The Role of BmTMED6 in Female Reproduction in Silkworm, Bombyx mori.

Transmembrane emp24 domain (TMED) proteins have been extensively studied in mammalian embryonic development, immune regulation, and signal transduction. However, their role in insects, apart from Drosophila melanogaster, remains largely unexplored. Our previous study demonstrated the abundant expression of BmTMED6 across all stages and tissues of the silkworm. In this study, we investigate the function of BmTMED6 in reproduction. We observe significant differences in the expression of BmTMED6 between male and female silkworms, particularly in the head and fatboby, during the larval stage. Furthermore, qRT-PCR and WB analysis reveal substantial variation in BmTMED6 levels in the ovaries during pupal development, suggesting a potential association with silkworm female reproduction. We find that reducing TMED6 expression significantly decreases the number of eggs laid by female moths, leading to an accumulation of unlaid eggs in the abdomen. Moreover, downregulation of BmTMED6 leads to a decrease in the expression of BmDop2R1 and BmDop2R2, while overexpression of BmTMED6 in vitro has the opposite effect. These indicate that BmTMED6 plays a role in oviposition in female moths, potentially through the dopamine signaling pathway. This study provides a new regulatory mechanism for female reproduction in insects.

[Analyzing the evolution of insect TMED gene and the expression pattern of silkworm TMED gene].

Transmembrane emp24 domain (TMED) gene is closely related to immune response, signal transduction, growth and disease development in mammals. However, only the Drosophila TMED gene has been reported on insects. We identified the TMED family genes of silkworm, Tribolium castaneum, tobacco moth and Italian bee from their genomes, and found that the TMED family gene composition patterns of one α-class, one ß-class, one δ-class and several γ-classes arose in the common ancestor of pre-divergent Hymenoptera insects, while the composition of Drosophila TMED family members has evolved in a unique pattern. Insect TMED family γ-class genes have evolved rapidly, diverging into three separate subclasses, TMED6-like, TMED5-like and TMED3-like. The TMED5-like gene was lost in Hymenoptera, duplicated in the ancestors of Lepidoptera and duplicated in Drosophila. Insect TMED protein not only has typical structural characteristics of TMED, but also has obvious signal peptide. There are seven TMED genes in silkworm, distributed in six chromosomes. One of seven is single exon and others are multi-exons. The complete open reading frame (ORF) sequences of seven TMED genes of silkworm were cloned from larval tissues and registered in GenBank database. BmTMED1, BmTMED2 and BmTMED6 were expressed in all stages and tissues of the silkworm, and all genes were expressed in the 4th and 5th instar and silk gland of the silkworm. The present study revealed the composition pattern of TMED family members, their γ class differentiation and their evolutionary history, providing a basis for further studies on TMED genes in silkworm and other insects.

A comprehensive review of recent advances in silk sericin: Extraction approaches, structure, biochemical characterization, and biomedical applications.

Silks are natural polymers that have been widely used for centuries. Silk consists of a filament core protein, termed fibroin, and a glue-like coating substance formed of sericin (SER) proteins. This protein is extracted from the silkworm cocoons (particularly Bombyx mori) and is mainly composed of amino acids like glycine, serine, aspartic acid, and threonine. Silk SER can be obtained using numerous methods, including enzymatic extraction, high-temperature, autoclaving, ethanol precipitation, cross-linking, and utilizing acidic, alkali, or neutral aqueous solutions. Given the versatility and outstanding properties of SER, it is widely fabricated to produce sponges, films, and hydrogels for further use in diverse biomedical applications. Hence, many authors reported that SER benefits cell proliferation, tissue engineering, and skin tissue restoration thanks to its moisturizing features, antioxidant and anti-inflammatory properties, and mitogenic effect on mammalian cells. Remarkably, SER is used in drug delivery depending on its chemical reactivity and pH-responsiveness. These unique features of SER enhance the bioactivity of drugs, facilitating the fabrication of biomedical materials at nano- and microscales, hydrogels, and conjugated molecules. This review thoroughly outlines the extraction techniques, biological properties, and respective biomedical applications of SER.

Adaptive Changes in Detoxification Metabolism and Transmembrane Transport of Bombyx mori Malpighian Tubules to Artificial Diet.

