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Apolipophorin-III (ApoLp-III) is an abundant hemolymph protein mainly involved in lipid transport and innate immunity in insects. In the present study, the gene Samia cynthia ricini ApoLp-III (ScApoLp-III) was identified from a transcriptome database, and contained 790 nucleotides with a putative open reading frame (ORF) of 561 bp encoding 186 amino acid residues. Phylogenetic analysis revealed that ScApoLp-III had significant homology with ApoLp-III protein from Antheraea pernyi. Higher ScApoLp-III expression levels were found in the fat body and silk gland by reverse transcription quantitative PCR (RT-qPCR). Injection of Staphylococcus aureus induced up-regulation of ScApoLp-III in the midgut, fat body and hemocytes. However, ScApoLp-III was down-regulated in the midgut and fat body after Pseudomonas aeruginosa injection, indicating that ScApoLp-III may contribute to the host’s defense against invading pathogens. Additionally, recombinant ScApoLp-III was found to bind different bacteria, including E. coli, P. aeruginosa, S. aureus and B. subtilis. Bactericidal tests showed that recombinant ScApoLp-III strongly inhibited Gram-negative bacteria, including Escherichia coli and P. aeruginosa. However, it had no obvious influence on Gram-positive bacteria. Taken together, our results suggest that the ScApoLp-III might play an important role in the innate immunity of S. c. ricini.

1-Deoxynojirimycin (DNJ) is a natural d -glucose analogue from mulberry with promising physiological activity in vivo. Up to the present, the antidiabetic effects of DNJ on lowering blood sugar and accelerating lipid metabolism in mammals were broadly reported, but the specific character of DNJ against insects was vastly ignored. In this study, a toxicological test of DNJ againgst eri-silkworm, Samia cynthia ricini was carried out to investigate the potential of DNJ in insect management. Further, a method of nuclear magnetic resonance (NMR) metabonomics and real-time qPCR (RT-qPCR) were performed to analyze the alteration in midgut of eri-silkworm caused by DNJ. The result of toxicology showed that 5% and 10% DNJ could significantly inhibit the development of third-instar larvae on day 1–5, and mass deaths happened in DNJ groups on day 3–5. The quantitative analysis of 1H NMR in fifth-instar larvae showed that trehalose level increased in midgut of 0, 6 and 12 h DNJ groups, while the concentrations of glucose, lactate, alanine, pyruvate, α-ketoglutarate and fumarate were reduced in varying degrees. Meanwhile, principal component analysis (PCA) indicated that there were significant differences in the metabolic profiles among 12 h DNJ groups and the control group. In addition, RT-qPCR results displayed that four genes coding α-glucosidase, trehalase (THL) and lactate dehydrogenase (LDH) were lowered in expression of 12 h DNJ groups. Simultaneously, THL activity was significantly lowerd in 12 h DNJ groups. These mutually corroborated results indicated that the backbone pathways of energy metabolism, including hydrolysis of trehalose and glycogens, glycolysis and tricarboxylic acid (TCA) cycle were significantly inhibited by DNJ. Thus, the specific mechanism of DNJ efficiently suppressing the growth and energy metabolism of eri-silkworm was explored in this study, providing the potential of DNJ as to the production of botanical insecticide.

Ferritin is a ubiquitous iron storage protein that plays an important role in host defence against pathogen infections. In the present study, native ferritin was isolated from the hemolymph of Bombyx mori using native-polyacrylamide gel electrophoresis (native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that ferritin consisted of two subunits, designated as BmFerHCH and BmFerLCH. Previously integrated previous transcriptome and iTRAQ data showed that the two subunits were down-regulated in resistant silkworm strain BC9 and there was no obvious change in the expression levels of the subunits in susceptible silkworm strain P50 after BmNPV infection. Virus overlay assays revealed that B. mori ferritin as the form of heteropolymer had an interaction with B. mori nucleopolyhedrovirus (BmNPV), but it can't interact with BmNPV after depolymerisation. What's more, reverse transcription quantitative PCR (RT-qPCR) analysis suggested that BmFerHCH and BmFerLCH could be induced by bacteria, virus and iron. This is the first study to extract B. mori ferritin successfully and confirms their roles in the process of BmNPV infection. All these results will lay a foundation for further research the function of B. mori ferritin.

