two key genes, KWMTBOMO10339 and KWMTBOMO16553, were screened out according to the annotation results, which were involved in amino acid transport and ion exchange function, respectively. The interaction analysis between ncRNA and target genes showed that there were five miRNAs regulating these two genes. The qPCR analysis showed that the I gene was down-regulated, and the miRNA expression profiles were most up-regulated. Therefore, during the yellow and white cocoon formation, KWMTBOMO10339 and KWMTBOMO16553 may be regulated by miRNA, resulting in the non-expression of KWMTBOMO10339 and KWMTBOMO16553 in yellow cocoon silkworm, and the pigment molecules can enter hemolymph from the midgut to form yellow blood, then transport to the middle silk gland to finally form yellow cocoons.

Novel silkworm (Bombyx mori) sulfotransferase swSULT ST3 is involved in metabolism of polyphenols from mulberry leaves.

Polyphenols in plants are important for defense responses against microorganisms, insect herbivory, and control of feeding. Owing to their antioxidant, anti-cancer, and anti-inflammatory activities, their importance in human nutrition has been acknowledged. However, metabolism of polyphenols derived from mulberry leaves in silkworms (Bombyx mori) remains unclear. Sulfotransferases (SULT) are involved in the metabolism of xenobiotics and endogenous compounds. The purpose of this study is to investigate the metabolic mechanism of polyphenols mediated by B. mori SULT. Here, we identified a novel SULT in silkworms (herein, swSULT ST3). Recombinant swSULT ST3 overexpressed in Escherichia coli effectively sulfated polyphenols present in mulberry leaves. swSULT ST3 showed high specific activity toward genistein among the polyphenols. Genistein-7-sulfate was produced by the activity of swSULT ST3. Higher expression of swSULT ST3 mRNA was observed in the midgut and fat body than in the hemocytes, testis, ovary, and silk gland. Polyphenols inhibited the aldo-keto reductase detoxification of reactive aldehydes from mulberry leaves, and the most noticeable inhibition was observed with genistein. Our results suggest that swSULT ST3 plays a role in the detoxification of polyphenols, including genistein, and contributes to the effects of aldo-keto reductase in the midgut of silkworms. This study provides new insight into the functions of SULTs and the molecular mechanism responsible for host plant selection in lepidopteran insects.

Global Profiling of Genes Expressed in the Silk Glands of Philippine-Reared Mulberry Silkworms (Bombyx mori).

Simple SummaryThe sericulture industry in the Philippines remains to be enhanced through efforts to improve local strains of the silkworm Bombyx mori, such as selective hybridization. Genetic trait markers could help improve breeding; however, there is a scarcity of genetic data on local strains. This study aims to bridge this gap by analyzing the gene expression profiles of Philippine-reared silkworm strains through next-generation sequencing. Transcriptome assemblies were generated, and gene expressions were compared between silkworm strains reared in different temperatures, revealing genes that may be important for development in a tropical environment. This study is the first to provide transcriptome datasets for the Philippine-reared B. mori strains, which may serve as a resource for improving local strains and increasing silk production.RNA sequencing was used to assemble transcriptome data for Philippine-reared silkworm and compare gene expression profiles of strains reared in high- and low-temperature environments. RNA was isolated from the silk glands of fifth instar larvae and mRNA-enriched libraries were sequenced using Illumina NextSeq 500. Transcriptome reads were assembled using reference-based and de novo assemblers, and assemblies were evaluated using different metrics for transcriptome quality, including the read mapping rate, E90N50, RSEM-eval, and the presence of single-copy orthologs. All transcriptome assemblies were able to reconstruct >40,000 transcripts. Differential expression analysis found 476 differentially expressed genes (DEGs; 222 upregulated, 254 downregulated) in strains reared in different temperatures. Among the top DEGs were myrosinase, heat shock proteins, serine protease inhibitors, dehydrogenases, and regulators of the juvenile hormone. Validation of some of the top DEGs using qPCR supported the findings of the in silico analysis. GO term enrichment analysis reveals an overrepresentation of GO terms related to nucleotide metabolism and biosynthesis, lipid and carbohydrate metabolic processes, regulation of transcription, nucleotide binding, protein binding, metal binding, catalytic activity, oxidoreductase activity, and hydrolase activity. The data provided here will serve as a resource for improving local strains and increasing silk production of Philippine-reared B. mori strains.

