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Phospholipase A2 (PLA2) is widely distributed in animals, plants, and microorganisms, and it plays an important role in many physiological activities. In a previous study, we have identified a secretory PLA2 in Bombyx mori ( BmsPLA2-1-1 ). In this study, we further identified four new sPLA2 genes ( BmsPLA2-1-2 , BmsPLA2-2 , BmsPLA2-3 , and BmsPLA2-4 ) in B . mori genome. All four genes exhibits the characteristic features of sPLA2, including the sPLA2 domain, metal binding sites, and highly conserved catalytic domain. This study completed the cloning, in vitro expression, and expression pattern analysis of the BmsPLA2-4 gene in B . mori . The full length of BmsPLA2-4 is 585 bp, and the recombinant protein obtained through prokaryotic expression has an estimated size of 25 kDa. qRT-PCR analysis revealed that the expression level of BmsPLA2-4 reached its peak on the first day of the fifth instar larval stage. Tissue expression profiling analysis showed that BmsPLA2-4 had the highest expression level in the midgut, followed by the epidermis and fat body. Western blotting analysis results were consistent with those of qRT-PCR. Furthermore, after infecting fifth instar 1-day-old larvae with Escherichia coli and Staphylococcus aureus , the expression level of the BmsPLA2-4 gene significantly increased in 24 h. The findings of this study provides a theoretical basis and valuable experimental data for future related research.

Lipases play crucial roles in food digestion by degrading dietary lipids into free fatty acids and glycerols. The domesticated silkworm (Bombyx mori) has been widely used as an important Lepidopteran model for decades. However, little is known about the lipase gene family in the silkworm, especially their hydrolytic activities as digestive enzymes. In this study, a total of 38 lipase genes were identified in the silkworm genome. Phylogenetic analysis indicated that they were divided into three major groups. Twelve lipases were confirmed to be expressed in the midgut at both transcriptional and translational levels. They were grouped into the same gene cluster, suggesting that they could have similar physiological functions. Quantitative real-time PCR (qRT-PCR) analyses indicated that lipases were mainly expressed in anterior and middle midgut regions, and their expression levels varied greatly along the length of midgut. A majority of lipases were down-regulated in the midgut when larvae stopped feeding. However, a unique lipase gene (Bmlip10583) showed low expression level during feeding stage, but it was significantly up-regulated during the larvae-pupae transition. These results demonstrated that expression of silkworm lipases was spatially and temporally regulated in the midgut during larval development. Taken together, our results provide a fundamental research of the lipase gene family in the silkworm., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Silk gland is an organ that produces and secretes silk proteins. The development of the silk gland is essential for high silk production yield and silk quality. Although Sage reportedly plays a pivotal role in embryonic silk gland development, the mechanism underlying its action remains unclear. Our study aimed to determine the genes downstream of Sage through which it regulates the development of the silk gland. After chromatin immunoprecipitation and sequencing, Dfd was identified as a downstream target gene of Sage and it was confirmed that Sage could inhibit Dfd expression by competing with SGF1. When Dfd was knocked down through RNA interference (RNAi), the number of cells in the middle silk gland decreased, and the posterior silk gland was straightened. Simultaneously, the expression of Ser1 and silk fibroin genes was no longer strictly regional. These changes eventually led to an alteration in the composition of the Dfd RNAi cocoon. In conclusion, our research contributes to a deeper understanding of the development of silk glands., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Temperature stress disrupts protein homeostasis in biological cells. High temperature causes death of the silkworm Bombyx mori, while low temperature leads to slow growth of silkworm in sericulture. Small heat shock proteins (sHsps) play a crucial role in maintaining protein homeostasis and protecting cells against various harmful conditions. In this study, the expression patterns of Bmhsp19.9 were analyzed by reverse transcription (RT)-PCR and quantitative PCR. The promoters hsp19.9P1 and hsp19.9P2 of Bmhsp19.9 were cloned, quantitative PCR and western blotting confirmed that the longer promoter of hsp19.9P1 showed higher activity compared to hsp19.9P2. Overexpression of Bmhsp19.9 increased the viability of BmE cells at high temperatures. The transgenic vector pb-H19.9 was constructed, in which Bmhsp19.9 was overexpressed under the control of hsp19.9P1. Two transgenic lines, H19.9A and H19.9B, were generated via microinjection of P50 silkworm embryos. Quantitative PCR and western blotting revealed that the content of Bmhsp19.9 in transgenic silkworms was higher than that in the control. After exposure to 35 °C for 49 h, the survival rate was increased by approximately 40% in transgenic silkworms compared to non-transgenic P50. When P50 and H19.9A were reared at 13 °C, growth was faster and the survival rate and surviving larval weight were significantly higher in H19.9A compared to P50. These results suggest that overexpression of Bmhsp19.9 increased the resistance of transgenic silkworms to extreme temperatures, which may be useful for sericulture in regions and seasons with no optimal average temperatures for silkworm., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Phosphoserine phosphatase (PSP) catalyses the synthesis of l-serine via the phosphorylated pathway by facilitating the dephosphorylation of phosphoserine. A cDNA encoding PSP from the silkworm Bombyx mori (bmPSP) was isolated using reverse transcription-PCR and then sequenced. The resulting clone encoded 236 amino acids with a molecular weight of 26 150, exhibiting 14-60% sequence identity with other PSPs. The recombinant PSP was overexpressed in Escherichia coli and purified. Kinetic studies showed that bmPSP possessed activity toward l-phosphoserine, and Asp20, Asp22 and Asp204 in bmPSP were found to be critical for modulating bmPSP activity. Real-time PCR analysis provided evidence that the amount of bmpsp transcript was reduced in middle silk glands of a sericin-deficient silkworm strain. These findings revealed that bmPSP may play important roles in synthesizing one-carbon donors of l-serine, which is abundant in silk, as well as other cell metabolites in B. mori., (© 2019 The Royal Entomological Society.)