Bombyx mori nucleopolyhedrovirus (BmNPV) has been identified as a major pathogen responsible for severe economic loss. Most silkworm strains are susceptible to BmNPV, with only a few highly resistant strains thus far identified. Here we investigated the molecular basis of silkworm resistance to BmNPV using susceptible (the recurrent parent P50) and resistant (near-isogenic line BC9) strains and a combination of iTRAQ-based quantitative proteomics, reverse-transcription quantitative PCR and Western blotting. By comparing the proteomes of infected and non-infected P50 and BC9 silkworms, we identified 793 differentially expressed proteins (DEPs). By gene ontology and KEGG enrichment analyses, we found that these DEPs are preferentially involved in metabolism, catalytic activity, amino sugar and nucleotide sugar metabolism and carbon metabolism. 114 (14.38%) DEPs were associated with the cytoskeleton, immune response, apoptosis, ubiquitination, translation, ion transport, endocytosis and endopeptidase activity. After removing the genetic background and individual immune stress response proteins, we identified 84 DEPs were found that are potentially involved in resistance to BmNPV. Further studies showed that a serine protease was down-regulated in P50 and up-regulated in BC9 after BmNPV infection. Taken together, these results provide insights into the molecular mechanism of silkworm response to BmNPV. Biological significance Bombyx mori nucleopolyhedrovirus (BmNPV) is highly pathogenic, causing serious losses in sericulture every year. However, the molecular mechanisms of BmNPV infection and host defence remain unclear. Here we combined quantitative proteomic, bioinformatics, RT-qPCR and Western blotting analyses and found that BmNPV invasion causes complex protein alterations in the larval midgut, and that these changes are related to cytoskeleton, immune response, apoptosis, ubiquitination, translation, ion transport, endocytosis and endopeptidase activity. Five important differentially expression proteins were validation by independent approaches. These finding will help address the molecular mechanisms of silkworm resistance to BmNPV and provide a molecular target for resisting BmNPV.

DNA modification is a naturally occurring DNA modification in prokaryotic and eukaryotic organisms and is involved in several biological processes. Although genome-wide methylation has been studied in many insects, the understanding of global and genomic DNA methylation during insect early embryonic development, is lacking especially for insect diapause. In this study, we analyzed the relationship between DNA methylomes and transcriptomes in diapause-destined eggs compared to diapause-terminated eggs in the silkworm, Bombyx mori (B. mori). The results revealed that methylation was sparse in this species, as previously reported. Moreover, methylation levels in diapause-terminated eggs (HCl-treated) were 0.05% higher than in non-treated eggs, mainly due to the contribution of CG methylation sites. Methylation tends to occur in the coding sequences and promoter regions, especially at transcription initiation sites and short interspersed elements. Additionally, 364 methylome- and transcriptome-associated genes were identified, which showed significant differences in methylation and expression levels in diapause-destined eggs when compared with diapause-terminated eggs, and 74% of methylome and transcriptome associated genes showed both hypermethylation and elevated expression. Most importantly, Kyoto Encyclopaedia of Genes and Genomes (KEGG) analyses showed that methylation may be positively associated with Bombyx mori embryonic development, by regulating cell differentiation, metabolism, apoptosis pathways and phosphorylation. Through analyzing the G2/M phase-specific E3 ubiquitin-protein ligase (G2E3), we speculate that methylation may affect embryo diapause by regulating the cell cycle in Bombyx mori. These findings will help unravel potential linkages between DNA methylation and gene expression during early insect embryonic development and insect diapause.

Ferritin is a ubiquitous and conserved iron storage protein that plays a significant role in host detoxification, iron storage, and immune response. Although ferritin has been studied in many species, little is known about its role in the Eri-silkworm (Samia cynthia ricini). In this study, the ferritin light-chain subunit gene, named ScFerLCH, was identified from S. c. ricini. The full-length gene, ScFerLCH, was 1,155 bp and encoded a protein consisting of 231 amino acids with a deduced molecular weight of 26.38 kDa. Higher ScFerLCH expression levels were found in the midgut, silk gland, and fat body by quantitative reverse-transcription polymerase chain reaction and western blot analysis. Injection of Staphylococcus aureus and Pseudomonas aeruginosa could induce upregulation of ScFerLCH in the hemolymph, fat body, and midgut, indicating that ScFerLCH may contribute to the host defense against invading pathogens. In addition, the native ferritin protein was isolated from S. c. ricini by native polyacrylamide gel electrophoresis and its two subunits, ferritin heavy-chain subunit (ScFerHCH) and ferritin light-chain subunit (ScFerLCH), were identified by mass spectrometry. Specifically, we found that recombinant ferritin subunits could self-assemble into a protein complex in vitro; moreover, both recombinant subunits and the protein complex were found to bind different bacteria, including Escherichia coli, P. aeruginosa, S. aureus, and Bacillus subtilis. However, bactericidal tests showed that the protein complex could not inhibit the growth of bacteria directly. Taken together, our results suggest that ScFerritin might play an important role in mediating molecular interaction with pathogens.