Diet and ontogeny drastically alter the larval microbiome of the invertebrate model Galleria mellonella.

Larvae of the greater wax moth (Galleria mellonella) are an emerging animal model to study the innate immune response and biodegradation of plastic polymers. Both of these complex biological processes are likely impacted by the plasticity of host-microbe interactions, which remains understudied in lepidopterans. Consequently, we carried out 16S rRNA sequencing to explore the effect diet (natural, artificial) has on the bacterial assemblages of G. mellonella in different tissues (gut, fat bodies, silk glands) throughout development (eggs, six instar stages, adults). The microbiome was rich in diversity, with Proteobacteria and Firmicutes being the most represented phyla. Contrary to other lepidopterans, G. mellonella appears to possess a resident microbiome dominated by Ralstonia. As larvae progress through development, the bacterial assemblages become increasingly shaped by the caterpillar's diet. In particular, a number of bacteria genera widely associated with the G. mellonella microbiome (e.g., Enterococcus and Enterbacter) were significantly enriched on an artificial diet. Overall, these results indicate that the G. mellonella microbiome is not as simplistic and homogenous as previously described. Rather, its bacterial communities are drastically affected by both diet and ontogeny, which should be taken into consideration in future studies planning to use G. mellonella as model species.

Bombyx mori Vps13d is a key gene affecting silk yield

Bombyx mori is an important economic insect, its economic value mainly reflected in the silk yield. The major functional genes affecting the silk yield of B. mori have not been determined yet. Bombyx mori vacuolar protein sorting-associated protein 13d (BmVps13d) has been identified, but its function is not reported. In this study, BmVps13d protein shared 30.84% and 34.35% identity with that of in Drosophila melanogaster and Homo. sapiens, respectively. The expressions of BmVps13d were significantly higher in the midgut and silk gland of JS (high silk yield) than in that of L10 (low silk yield). An insertion of 9 bp nucleotides and two deficiencies of adenine ribonucleotides in the putative promoter region of BmVps13d gene in L10 resulted in the decline of promoter activity was confirmed using dual luciferase assay. Finally, the functions of BmVps13d in B. mori were studied using the CRISPR/Cas9 system, and the mutation of BmVps13d resulted in a 24.7% decline in weight of larvae, as well as a 27.1% (female) decline and a 11.8% (male) decline in the silk yield. This study provides a foundation for studying the molecular mechanism of silk yield and breeding the silkworm with high silk yield.

Bioengineered Silkworm for Producing Cocoons with High Fibroin Content for Regenerated Fibroin Biomaterial-Based Applications.

Silk fibroin exhibits high biocompatibility and biodegradability, making it a versatile biomaterial for medical applications. However, contaminated silkworm-derived substances in remnant sericin from the filature and degumming process can result in undesired immune reactions and silk allergy, limiting the widespread use of fibroin. Here, we established transgenic silkworms with modified middle silk glands, in which sericin expression was repressed by the ectopic expression of cabbage butterfly-derived cytotoxin pierisin-1A, to produce cocoons composed solely of fibroin. Intact, nondegraded fibroin can be prepared from the transgenic cocoons without the need for sericin removal by the filature and degumming steps that cause fibroin degradation. A wide-angle X-ray diffraction analysis revealed low crystallinity in the transgenic cocoons. However, nondegraded fibroin obtained from transgenic cocoons enabled the formation of fibroin sponges with varying densities by using 1–5% (v/v) alcohol. The effective chondrogenic differentiation of ATDC5 cells was induced following their cultivation on substrates coated with intact fibroin. Our results showed that intact, allergen-free fibroin can be obtained from transgenic cocoons without the need for sericin removal, providing a method to produce fibroin-based materials with high biocompatibility for biomedical uses.