In this study, we identified and characterized a phosphoserine aminotransferase (bmPSAT) from Bombyx mori (B. mori) that is responsible for l-serine biosynthesis. A complementary DNA that encodes bmPSAT was cloned by reverse transcriptase polymerase reaction and sequenced. The presumed amino acid sequence revealed 47-87% identity with known PSATs from insects, humans, plants, and bacteria. Through phylogenetic analysis, we found that bmPSAT is evolutionary related to insect PSATs. Recombinant bmPSAT was produced in Escherichia coli by using a cold-shock promotor and purified to homogeneity. This enzyme utilizes phosphohydroxypyruvate and glutamate for transamination. bmPSAT messenger RNA (mRNA) was expressed at higher levels in several tissues of standard strain silkworm including the silk gland, whereas a sericin-deficient silkworm strain exhibited a diminished expression of bmPSAT mRNA in the silk gland. These findings indicate that bmPSAT may play an important role in synthesizing and supplying l-serine in the larva of B. mori., (© 2019 Wiley Periodicals, Inc.)

The arylalkylamine N-acyltransferases (AANATs) are enzymes that catalyze the acyl-CoA-dependent formation of N-acylarylalkylamides: acyl-CoA + arylalkylamine → N-acylarylalkylamides + CoA-SH. Herein, we describe our study of a previously uncharacterized AANAT from Bombyx mori: Bm-iAANAT3. Bm-iAANAT3 catalyzes the direct formation of N-acylarylalkylamides and accepts a broad range of short-chain acyl-CoA thioesters and amines as substrates. Acyl-CoA thioesters possessing an acyl chain length >10 carbon atoms are not substrates for Bm-iAANAT3. We report that Bm-iAANAT3 is a "versatile generalist", most likely, functioning in amine acetylation - a reaction in amine inactivation/excretion, cuticle sclerotization, and melanism. We propose a kinetic and chemical mechanism for Bm-iAANAT3 that is consistent with our steady-state kinetic analysis, dead-end inhibition studies, determination of the pH-rate profiles, and site-directed mutagenesis of a catalytically important amino acid in Bm-iAANAT3. These mechanistic studies of Bm-iAANAT3 will foster the development of novel compounds targeted against this enzyme and other insect AANATs for the control of insect pests., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aldose reductase (AR) is a rate-limiting enzyme in the polyol pathway and is also the key enzyme involved in diabetic complications. The silkworm purple quail-like mutant (q-l p ) exhibits pigmented dots on its epidermis. The q-l p mutant also shows developmental abnormalities and decreased vitality. In this study, fat bodies from the q-l p mutant and the wildtype 932VR strain were subjected to two-dimensional gel electrophoresis (2-DE) analysis, and the Bombyx mori AR (BmAR) protein was found to be significantly downregulated in the q-l p mutant. The expression of BmAR at the mRNA level was also significantly downregulated, as verified through quantitative reverse transcription PCR (qRT-PCR). Knockdown of the expression of BmAR via RNAi resulted in a reduction of silkworm weight. The sorbitol level in q-l p was significantly lower than in the wildtype. These results suggested that the BmAR gene is closely related to the development of the q-l p mutant. Investigation of the cause of BmAR downregulation in the q-l p mutant could contribute to revealing the function of AR in insects and offers a new method of identifying AR inhibitors for the treatment of diabetic complications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The wild silkworm Bombyx mandarina was domesticated to produce silk in China approximately 5000 years ago. Silk production is greatly improved in the domesticated silkworm B. mori, but the molecular basis of the functional evolution of silk gland remains elusive. We performed shotgun proteomics with label-free quantification analysis and identified 1012 and 822 proteins from the posterior silk glands (PSGs) of wild silkworms on the third and fifth days of the fifth instar, respectively, with 128 of these differentially expressed. Bioinformatics analysis revealed that, with the development of the PSG, the up-regulated proteins were mainly involved in the ribosome pathway, similar to what we previously reported for B. mori. Additionally, we screened 50 proteins with differential expression between wild and domesticated silkworms that might be involved in domestication at the two stages. Interestingly, the up-regulated proteins in domesticated compared to wild silkworms were enriched in the ribosome pathway, which is closely related to cell size and translation capacity. Together, these results suggest that functional evolution of the PSG during domestication was driven by reinforcing the advantageous pathways to increase the synthesis efficiency of silk proteins in each cell and thereby improve silk yield.