Sanguinarine (Sang) is a natural alkaloid and distributed in several plants of Papaveraceae. The antitumor, antioxidant, antimicrobial and anti-inflammatory effects of Sang were extensively reported, but its speciality and mechanism against Lepidoptera insects were still unknown. In this study, detailed toxicological parameters of Sang against silkworms, Bombyx mori (B. mori), were determined by a toxicological test. Then, a nuclear magnetic resonance-based (NMR) metabolomics method was adopted to analyze the changes in hemolymph metabolites of silkworms after feeding Sang. The growth of fourth-instar larvae was significantly ceased by the oral administration of 0.05–0.3% Sang and vast deaths appeared in 0.3% Sang group on Day 4 and Day 5. The quantitative analysis of metabolites indicated that trehalose and citrate levels in hemolymph were increased after 24 h of feeding 0.3% Sang, whereas the concentrations of pyruvate, succinate, malate and fumarate were decreased. In addition, the enzymatic determination and reverse transcription quantitative PCR (RT-qPCR) showed that the trehalase (THL) activity and the transcriptional level of one gene coding THL were uniformly weakened by 0.3% Sang. One of the important mechanisms of Sang against silkworms might be interpreted as follows. Sang impaired trehalose hydrolysis, reduced THL activity and transcription, and led to the inhibition of energy metabolism, consequent antigrowth and high lethality in larvae of B. mori. Our findings offered new insights into the insecticidal effect of Sang from the perspective of energy metabolism and provided the basis for the application of Sang in the control of Lepidoptera pests.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary silkworm pathogen, and the molecular mechanism of silkworm defense to BmNPV infection is still unclear. Herein, comparative metabolomics was adopted to analyze the variations in the hemolymph metabolites of different resistant silkworm strains following BmNPV inoculation using a

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious pathogenic microorganism that causes tremendous loss to sericulture. Previous studies have found that some proteins of serine protease family in the digestive juice of B. mori larvae have anti-BmNPV activity. In our previous publication about proteome analysis of the digestive juice of B. mori larvae, the digestive enzyme trypsin, alkaline A (BmTA) was filtered as a differentially expressed protein possibly involved in BmNPV resistance. Here, the biological characteristics and anti-BmNPV functions of BmTA were comprehensively analysed. The cDNA sequence of BmTA had an ORF of 768 nucleotides encoding 255 amino acid residues. Domain architecture analysis showed that BmTA contained a signal peptide and a typical Tryp_SPc domain. Quantitative real-time PCR analysis showed that BmTA was highly expressed in the larval stages and specifically expressed in the midgut of B. mori larvae. The expression level of BmTA in BmNPV resistant strain A35 was higher than that in susceptible strain P50. After BmNPV infection, the expression of BmTA increased in both strains from 24 to 72 h. Virus amplification analysis showed that the relative levels of VP39 in B. mori larvae and BmN cells infected with the appropriate concentration of recombinant-BmTA-treated BmNPV were significantly lower than in the control groups. Moreover, overexpression of BmTA in BmN cells significantly inhibited the amplification of BmNPV. Taken together, the results of this study indicated that BmTA possessed anti-BmNPV activity in B. mori, which broadens the horizon for virus-resistant breeding of silkworms.

The innate immune system is conserved among different insect species in its response to microorganism infection. The transmembrane receptors of the Toll superfamily play an important role in activating immune response, however, the function of silkworm Toll family member 18 Wheeler (18 W) remained unclear. Here, the 18w gene in silkworm was characterized. A relatively high transcription level of Bm18w mRNA was found in Malpighian tubules, and in eggs, larvae pre-molt to fourth instar, pupae and adults. When silkworm larvae were infected with E. coli or S. aureus, Bm18w showed a significant response, especially to E. coli, but did not have antibacterial activity. To further identify the downstream antimicrobial peptide genes of Bm18w, expression of Bm18w was knocked down with siRNA in vitro, resulting in significant decreases of cecropin-A, gloverin 2, and moricin B3. The overexpression of Bm18w was carried out using pIZT/V5-His-mCherry insect vector in BmN cells and significant upregulation of cecropin-A and gloverin 2 was detected, as well as upregulation of attacin and defensin. Based on the results, we concluded that Bm18w is involved in response to bacterial infection by selectively inducing the expression of antimicrobial peptide genes, especially cecropin-A and gloverin 2. This study provides valuable data to supplement understanding of the immune pathway of the silkworm.