Mulberry silkworm (Bombyx mori L.) hybrids in Uzbek selection and, viability, cocoon productivity, and reproductive indicators in summer and fall seasons

The demand for silk products with high metric number (thin) in the world market is growing every year. The main demand of cocoon processing enterprises in China, India, South Korea, Italy, France and Japan, the leading countries producing silk products, is focused on silk yarns of type 3A, 4A, and 5A. Due to the lack of special mulberries for re-worm feeding, summer-fall worm feeding usually begins after the mulberry trees and mulberries, whose seedlings have been cut down for spring worm feeding, have harvested enough leaf produce. Nutritional degradation of the leaves to worms and hot weather together make it difficult for the worms to eat and digest nutrients during repeated feeding, and the worms slow down the synthesis of silk substance in the silk gland. Therefore, in summer and fall, small, silky-thin cocoons are obtained. At the same time, it is important to try and select breeds and hybrids that are resistant to hot weather conditions and create new breeds and hybrids suitable for the summer and fall seasons. This article describes the results of research conducted on the testing of silkworm breeds and hybrids in the conditions of repeated worm feeding and the selection of the required level in terms of productivity characteristics. In separate experiments conducted during the summer and fall seasons of 2016-2018, the worm viability, cocoon productivity, and germination traits of each breed, system, and breeding hybrid were studied comparatively.

Effect of Micronutrient Supplemented Mulberry Leaves on Larvae of Silk Worm Bombyx mori

The present study explores the effect of micronutrient supplemented mulberry leaves on PM×CSR2 hybrid variety of Bombyx mori L. larvae. The field experiment with twelve treatments including a control (T0 to T11), replicated thrice, was supplemented with the desired quantity of the respective micronutrient in individual or in combination. Twenty early B. mori fourth and fifth instar larvae/ replication were fed with the 5-6 fully mature mulberry leaves (T0 to T11) twice a day (morning and evening). The results showed that food consumption increased to a maximum of 31.56 and 22.98%, and assimilation increased by 51.38 and 34.56% over control (4th and 5th instar, respectively) with treatment T9 (CuSO4 15 kg/ ha +ZnSO4 15 kg/ ha + FeSO4 30 kg/ ha). With the same treatment, total growth increased by 94.95 and65.67%, and approximate digestibility by 15.14 and 9.33%; tissue growth efficiency also increased by 65.35and 49.61% with this treatment, while maximum ecological growth efficiency increased by 49.58% and52.0% over control in T11 (CuSO4 25 kg/ ha + ZnSO4 25 kg/ ha + FeSO4 25 kg/ ha). Larval duration wasmaximum with T1 (FeSO4 10 kg/ ha alone) (7.63± 19.16 days) as against when fed with the control (T0).Larval length increased by 18.0 and 18.51% in T9, with the larval width increasing by 50.0% in morethan one treatment. Maximum chawki larval weight and larval weight increased by 16.78 and 29.44%,while maximum silk gland weight was 2.14± 0.05g in T9.

Spider Origins: a Palaeontological Perspective

Spiders (Araneae) can be placed within a series of nested clades within Arachnida. Arachnopulmonata encompasses all arachnids with book lungs. Pantetrapulmonata encompasses arachnids with four pairs of lungs in their ground pattern. Tetrapulmonata potentially encompasses pantetrapulmonate arachnids which have reduced the number of lateral eye lenses and endites on the coxae of the first pair of walking legs. Serikodiastida encompasses arachnids with opisthosomal silk glands. Several spider-like fossils, some of which were previously interpreted as spiders, may belong either here or in the wider tetrapulmonate group. Araneida encompasses arachnids with spinnerets and a male palpal organ, and fossils reveal that the common ancestor of spiders would have retained a flagelliform telson. Araneae can thus be defined as spiders which have lost the telson, something which appears to have occurred multiple times among arachnids. The oldest fossil example of each of these clades is documented, and compared to molecular estimates of origination dates where available, to yield a broad temporal and phylogenetic framework for spider origins.

Eco-Friendly Bio-Hydrogels Based on Antheraea Pernyi Silk Gland Protein for Cell and Drug Delivery.