The high adaptability of insects to food sources has contributed to their ranking among the most abundant and diverse species on Earth. However, the molecular mechanisms underlying the rapid adaptation of insects to different foods remain unclear. We explored the changes in gene expression and metabolic composition of the Malpighian tubules as an important metabolic excretion and detoxification organ in silkworms (Bombyx mori) fed mulberry leaf and artificial diets. A total of 2436 differentially expressed genes (DEGs) and 245 differential metabolites were identified between groups, with the majority of DEGs associated with metabolic detoxification, transmembrane transport, and mitochondrial function. Detoxification enzymes, such as cytochrome P450 (CYP), glutathione-S-transferase (GST), and UDP-glycosyltransferase, and ABC and SLC transporters of endogenous and exogenous solutes were more abundant in the artificial diet group. Enzyme activity assays confirmed increased CYP and GST activity in the Malpighian tubules of the artificial diet-fed group. Metabolome analysis showed increased contents of secondary metabolites, terpenoids, flavonoids, alkaloids, organic acids, lipids, and food additives in the artificial diet group. Our findings highlight the important role of the Malpighian tubules in adaptation to different foods and provide guidance for further optimization of artificial diets to improve silkworm breeding.

Analyzing the evolution of insect TMED gene and the expression pattern of silkworm TMED gene / 生物工程学报

Transmembrane emp24 domain (TMED) gene is closely related to immune response, signal transduction, growth and disease development in mammals. However, only the Drosophila TMED gene has been reported on insects. We identified the TMED family genes of silkworm, Tribolium castaneum, tobacco moth and Italian bee from their genomes, and found that the TMED family gene composition patterns of one α-class, one β-class, one δ-class and several γ-classes arose in the common ancestor of pre-divergent Hymenoptera insects, while the composition of Drosophila TMED family members has evolved in a unique pattern. Insect TMED family γ-class genes have evolved rapidly, diverging into three separate subclasses, TMED6-like, TMED5-like and TMED3-like. The TMED5-like gene was lost in Hymenoptera, duplicated in the ancestors of Lepidoptera and duplicated in Drosophila. Insect TMED protein not only has typical structural characteristics of TMED, but also has obvious signal peptide. There are seven TMED genes in silkworm, distributed in six chromosomes. One of seven is single exon and others are multi-exons. The complete open reading frame (ORF) sequences of seven TMED genes of silkworm were cloned from larval tissues and registered in GenBank database. BmTMED1, BmTMED2 and BmTMED6 were expressed in all stages and tissues of the silkworm, and all genes were expressed in the 4th and 5th instar and silk gland of the silkworm. The present study revealed the composition pattern of TMED family members, their γ class differentiation and their evolutionary history, providing a basis for further studies on TMED genes in silkworm and other insects.

Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth.

Traditional sanitation practices remain the main strategy for controlling Bombyx mori infections caused by microsporidia Nosema bombycis. This actualizes the development of new approaches to increase the silkworm resistance to this parasite. Here, we constructed a mouse scFv library against the outer loops of N. bombycis ATP/ADP carriers and selected nine scFv fragments to the transporter, highly expressed in the early stages of the parasite intracellular growth. Expression of selected scFv genes in Sf9 cells, their infection with different ratios of microsporidia spores per insect cell, qPCR analysis of N. bombycis PTP2 and Spodoptera frugiperda COXI transcripts in 100 infected cultures made it possible to select the scFv fragment most effectively inhibiting the parasite growth. Western blot analysis of 42 infected cultures with Abs against the parasite ß-tubulin confirmed its inhibitory efficiency. Since the VL part of this scFv fragment was identified as a human IgG domain retained from the pSEX81 phagemid during library construction, its VH sequence should be a key antigen-recognizing determinant. Along with the further selection of new recombinant Abs, this suggests the searching for its natural mouse VL domain or "camelization" of the VH fragment by introducing cysteine and hydrophilic residues, as well as the randomization of its CDRs.

Comparative Transcriptome Analysis Identifies Key Defense Genes and Mechanisms in Mulberry (Morus alba) Leaves against Silkworms (Bombyx mori).