Proteins p38 map kinase and ribosomal S6 kinase (S6K) as members of mitogen-activated protein kinases (MAPKs) play important roles against pathogens. In this study, Bmp38 and BmS6K were identified as differentially expressed proteins from iTRAQ database. Bmp38 and BmS6K were expressed, and recombinant proteins were purified. The bioinformatics analysis showed that both proteins have serine/threonine-protein kinases, catalytic domain (S_TKc) with 360 and 753 amino acids, respectively. The real-time quantitative polymerase chain reaction (RT-qPCR) results suggest that Bmp38 and BmS6K had high expression in the midgut and hemolymph. The comparative expression level of Bmp38 and BmS6K in BC9 was upregulated than in P50 in the midgut after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Western bolt results showed a positive correlation between RT-qPCR and iTRAQ data for Bmp38, but BmS6K data showed partial correlation with iTRAQ. Injection of anti-Bmp38 and anti-BmS6K serum suggested that Bmp38 may be involved against BmNPV infection, whereas BmS6K may require phosphorylation modification to inhibit BmNPV infection. Taken together, our results suggest that Bmp38 and BmS6k might play an important role in innate immunity of silkworm against BmNPV.

DNA methylation is one of the most widespread epigenetic marks and has been linked to insect development, especially influencing embryonic development. However, the regulation of DNA methylation in silkworm embryonic development and diapause remain to investigate. In this study, reverse-transcription quantitative polymerase chain reaction was performed to identify the expression level of Bombyx mori DNA methyltransferases (BmDNMTs) 1 and 2 ( BmDnmt1 and BmDnmt2) in different tissues, different embryonic developmental stages, and different strains of the silkworm. The results showed that BmDNMTs were the most highly expressed during embryonic development, especially at early embryonic stages. In particular, the expression of BmDNMTs was significantly upregulated in diapause-terminated eggs by HCl treatment. Moreover, tissue distribution showed that BmDnmt2 was highly expressed in testis and ovary, and BmDnmt1 was highly expressed in testis. This study contributes to understanding the correlation of DNA methylation occurs with embryogenesis and gametogenesis in insect, meanwhile, it provides a research orientation to further analyze the role of DNA methylation in diapause initiation and termination in insect embryonic development.

Samia cynthia ricini (Lepidoptera: Saturniidae) is an important commercial silk-producing insect; however, in contrast to the silkworm, mulberry leaves are toxic to this insect because the leaves contain the component 1-deoxynojirimycin (DNJ). A transcriptomic analysis of eri-silkworm haemolymph was conducted to examine the genes related to different metabolic pathways and to elucidate the molecular mechanism underlying eri-silkworm haemolymph responses to DNJ. Eight hundred sixty-five differentially expressed genes (DEGs) were identified, among which 577 DEGs were up-regulated and 288 DEGs were down-regulated in the 2% DNJ group compared to control (ddH2O) after 12h. Based on the results of the functional analysis, these DEGs were associated with ribosomes, glycolysis, N-glycan biosynthesis, and oxidative phosphorylation. In particular, according to the KEGG analysis, 138 DEGs were involved in energy metabolism, glycometabolism and lipid metabolism, and the changes in the expression of nine DEGs were confirmed by reverse transcription quantitative PCR (RT-qPCR). Thus, DNJ induced significant metabolic alterations in eri-silkworm haemolymph. These results will lay the foundation for research into the toxic effects of DNJ on eri-silkworm as a model and provide a reference for the exploitation of new drugs in humans.

The velvet antler is a special organ that has important biological significance for deer, and its growth is a complicated biological metabolism process. Growing evidence suggests that genetics factors play essential roles in the weight of velvet antlers. In this study, we investigated five sika deer (Cervus nippon) populations under the same feeding condition, and screened genetic variations in the 100 samples (including 50 heavy and 50 light velvet antler weight samples) by whole genome re-sequencing. The results showed that 94 genetic variations were related to the velvet antler weight, among which two single nucleotide polymorphism (SNP) sites were located on the exon regions of OAS2 and ALYREF/THOC4, respectively. Furthermore, ALYREF/THOC4 is highly expressed in the velvet antler. The biological functions of these genetic variations were highly related to the growth and development of deer velvet antlers. Collectively, we screened genes related to the velvet antler weight in sika deer populations by whole genome re-sequencing and identified 94 sites as candidate genetic variations related to the velvet antler weight. We hope that it will contribute to further mechanistic studies of velvet antler development and weight variations.