The Antheraea Pernyi silk gland protein originates from natural organisms and synthesized by tussah silk glands and has widely potential biomaterial applications due to the superior biocompatibility. This study investigates the Antheraea Pernyi silk gland protein-based drug-loaded bio-hydrogels for bioengineered tissue fabricated by using an eco-friendly method without the harsh extracting process and the usage of toxic chemicals. The drug-loaded bio-hydrogels exhibited a porous structure and interconnected pore walls. The swelling ratio and water absorption of drug-loaded bio-hydrogels were, respectively, above 95% and 1.5 × 103%. The cumulative release of drug loaded hydrogels all reached more than 90% within 4 h, and this indicates the potential of drug-loaded hydrogels as future drug-carrying biomaterials. RSC96 Schwann cells cultured on drug-loaded hydrogels for 72 h under cell culture medium show no toxic effects and more pro-proliferative effects. The results suggest the suitability of drug-loaded bio-hydrogels as natural biopolymer for the potential in vitro RSC96 cell culture platform and other biomaterial applications.

The Evaluation of the Biological Effects of Melanin by Using Silkworm as a Model Animal.

Melanin has been reported to have potential applications in industries such as cosmetics and food due to its anti-UV and antioxidative qualities. However, the corresponding data on its safety evaluation or biological consequences are fairly limited; such data are critical given its widespread use. The effect of different concentrations (1, 2, 3, and 4%) of melanin on growth status (larvae length and weight, cocoon weight, and morphology), the microstructure of the various tissues (fat body, silk gland, and midgut), and silk properties was studied by using the silkworm (bombyx mori) as the model organism. The weight and length of silkworm larvae fed with melanin were lower than the control, indicating that melanin appears to have a negative effect on the growth status of silkworms; however, the histophysiology analysis indicates that the cell morphologies are not changed, the XRD and FTIR spectra indicate that the secondary and crystalline structures of silks are also well preserved, and the thermogravimetric analysis and tensile test indicate that the thermal stability and mechanical properties are well maintained and even improved to some extent. Generally, it indicates that melanin has a certain inhibitory effect on the growth of silkworm larva but causes no harm to the cell microstructures or silk properties; this demonstrates that the safety of melanin as a food addictive should be considered seriously. The increase of thermal stability and mechanical properties shows that melanin may be a good chemical modifier in textile industries.

Comparative transcriptome and proteome reveal synergistic functions of differentially expressed genes and proteins implicated in an over-dominant silkworm heterosis of increased silk yield.

We previously observed an over-dominant silkworm heterosis of increased yield in a cross of Bombyx mori nuclear polyhydrosis virus-resistant strain NB with a susceptible strain 306. In the present study, we found that heterosis also exists in crosses of NB with other susceptible strains, indicating it is a more general phenomenon. We performed comparative transcriptome and proteome and identified 1624 differentially expressed genes (DEGs) and 298 differentially expressed proteins (DEPs) in silk glands between parents and F1 hybrids, of which 24 DEGs/DEPs showed consistent expression at mRNA and protein levels revealed by Venn joint analysis. Their expressions are completely non-additive, mainly transgressive and under low-parent, suggesting recombination of parental genomes may be the major genetic mechanism for the heterosis. GO and KEGG analyses revealed that they may function in generally similar but distinctive aspects of metabolisms and processes with signal transduction and translation being most affected. Notably, they may not only up-regulate biosynthesis and transport of silk proteins but also down-regulate other unrelated processes, synergistically and globally remodelling the silk gland to increase yield and cause the heterosis. Our findings contribute insights into the understanding of silkworm heterosis and silk gland development and provide targets for transgenic manipulation to further increase the silk yield.

A single-cell transcriptomic atlas characterizes the silk-producing organ in the silkworm

The silk gland of the domesticated silkworm Bombyx mori, is a remarkable organ that produces vast amounts of silk with exceptional properties. Little is known about which silk gland cells execute silk protein synthesis and its precise spatiotemporal control. Here, we use single-cell RNA sequencing to build a comprehensive cell atlas of the silkworm silk gland, consisting of 14,972 high-quality cells representing 10 distinct cell types, in three early developmental stages. We annotate all 10 cell types and determine their distributions in each region of the silk gland. Additionally, we decode the developmental trajectory and gene expression status of silk gland cells. Finally, we discover marker genes involved in the regulation of silk gland development and silk protein synthesis. Altogether, this work reveals the heterogeneity of silkworm silk gland cells and their gene expression dynamics, affording a deeper understanding of silk-producing organs at the single-cell level.

Characterization of the genome and silk-gland transcriptomes of Darwin's bark spider (Caerostris darwini).