As a consequence of long-term coevolution and natural selection, the leaves of mulberry (Morus alba) trees have become the best food source for silkworms (Bombyx mori). Nevertheless, the molecular and genomic basis of defense response remains largely unexplored. In the present study, we assessed changes in the transcriptome changes of mulberry in response to silkworm larval feeding at 0, 3, and 6 h. A total of 4709 (up = 2971, down = 1738) and 3009 (up = 1868, down = 1141) unigenes were identified after 3 and 6 h of silkworm infestation, respectively. MapMan enrichment analysis results show structural traits such as leaf surface wax, cell wall thickness and lignification form the first physical barrier to feeding by the silkworms. Cluster analysis revealed six unique temporal patterns of transcriptome changes. We predicted that mulberry promoted rapid changes in signaling and other regulatory processes to deal with mechanical damage, photosynthesis impairment, and other injury caused by herbivores within 3-6 h. LRR-RK coding genes (THE1, FER) was predicted participated in perception of cell wall perturbation in mulberry responding to silkworm feeding. Ca2+ signal sensors (CMLs), ROS (OST1, SOS3), RBOHD/F, CDPKs, and ABA were part of the regulatory network after silkworm feeding. Jasmonic acid (JA) signal transduction was predicted to act in silkworm feeding response, 10 JA signaling genes (such as OPR3, JAR1, and JAZ1) and 21 JA synthesis genes (such as LOX2, AOS, and ACX1) were upregulated after silkworm feeding for 3 h. Besides, genes of "alpha-Linolenic acid metabolism" and "phenylpropanoid biosynthesis" were activated in 3 h to reprogram secondary metabolism. Collectively, these findings provided valuable insights into silkworm herbivory-induced regulatory and metabolic processes in mulberry, which might help improve the coevolution of silkworm and mulberry.

Haemolymph metabolomic differences in silkworms (Bombyx mori L.) under mulberry leaf and two artificial diet rearing methods.

The new technology of silkworm (Bombyx mori L.) artificial feed breeding has many characteristics and advantages. This study assessed silkworm rearing with mulberry leaf at all instars (MF) as the control, and used metabolomics to explore the differences in haemolymph metabolism of fifth instar silkworms under two modes of rearing with an artificial diet at all instars (AF) and rearing with an artificial diet during first to third instars and mulberry leaf during the fourth and fifth instars (AMF). The results show that, compared with silkworms of the MF group, the amount and fold change of various metabolites were higher in the haemolymph of AF group silkworms, and the metabolism of amino acids and uric acid, carbohydrates, lipids, and vitamins were changed. These changes may be the reasons for the poor performance of the AF silkworms. However, the amount and fold change of the various metabolites of silkworms in the AMF group were lower, and some metabolic pathways were more active. The amount of material and energy supply were greater. These changes could explain the high efficiency growth of body weight of silkworms after the conversion from artificial diet rearing to mulberry leaf rearing. These findings provide an important theoretical basis for the optimisation of artificial diet rearing technology for silkworms.

Evaluation and Comparison of the Effects of Mature Silkworm (Bombyx mori) and Silkworm Pupae Extracts on Schwann Cell Proliferation and Axon Growth: An In Vitro Study.

Background: Silkworm products were first used by physicians more than 8500 years ago, in the early Neolithic period. In Persian medicine, silkworm extract has several uses for treating and preventing neurological, cardiac, and liver diseases. Mature silkworms (Bombyx mori) and their pupae contain a variety of growth factors and proteins that can be used in many repair processes, including nerve regeneration. Objectives: The study aimed to evaluate the effects of mature silkworm (Bombyx mori), and silkworm pupae extract on Schwann cell proliferation and axon growth. Methods: Silkworm (Bombyx mori) and silkworm pupae extracts were prepared. Then, the concentration and type of amino acids and proteins in the extracts were evaluated by Bradford assay, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and liquid chromatograph-mass spectrometer (LC-MS/MS). Also, the regenerative potential of extracts for improving Schwann cell proliferation and axon growth was examined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay, electron microscopy, and NeuroFilament-200 (NF-200) immunostaining. Results: According to the results of the Bradford test, the total protein content of pupae extract was almost twice that of mature worm extract. Also, SDS-PAGE analysis revealed numerous proteins and growth factors, such as bombyrin and laminin, in extracts that are involved in the repair of the nervous system. In accordance with Bradford's results, the evaluation of extracts using LC-MS/MS revealed that the number of amino acids in pupae extract was higher than in mature silkworm extract. It was found that the proliferation of Schwann cells at a concentration of 0.25 mg/mL in both extracts was higher than the concentrations of 0.01 and 0.05 mg/mL. When using both extracts on dorsal root ganglion (DRGs), an increase in length and number was observed in axons. Conclusions: The findings of this study demonstrated that extracts obtained from silkworms, especially pupae, can play an effective role in Schwann cell proliferation and axonal growth, which can be strong evidence for nerve regeneration, and, consequently, repairing peripheral nerve damage.