Ferritins are conserved iron-binding proteins that are primarily involved in iron storage, detoxification and the immune response. Despite the importance of ferritin in organisms, little is known about their roles in the eri-silkworm (Samia cynthia ricini). We previously identified a ferritin heavy chain subunit named ScFerHCH in the S. c. ricini transcriptome database. The full-length S. c. ricini ferritin heavy chain subunit (ScFerHCH) was 1863 bp and encoded a protein of 231 amino acids with a deduced molecular weight of 25.89 kDa. Phylogenetic analysis revealed that ScFerHCH shared a high amino acid identity with the Bombyx mori and Danaus plexippus heavy chain subunits. Higher ScFerHCH expression levels were found in the silk gland, fat body and midgut of S. c. ricini by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Injection of Staphylococcus aureus and Pseudomonas aeruginosa was associated with an upregulation of ScFerHCH in the midgut, fat body and hemolymph, indicating that ScFerHCH may contribute to the host’s defense against invading pathogens. In addition, the anti-oxidation activity and iron-binding capacity of recombinant ScFerHCH protein were examined. Taken together, our results suggest that the ferritin heavy chain subunit from eri-silkworm may play critical roles not only in innate immune defense, but also in organismic iron homeostasis.

The molecular mechanism of silkworm resistance to Bombyx mori nucleopolyhedrovirus (BmNPV) infection remains largely unclear. Accumulating evidence suggests that subcellular fractionation combined with proteomics is an ideal technique to analyse host antiviral mechanisms. To clarify the anti-BmNPV mechanism of the silkworm, the near-isogenic line BC9 (resistant strain) and the recurrent parent P50 (susceptible strain) were used in a comparative subcellular proteomics study. Two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MS) was conducted on proteins extracted from the cytosol, mitochondria, and microsomes of BmNPV-infected and control larval midguts. A total of 87 proteins were successfully identified from the three subcellular fractions. These proteins were primarily involved in energy metabolism, protein metabolism, signalling pathways, disease, and transport. In particular, disease-relevant proteins were especially changed in microsomes. After infection with BmNPV, differentially expressed proteins (DEPs) primarily appeared in the cytosolic and microsomal fractions, which indicated that these two fractions might play a more important role in the response to BmNPV infection. After removing genetic background and individual immune stress response proteins, 16 proteins were identified as potentially involved in repressing BmNPV infection. Of these proteins, the differential expression patterns of 8 proteins according to reverse transcription quantitative PCR (RT-qPCR) analyses were consistent with the 2-DE results.

The ATP-binding cassette (ABC) transporters belong to a superfamily of genes involved in the transport of specific molecules across lipid membranes, as well as insecticide resistance, present in all living organisms. In this study, we combined the Cnaphalocrocis medinals transcriptome database with a bioinformatics approach to identify four C. medinals ABCs (CmABCs), including CmABCG1, CmABCG4, CmABCC2 and CmABCC3. Tissue expression analysis showed that these genes had a tissue-specific expression pattern. CmABCG1 had significantly higher expression in the haemolymph and head compared to the other tissues. The expression of CmABCG4, CmABCC2 and CmABCC3 was highest in the midgut, followed by expression in the fat body. The developmental stage expression analysis showed that CmABCG1, CmABCG4, CmABCC2 and CmABCC3 were mainly expressed in adults. The transcription of CmABCG1, CmABCG4 and CmABCC2 was significantly induced by chlorpyrifos. Taken together, the results of our study provided useful information for understanding of the detoxification system of C. medinalis.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a major pathogen causing severe economic loss. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity. In this study, antiviral activity examination of different resistant strains showed that the digestive juice of the resistant strain (A35) had higher inhibition to virus than the susceptible strain (P50). Subsequently, the label-free quantitative proteomics was used to study the midgut digestive juice response to BmNPV infection in P50 and A35 strains. A total of 98 proteins were identified, of which 80 were differentially expressed proteins (DEPs) with 54 enzymes and 26 nonenzymatic proteins by comparing the proteomes of infected and non-infected P50 and A35 silkworms. These DEPs are mainly involved in metabolism, proteolysis, neuroactive ligand receptor interaction, starch and sucrose metabolism and glutathione metabolism. After removing the genetic background and individual immune stress response proteins, 9 DEPs were identified potentially involved in resistance to BmNPV. Further studies showed that a serine protease, an alkaline phosphatase and serine protease inhibitor 2 isoform X1 were differentially expressed in A35 compared to P50 or post BmNPV infection. Taken together, these results provide insights into the potential mechanisms for silkworm digestive juice to provide resistance to BmNPV infection. Signifcance: Bombyx mori nucleopolyhedrovirus (BmNPV) is highly pathogenic, which has a great impact on the sericulture. BmNPV entered the midgut lumen and exposed to digestive juices after oral infection. Previous studies have revealed that some proteins in silkworm digestive juice show antiviral activity, however, current information on the digestive juice proteome of high resistant silkworm strain after BmNPV challenge compared to susceptible strain is incomprehensive. Here, we combined label-free quantification method, bioinformatics, RT-qPCR and western blot analysis and found that BmNPV infection causes some protein changes in the silkworm midgut digestive juice. The DEPs were identified in the digestive juices of different resistant strains following BmNPV infection, and screened out some proteins potentially related to resistance to BmNPV. Three important differentially expression proteins were validated by independent approaches. These findings uncover the potential role of silkworm digestive juice in providing resistance to BmNPV and supplemented the profile of the proteome of the digestive juices in B. mori., Graphical abstract Unlabelled Image, Highlights • The digestive juice antiviral activity of resistant strain was higher than susceptible strain. • Silkworm digestive juice was analyzed by label-free quantitative proteomics. • Eighty DEPs were identified in different resistant strains following BmNPV infection. • Some DEPs were validated by RT-qPCR and western blot. • These results provide insights into the potential mechanisms for B. mori resistant to BmNPV infection.