Natural silks crafted by spiders comprise some of the most versatile materials known. Artificial silks-based on the sequences of their natural brethren-replicate some desirable biophysical properties and are increasingly utilized in commercial and medical applications today. To characterize the repertoire of protein sequences giving silks their biophysical properties and to determine the set of expressed genes across each unique silk gland contributing to the formation of natural silks, we report here draft genomic and transcriptomic assemblies of Darwin's bark spider, Caerostris darwini, an orb-weaving spider whose dragline is one of the toughest known biomaterials on Earth. We identify at least 31 putative spidroin genes, with expansion of multiple spidroin gene classes relative to the golden orb-weaver, Trichonephila clavipes. We observed substantial sharing of spidroin repetitive sequence motifs between species as well as new motifs unique to C. darwini. Comparative gene expression analyses across six silk gland isolates in females plus a composite isolate of all silk glands in males demonstrated gland and sex-specific expression of spidroins, facilitating putative assignment of novel spidroin genes to classes. Broad expression of spidroins across silk gland types suggests that silks emanating from a given gland represent composite materials to a greater extent than previously appreciated. We hypothesize that the extraordinary toughness of C. darwini major ampullate dragline silk may relate to the unique protein composition of major ampullate spidroins, combined with the relatively high expression of stretchy flagelliform spidroins whose union into a single fiber may be aided by novel motifs and cassettes that act as molecule-binding helices. Our assemblies extend the catalog of sequences and sets of expressed genes that confer the unique biophysical properties observed in natural silks.

The influence of an analog of juvenile hormone on the silk gland of Africanized Apis mellifera larvae reared in vitro

As the larval silk glands of Africanized honey bees are well characterized in hive conditions, we compared the silk glands of larvae of Africanized honey bees from the hive (in vivo - literature information) with larvae reared under laboratory conditions according to a specific bioassay (in vitro rearing of Africanized honey bees). We also continuously exposed the larva to a juvenile hormone analog (JHA), the pyriproxyfen, in three different concentrations: 12.5 mg/L, 25 mg/L, and 50 mg/L. Concerning morphological and histochemical analyses, there were no characteristics in glands that indicated the acceleration of the cocoon spinning time and no features of cell death. The glands seemed to be in secretory activity. Pyriproxyfen-treated larvae had a slightly shorter silk synthesis period than control, although only in the 50 mg/L experimental group, the tactoids from silk were clearly visible in optical microscopy, probably due to a higher secretion activity as the response to JHA. We observed that the presence of lipids under artificial feeding was independent of the nurse bee presence. Thus, we found that pyriproxyfen alters the functioning of the silk gland, under laboratory conditions in morphological, histological, and/or morphometric aspects.

Combined analysis of silk synthesis and hemolymph amino acid metabolism reveal key roles for glycine in increasing silkworm silk yields

Rearing silkworms (Bombyx mori) using formula feed has revolutionized traditional mulberry feed strategies. However, low silk production efficiencies persist and have caused bottlenecks, hindering the industrial application of formula feed sericulture. Here, we investigated the effects of formula feed amino acid composition on silk yields. We showed that imbalanced amino acids reduced DNA proliferation, decreased Fib-H, Fib-L, and P25 gene expression, and caused mild autophagy in the posterior silk gland, reducing cocoon shell weight and ratio. When compared with mulberry leaves, Gly, Ala, Ser, and Tyr percentages of total amino acids in formula feed were decreased by 5.26%, while Glu and Arg percentages increased by 9.56%. These changes increased uric acid and several amino acids levels in the hemolymph of silkworms on formula feed. Further analyses showed that Gly and Thr (important synthetic Gly sources) increased silk yields, with Gly increasing amino acid conversion efficiencies to silk protein, and reducing urea levels in hemolymph. Also, Gly promoted endomitotic DNA synthesis in silk gland cells via phosphoinositide 3-kinase (PI3K)/Akt/target of rapamycin (TOR) signaling. In this study, we highlighted the important role of Gly in regulating silk yields in silkworms.

Functional analysis of nuclear receptor HR96 gene in Bombyx mori exposed to phoxim.