Scavenger receptor C regulates antimicrobial peptide expression by activating toll signaling in silkworm, Bombyx mori.

Scavenger receptor is pattern-recognition receptor (PRR) that plays a crucial function in host defense against pathogens. Scavenger receptor C (SR-C) is present only in invertebrates and its function has not been studied in detail. In this study, an SR-C homologous gene from the silkworm, Bombyx mori, was identified and characterized. SR-C was largely expressed in hemocytes and Malpighian tubules, with continuous expression in hemocytes. The peak expression was observed in hemocytes during molting and wandering stages both at mRNA and protein levels. Furthermore, immunofluorescence demonstrated it to be mainly distributed in the cell membranes of hemocytes, including oenocytoids and granulocytes. The recombinant SR-C protein (rSR-C) could bind to different types of bacteria and pathogen-associated molecular patterns (PAMPs), with strong binding to gram-positive bacteria and Lys-type peptidoglycans. The overexpression of SR-C induced the expression of genes related to the Toll pathway and antibacterial peptides. While the knockdown of SR-C reduced the expression of AMPs and inhibited the Toll pathway, it impaired the bacterial clearance ability of silkworm larvae, thus decreasing silkworm larvae's survival rate. Altogether, SR-C is a PRR that protect silkworms against bacterial pathogens by enhancing the expression of AMPs expression via the Toll pathway in hemocytes.

Role of Bm30kc6 gene in cell apoptosis and the silk gland degradation signaling pathway in Bombyx mori L.

Apoptosis is a process of programmed cell death that is regulated by genes independently. The Bm30kc6 gene is a kind of small molecular lipoprotein about 30 kDa, expressed highly in the late stage of the silkworm hemolymph. Our study showed that overexpression of Bm30kc6 could decrease caspase-3 activation. Meanwhile, activation of caspase-3 increased when Bm30kc6 expression was disturbed by small interfering RNA (siRNA). Cell apoptosis was decreased when Bm30kc6 was overexpressed under UV treatment. The apoptosis rate induced by actinomycin D is similar to the trend by UV. It was inferred that Bm30kc6 has an inhibitory effect on the apoptosis of silkworm cells. The apoptosis-related genes, such as BmFadd, BmDredd, and BmDaxx were increased after overexpression of Bm30kc6 or decreased after interference of siRNA. It was speculated that there was an interactive relationship between Bm30kc6, BmDaxx, BmFadd, and BmDredd in the apoptosis signaling pathways. We investigated the transcription expression of the Bm30kc6 gene in different growth stages and tissues of the silkworm. The results showed that Bm30kc6 reached its peak in the hemolymph during the 6th to 7th days of the 5th instar, or in spinning post 24 h of the silk gland. In the silkworm BmN cells treated with caspase-3/7 inhibitor, the caspase-3 enzyme activity, and the expression levels of Bm30kc6, BmFadd, BmDredd, and BmDaxx were significantly reduced. The expression levels of Bm30kc6 increased sharply when silkworms were treated by molting hormone at Day 3 or 5 of the 5th instar. The results indicated that the expression of the Bm30kc6 gene was affected by the molting hormone and was likely to be its downstream target. In conclusion, the results suggest that the Bm30kc6 gene is involved in the regulation of the apoptotic signaling pathway and plays a role in the apoptotic process.

[Comparison of chemical constituents of wild silkworm cocoon and domestic silkworm cocoon by UHPLC-MS technology].

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.

High-quality genome assembly of the silkworm, Bombyx mori.

In 2008, the genome assembly and gene models for the domestic silkworm, Bombyx mori, were published by a Japanese and Chinese collaboration group. However, the genome assembly contains a non-negligible number of misassembled and gap regions due to the presence of many repetitive sequences within the silkworm genome. The erroneous genome assembly occasionally causes incorrect gene prediction. Here we performed hybrid assembly based on 140 × deep sequencing of long (PacBio) and short (Illumina) reads. The remaining gaps in the initial genome assembly were closed using BAC and Fosmid sequences, giving a new total length of 460.3 Mb, with 30 gap regions and an N50 comprising 16.8 Mb in scaffolds and 12.2 Mb in contigs. More RNA-seq and piRNA-seq reads were mapped on the new genome assembly compared with the previous version, indicating that the new genome assembly covers more transcribed regions, including repetitive elements. We performed gene prediction based on the new genome assembly using available mRNA and protein sequence data. The number of gene models was 16,880 with an N50 of 2154 bp. The new gene models reflected more accurate coding sequences and gene sets than old ones. The proportion of repetitive elements was also reestimated using the new genome assembly, and was calculated to be 46.8% in the silkworm genome. The new genome assembly and gene models are provided in SilkBase (http://silkbase.ab.a.u-tokyo.ac.jp).