Serpins are a family of serine protease inhibitors that are found widely in insects. They play an important role in insect physiological responses, such as innate immunity and development. In this study, we obtained the Bombyx mori serpin6 (BmSerpin6) sequence from National Center for Biotechnology Information (NCBI) and the silkworm genome database (SilkDB). Reverse transcription PCR (RT-PCR) results showed that BmSerpin6 was expressed highly in hemocytes, the midgut, and the fat body. After challenging with Micrococcus luteus (Mi) and Serratia marcescens (Sm), the BmSerpin6 expression level was induced significantly. Transcript levels of gloverin2 and prophenoloxidase (PPO) activity were reduced significantly in the fat body and hemocytes after injecting the recombinant BmSerpin6 protein into silkworm larvae. A BmSerpin6 recombinant plasmid (BmSerpin6-pAC 5.1) was constructed successfully and transfected into Drosophila S2 cells, which resulted in significantly reduced expression of the drosomycin protein. These results indicated that BmSerpin6 might regulate silkworm immune responses.

The rice leaf roller (Cnaphalocrocis medinalis) is one of the most destructive agricultural pests. Due to its migratory behavior, it is difficult to control worldwide. To date, little is known about major genes of C. medinalis involved in chitin metabolism and insecticide detoxification. In order to obtain a comprehensive genome dataset of C. medinalis, we conducted de novo transcriptome sequencing which focused on the major feeding stage of fourth-instar larvae, and our work revealed useful information on chitin metabolism and insecticide detoxification and target genes of C. medinalis. We acquired 29,367,797 Illumina reads and assembled these reads into 63,174 unigenes with an average length of 753 bp. Among these unigenes, 31,810 were annotated against the National Center for Biotechnology Information non-redundant (NCBI nr) protein database, resulting in 24,246, 8669 and 18,176 assigned to Swiss-Prot, clusters of orthologous group (COG), and gene ontology (GO), respectively. We were able to map 10,043 unigenes into 285 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). Specifically, 16 genes, including five chitin deacetylases, two chitin synthases, five chitinases and four other related enzymes, were identified to be putatively involved in chitin biosynthesis and degradation, whereas 360 genes, including cytochrome P450s, glutathione S-transferases, esterases, and acetylcholinesterases, were found to be potentially involved in insecticide detoxification or as insecticide targets. The reliability of the transcriptome data was determined by reverse transcription quantitative PCR (RT-qPCR) for the selected genes. Our data serves as a new and valuable sequence resource for genomic studies on C. medinalis. The findings should improve our understanding of C. medinalis genetics and contribute to management of this important agricultural pest.