The nuclear receptor (NRs) gene family functions as ligand-dependent transcription factors in a variety of animals, which participates in a variety of biological processes, such as cell differentiation, metabolic regulation, reproduction, development, insect metamorphosis. In this study, a nuclear receptor HR96 gene in silkworm Bombyx mori (BmHR96) was identified, and the responses of BmHR96 gene to 20-hydroxyecdysone (20E), three insecticides, and two disinfectants were analyzed and its function in phoxim exposure was explored. Quantitative real-time polymerase chain reaction indicated that the expression of BmHR96 mRNA was the highest in ovary of 5th instar Day 3 silkworm larvae and in silk gland of the wandering stage. The expression patterns of BmHR96 gene in ovary, head, testis, and midgut of different stages were different. After injecting 20E into B. mori, the expression of BmHR96 mRNA had no significant difference compared with control. Three insecticides and two disinfectants were used to treat B. mori, respectively, and it was found that they had different influence patterns on the expression level of BmHR96. siRNA of BmHR96 was injected into silkworm larvae and the expression of BmHR96 was decreased significantly after injecting 72 h. After silencing of BmHR96, B. mori was fed with phoxim-treated leaves. The results showed that the mortality of B. mori after silencing of BmHR96 was significantly higher than the control. Our results indicated that HR96 plays an important role in regulating the stress response of phoxim.

Effects of Probiotic Supplemented diet on Growth Performance of Silkworm Bombyx mori and Improved Characteristics of Cocoon and Silk

Aims: The present work is to investigate the effect of probiotic namely normagut on the biochemical and commercial characteristics of silkworm, B. mori.
 Study Design: Normagut treated mulberry leaves were supplemented at three different concentrations: 1%, 2% and 3%. It was fed to B. mori larvae and biochemical parameters were analyzed.
 Place and Duration of Study: Experiments were performed at Muslim Arts College, Tamilnadu, India between June 2019 and May 2020.
 Methodology: The feeding supplementation started from first day of third instar to last day of fifth instar for every 24 h. The research was performed with control and experimental groups of 3 replications of 30 larvae each.
 Results: In our study, 2% concentration of probiotic normagut was very effective and maximum of fat body protein (31.18 µg/mg), glycogen (20.21 µg/mg) and lipid (255.31 µg/mg) was observed. Maximum of haemolymph protein (58.12 µg/ml), trehalose (365.06 µg/ml) and of lipid (41.06 µg/ml), free amino acid (34.32 µg/ml), protein (64.43 µg/mg ) and 15.27 µg/mg of lipid was recorded in silk gland of B. mori (p<0.001). The economic traits of larval weight (3869.23 mg), cocoon weight (2131.36 mg), shell weight (463.13 mg), shell ratio (21.72%) and filament length (912.24 m) was recorded in 2% normagut treated group.
 Conclusion: The probiotics enhanced the commercial characteristics of cocoon and silk.

Temporal analysis of N-acetylglucosamine extension of N-glycans in the middle silk gland of silkworm Bombyx mori

N-glycosylation of proteins is an important post-translational modification in eukaryotic cells. One of the key modifications in protein N-glycosylation is N-acetylglucosamine (GlcNAc) extension mediated by N-acetylglucosaminyltransferase I (GNTI), which triggers N-glycan maturation from high-mannose-type to hybrid- and complex-type structures in Golgi. However, the temporal contributions of GNTI to GlcNAc extension and the resultant N-glycan structures in insects have not been analyzed. Here, focusing on GlcNAc extension of N-glycan in the silkworm Bombyx mori, we analyzed the temporal N-glycan alterations in the middle silk gland (MSG) and characterized the property of key enzyme for complex-type N-glycan biosynthesis, B.mori GNTI (BmGNTI). N-glycan analysis of N-glycoproteins in the MSG demonstrated that BmGNTI identified and characterized in this study consistently contributed to GlcNAc extension of N-glycans, which led to the accumulation of GlcNAc-extended N-glycans as predominant structures throughout the MSG development. The expression profile of GlcNAc extension-related genes revealed that the enzymes contributing to the hydrolysis of GlcNAc showed stage-specific expressions, thereby resulting in accumulations of the end product N-glycans of the enzyme. These results lead to the speculation that not BmGNTI but rather glycosylhydrolases critically influenced the structural formations and the changes in the ratio of N-glycans with GlcNAc residue(s) in MSG.