Three vital RNA functions and interactions in the process of silk gland apoptosis in silkworm Bombyx mori.

The Silkworm Bombyx mori is an important insect in terms of economics and a model organism with a complete metamorphosis. The economic importance of silkworms is dependent on the functions of the silkgland, a specialized organ that synthesizes silk proteins. The silk gland undergoes massive degeneration during the larval to pupal stage, which involves in cell apoptosis. In this paper, high throughput sequencing was used to detect the expression of messenger RNA (mRNA), long noncoding RNA (lncRNA), and microRNA (miRNA) from silk glands of Day 3 in the fifth instar larvae (L5D3) and the spinning 36h (sp36h). We analyzed the Gene Ontology (GO) functions of target genes of the differentially expressed lncRNAs and miRNAs. We investigated the regulations of mRNA, lncRNA, and miRNA on silk gland apoptosis in L5D3 and sp36h. In total, 10,947 lncRNAs were detected in the silk gland and the index number TCONS-00021360 lncRNA may be involved in the process of apoptosis. In addition, 344 miRNAs targeted 285 mRNAs were related to the death process under GO entry. The results indicated that miRNAs play an important role in the molecular regulation of the silk gland apoptosis compared with that of lncRNAs. Finally, we screened 746 lncRNAs and 20 miRNAs that might interact with BmDredd, and drew an interaction network among them.

Comparison of chemical constituents of wild silkworm cocoon and domestic silkworm cocoon by UHPLC-MS technology / 生物工程学报

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.

Silver effects on silkworm, Bombyx mori.

Silver nanoparticles (Ag-NPs) are known as a noble metal, and owing to their exclusive properties, their use is widespread in consumer products and they are mostly incorporated into food packaging and food contact products. The aim of this work was to evaluate the effects of direct ingestion of Ag-NPs through food to assess their toxicity effects on the growth and development of silkworms at different concentrations (1 mg·L-1 to 100 mg·L-1), in addition to the examination of the distribution of Ag-NPs in the silkworm body and midgut histopathological analysis. RNA sequencing was performed to investigate the transcriptomic responses to Ag-NPs exposure. Our results show that the highest Ag-NPs' concentrations induced a significant increase in the silkworm body weight with histopathological changes in the midgut compared to the control group. The gene ontology (GO) and pathway enrichment analysis for differentially expressed genes showed that Ag-NPs altered the gene expressions and that they were significantly involved in carbohydrate metabolism, digestive system, and energy metabolism. These findings indicate that the Ag-NPs may induce harmful effects on the primary target organs (alimentary system) with energy deregulation and nutrition digestion and absorption imbalance. This study is an important step in understanding the molecular mechanisms of Ag-NPs' toxicity in vivo.

Transcriptome analysis of differentially expressed genes involved in innate immunity following Bacillus thuringiensis challenge in Bombyx mori larvae.

In this study, we describe RNA-seq expression profiling of larval Bombyx mori response to hemocoel injection of Bacillus thuringiensis (Bt). Two transcriptomes were generated from the hemocytes of the PBS- and Bt-injected B. mori larvae. More than 49 million 100-bp paired-end reads, encompassing over 7.3 Gb of sequence data, were generated for each library. After filtering the raw reads and removing the rRNA mapped reads, more than 89% of the reads in each library could be mapped to the silkworm genome reference sequence. Comparison of gene expression levels revealed that a total of 133 unigenes were upregulated while 84 unigenes were downregulated in PBS vs Bt. To further investigate the biological functions of different expression genes (DEGs), gene ontology (GO) and functional enrichment analysis were performed to map all the DEGs to terms in the GO, euKaryotic Ortholog Groups of proteins (KOG) and Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) database. Among these DEGs, many genes involved in immunity against Bt challenge were identified. These included genes participated in pattern recognition, antimicrobial peptides, insecticide resistance or detoxification, immune melanization, cytoskeleton reorganization and many other immune effectors. To confirm the gene expression patterns identified by the RNA-seq data, the transcript levels of 10 immune related DEGs were examined by quantitative real-time PCR (qRT-PCR). The results showed that the DEGs obtained from the deep sequencing data were accurate and gene expression profiles from RNA-Seq data were reliable. Our studies provide insights into the to immune response of B. mori underling the stress of Bt, which is valuable to understand how Bt affects the innate immune system of silkworm and provide new approaches to control insect pests by using Bt as a biological insecticide.