The ENF peptide family, so termed after the consensus sequence in their amino termini (Glu-Asn-Phe-), is assumed to play multiple important roles in defense reactions, growth regulation, and homeostasis of Lepidopteran insects. The paralytic peptide of Bombyx mori (BmPP) is one such peptide that is involved in the paralytic and plasmatocyte-spreading activities in the hemocyte immune reaction. The growth-blocking peptide of Pseudaletia separata (PsGBP), which is also a member of the ENF peptide family, has similar functions that can reportedly be attenuated by the growth-blocking peptide-binding protein (GBP-BP). Using the fluorescent differential display (FDD) technique, the differential expression pattern of genes in highly susceptible silkworm strain 306 were analyzed, following infection with B. mori nuclear polyhedrosis virus (BmNPV), and a differential band (G12(782)) was obtained from the hemolymph RNA pools. Using 5'-RACE with a specially designed primer based on the FDD study, a 1,401 bp cDNA clone was obtained containing a 1,311 bp open reading frame (ORF, GenBank accession number DQ306881). The deduced protein was highly homologous in primary structure to GBP-BP and was termed B. mori paralytic peptide-binding protein (PP-BP). The B. mori PP-BP gene is organized into two exons and only one intron, using bioinformatics searches.Using RT-PCR analysis, it was found that the B. mori PP-BP gene was expressed almost exclusively in the hemolymph. Real-time quantitative PCR analysis indicated that the B. mori PP-BP mRNA level in B. mori strain 306 exposed to BmNPV was much higher than that in B. mori strain without the virus infection. This result implies that the B. mori PP-BP is related to the cellular immune response after BmNPV invades the hemolymph.

1-deoxynojirimycin (DNJ) is a natural D-glucose analogue and has a strong physiological activity in inhibiting α-glucosidase in vivo. The antidiabetic effects of DNJ in mice or other mammals were extensively explored, but the physiological and toxic roles of DNJ in insects was seldom reported. In this study, the biological effects of DNJ were examined in midgut extracts of fourth-instar larvae of Eri silkworm (Samia cynthia ricini, Saturniidae). Based on nuclear magnetic resonance (NMR) metabonomics technology, we analyzed the alterations of glycometabolism, lipids, and energy metabolism pathways in the midgut of S. cynthia ricini caused by DNJ. Pattern recognition analysis (partial least square-discriminant analysis, PLS-DA) showed that four groups of latex, 0.25% DNJ, 0.5% DNJ and the mixture of 0.5% DNJ and latex (1:1) were distinctly different from the control group. Moreover, several metabolic pathways of DNJ-mediated modulation in the midgut were identified. Compared with the control group, alanine, succinate, glutamate, and fumarate concentrations decreased in three groups of 0.5% DNJ, latex, and the mixture, choline levels increased in two DNJ groups, and trehalose levels increased in all experimental groups. Therefore, these results suggest that DNJ modulated lipid metabolism by limiting the hydrolysis pathways of phospholipids metabolism. Additionally, DNJ has a potent negative effect on energy metabolism by inhibiting the hydrolysis of trehalose, glycolysis and the tricarboxylic acid (TCA) cycle. Overall, DNJ, as a single-ingredient, is an efficient substance for modulating lipid metabolism and inhibiting energy metabolism.

Bombyx mori L. (Lepidoptera: Bombycidae) nucleopolyhedrovirus (BmNPV) is a highly pathogenic virus in the sericultural industry, often causing severe damage leading to large economic losses. The immune mechanisms of B. mori against this virus remain obscure. Previous studies had demonstrated Bmlipase-1, BmNox and Bmserine protease-2 showing antiviral activity in vitro , but data on the transcription levels of these proteins in different resistant strains were not reported. In order to determine the resistance level of the four different strains (P50, A35, A40, A53) and gain a better understanding of the mechanism of resistance to BmNPV in B. mori , the relative expression level of the genes coding the three antiviral proteins in larval haemolymph and midgut of different B. mori strains resistant to BmNPV was determined. The results showed that these genes expressed significantly higher in the resistant strains compared to the susceptible strain, and the differential expression levels were consistent with the LC50 values in different strains. The transcription level of the target genes almost all up-regulated in the larvae midgut and down-regulated in the haemolymph. The results indicate the correlation of these genes to BmNPV resistance in B. mori.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a primary pathogen of silkworm (B. mori) that causes severe economic losses each year. However, the molecular mechanisms of silkworm-BmNPV interactions, especially the silkworm proteins that can interact with the virus, are still largely unknown. In this study, the total and membrane proteins of silkworm midguts were displayed using one- and two-dimensional electrophoresis. A virus overlay assay was used to detect B. mori proteins that specifically bind to BmNPV particles. Twelve proteins were located and identified using mass spectrometry, and the different expression of the corresponding genes in BmNPV susceptible and resistant silkworm strains also indicated their involvement in BmNPV infection. The 12 proteins are grouped based on their potential roles in viral infection, for example, endocytosis, intracellular transportation, and host responses. Based on these results, we hypothesize the following: I) vacuolar ATP synthase catalytic subunit A and subunit B may be implicated in the process of the membrane fusion of virus and the release of the nucleocapsid into cytoplasm; II) actin, enolase and phosphoglycerate kinase are cytoskeleton associated proteins and may play an important role in BmNPV intracellular transportation; III) mitochondrial prohibitin complex protein 2, ganglioside-induced differentiation-associated protein, calreticulin, regucalcin-like isoform X1 and 60 kDa heat shock protein are involved in cell apoptosis regulation during BmNPV infection in larvae midguts; IV) ribosomal P0 may be associated with BmNPV infection by regulating gene expression of BmNPV; V) arginine kinase has a role in the antiviral activities against BmNPV. Our work should prove informative by providing multiple protein targets and a novel direction to investigate the molecular mechanisms of the interactions between silkworms and BmNPV.

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the primary pathogens causing severe economic losses in sericulture. However, the molecular mechanism of silkworm resistance to BmNPV remains largely unknown. Here, the recurrent parent P50 (susceptible strain) and the near-isogenic line BC9 (resistance strain) were used in a comparative transcriptome study examining the response to infection with BmNPV. A total of 14,300 unigenes were obtained from two different resistant strains; of these, 869 differentially expressed genes (DEGs) were identified after comparing the four transcriptomes. Many DEGs associated with protein metabolism, cytoskeleton, and apoptosis may be involved in the host response to BmNPV infection. Moreover, some immunity related genes were also altered following BmNPV infection. Specifically, after removing genetic background and individual immune stress response genes, 22 genes were found to be potentially involved in repressing BmNPV infection. These genes were related to transport, virus replication, intracellular innate immune, and apoptosis. Our study provided an overview of the molecular mechanism of silkworm resistance to BmNPV infection and laid a foundation for controlling BmNPV in the future.

The highly pathogenic Bombyx mori nucleopolyhedrovirus (BmNPV) has caused severe damages to sericulture in many countries, and the relationship between the pathogenicity of various BmNPV strains and their geographical evolution has been the topic of our interest. In this study, we isolated a new BmNPV strain from Thailand (BmNPV-Thai), based on the sequences of its conservative genes p10, p35, polh, egt and vp39. The BmNPV-Thai appears to have baculovirus repeated ORF (bro) genes different from four other well-known BmNPV strains of China (GD, CQ1), Japan (T3), and France (SC7); It only has bro-a, bro-c, and bro-d, but not bro-b and bro-e genes. These bro genes are localized only in the two subgroups highly homologous to their counterparts and their encoded BRO proteins differ mainly at their N-terminal amino acid residues. Phylogenetic analysis indicates that the evolution of the bro genes of the five BmNPV strains is not obviously associated with their geographic locations.

We aimed to investigate whether 1-deoxynojirimycin (DNJ) modulates glycometabolism and has toxicity in Eri silkworm (Samia cynthia ricini, Saturniidae). In this paper, hemolymph metabolites were used to explore metabolic changes after oral administration of DNJ or mulberry latex and to characterize the biological function of DNJ at the metabolic and systemic levels. Hemolymph samples were collected from fourth-instar larvae of Eri silkworm and ex-vivo high-resolution 1H nuclear magnetic resonance (NMR) spectra were acquired from the collected hemolymph samples. Then the obtained spectra were analyzed by principal component analysis (PCA) and independent-samples t-test. Metabolic pattern recognition analysis of hemolymph samples indicated that the groups of 0.25% DNJ, latex, and the mixture of 0.5% DNJ and latex (1:1) were significantly different from the control group. Moreover, compared to the control group, the groups of 0.25% DNJ, latex, and the mixture of 0.5% DNJ and latex (1:1) showed the decreased levels of citrate, succinate, fumarate, malate, and glutamine in hemolymph, the groups of 0.25% DNJ and the mixture of 0.5% DNJ and latex (1:1) showed the increased levels of trehalose and lactate. In addition, mulberry leaves exude latex was highly toxic to Eri silkworm because rich unidentified high-molecular-weight factor (s) acted as toxic substances. In our results, latex caused 20 deaths among 50 fourth-instar larvae of Eri silkmoth, but DNJ or the mixture did not caused death. All these results suggest that DNJ has a positive impact on the reverse glycometabolism by modulating glycometabolism and inhibiting glucogenesis and energy metabolism. DNJ is a secure substance as a single-ingredient antidiabetic medicine due to its nontoxicity and bioactivity.