Okaramine insecticidal alkaloids show similar activity on both exon 3c and exon 3b variants of glutamate-gated chloride channels of the larval silkworm, Bombyx mori.

The okaramine indole alkaloids were recently shown to be more selective than ivermectin in activating the glutamate-gated chloride channels of the silkworm larvae of Bombyx mori (BmGluCls). Those studies were carried out using the exon 3b variant as a representative of BmGluCls. However, it remains unknown whether okaramines are similarly effective on other silkworm GluCl variants and whether they share the same binding site as ivermectin on GluCls. To begin to address these questions, we examined the potency of four okaramines on the exon 3c variant of BmGluCls by two-electrode voltage clamp voltage recordings of glutamate-induced chloride currents. The potency of okaramines in activating the exon 3c BmGluCl agreed well with findings on the exon 3b BmGluCl and insecticidal potency. Okaramine B (10µM) reduced the maximum binding (Bmax) but not the dissociation constant (KD) of [3H]ivermectin in studies on plasma membrane fractions of HEK293 cells expressing the exon 3c variant. These findings indicate that activation of GluCls is important in the insecticidal actions of okaramines.

BMDREDD REGULATES THE APOPTOSIS COORDINATING WITH BMDAXX, BMCIDE-B, BMFADD, AND BMCREB IN BMN CELLS.

The apoptosis mechanisms in mammals were investigated relatively clearly. However, little is known about how apoptosis is achieved at a molecular level in silkworm cells. We cloned a caspase homologous gene named BmDredd (where Bm is Bombyx mori and Dredd is death-related ced-3/Nedd2-like caspase) in BmN cells from the ovary of Bm and analyzed its biological information. We constructed the N-terminal, C-terminal, and overexpression vector of BmDredd, respectively. Our results showed that the transcriptional expression level of BmDredd was increased in the apoptotic BmN cells. Furthermore, overexpression of BmDredd increased the caspase-3/7 activity. Simultaneously, RNAi of BmDredd could save BmN cells from apoptosis. The immunofluorescence study showed that BmDredd located at the cytoplasm in normal cell otherwise is found at the nucleus when cells undergo apoptosis. Moreover, we quantified the transcriptional expressions of apoptosis-related genes including BmDredd, BmDaxx (where Daxx is death-domain associated protein), BmCide-b (where Cide-b is cell death inducing DFF45-like effector), BmFadd (Fadd is fas-associated via death domain), and BmCreb (where Creb is cAMP-response element binding protein) in BmN cells with dsRNA interferences to detect the molecular mechanism of apoptosis. In conclusion, BmDredd may function for promoting apoptosis and there are various regulatory interactions among these apoptosis-related genes.

Intestinal microecology associated with fluoride resistance capability of the silkworm (Bombyx mori L.).

The silkworm (Bombyx mori L.) is an ideal model of Lepidoptera. However, the diversity and function of the intestinal microbiota in the gut of silkworm remain largely unknown. Changes in the intestinal microecology in fluoride-resistant strain T6 and fluoride-susceptible strain 734 of the silkworm in response to fluoride exposure were investigated. T6 and 734 were treated with 200 mg/kg fluoride (designated as T6-T and 734-T groups) and deionized water (designated as T6-C and 734-C groups). Culture-dependent approach revealed that the numbers of intestinal bacteria in the 734-T group significantly decreased compared with that in the 734-C group (4.8 ± 0.6 × 10(7) CFU/mL vs. 7.5 ± 0.7 × 10(7) CFU/mL; P < 0.05). Analyses of the intestinal content pH showed that the pH decreased in the 734-T group only. Additionally, SCFA concentrations significantly decreased in both treatment groups compared with the control groups. High-throughput sequencing indicated that the intestinal microbiota in the 734-T group was significantly more diverse than those in the other groups. The bacterial community was composed of two dominant groups (Firmicutes and Proteobacteria). Principal component analyses revealed a significant difference in the composition of the intestinal microbiota in the 734-T group compared with those in the other groups. Thaumarchaeota and Euryarchaeota were more abundant in the 734-T group, but they were less abundant in the other groups. This study enhances our understanding about the diversity and function of silkworm intestinal microbiota in response to fluoride exposure among silkworm strains with diverse resistance.