Genetically manipulated organisms with dysfunction of specific tissues are crucial for the study of various biological applications and mechanisms. However, the bioengineering of model organisms with tissue-specific dysfunction has not progressed because the challenges of expression of proteins, such as cytotoxins, in living cells of individual organisms need to be overcome first. Here, we report the establishment of a transgenic silkworm ( Bombyx mori ) with posterior silk glands (PSGs) that was designed to express the cabbage butterfly ( Pieris rapae ) cytotoxin pierisin-1A (P1A). P1A, a homolog of the apoptosis inducer pierisin-1, had relatively lower DNA ADP ribosyltransferase activity than pierisin-1; it also induced the repression of certain protein synthesis when expressed in B. mori -derived cultured cells. The transgene-derived P1A domain harboring enzymatic activity was successfully expressed in the transgenic silkworm PSGs. The glands showed no apoptosis-related morphological changes; however, an abnormal appearance was evident. The introduced truncated P1A resulted in the dysfunction of PSGs in that they failed to produce the silk protein fibroin. Cocoons generated by the silkworms solely consisted of the glue-like glycoprotein sericin, from which soluble sericin could be prepared to form hydrogels. Embryonic stem cells could be maintained on the hydrogels in an undifferentiated state and proliferated through stimulation by the cytokines introduced into the hydrogels. Thus, bioengineering with targeted P1A expression successfully produced silkworms with a biologically useful trait that has significant application potential., Competing Interests: The authors declare no conflict of interest.

A full-length cDNA of lebocin 5 (BmLeb5) was first cloned from silkworm, Bombyx mori , by rapid amplification of cDNA ends. The BmLeb5 gene is 808 bp in length and the open reading frame encodes a 179-amino acid hydroxyproline-rich peptide. Bioinformatic analysis results showed that BmLeb5 owns an O-glycosylation site and four RXXR motifs as other lebocins. Sequence similarity and phylogenic analysis results indicated that lebocins form a multiple gene family in silkworm as cecropins. Quantitative real-time PCR analysis revealed that BmLeb5 was highest expressed in the fat body. In the silkworm larvae infected by Beauveria bassiana , the expression level of BmLeb5 was upregulated in the fat body and hemolymph which are the most important immune tissues in silkworm. The recombinant protein of BmLeb5 was for the first time successfully expressed with prokaryotic expression system and purified. There are no reports so far that the expression of lebocins could be induced by entomopathogenic fungus. Our study suggested that BmLeb5 might play an important role in the immune response of silkworm to defend B. bassiana infection. The results also provided helpful information for further studying the lebocin family functioned in antifungal immune response in the silkworm., Competing Interests: The authors declare there is no conflict of interests.

To elucidate the mechanism for embryonic diapause or the breakdown of diapause in Bombyx mori, we biochemically analyzed nitric oxide synthase (NOS) during the embryogenesis of B. mori. The gene expression and enzyme activity of B. mori NOS (BmNOS) were examined in diapause, non-diapause, and HCl-treated diapause eggs. In the case of HCl-treated diapause eggs, the gene expression and enzyme activity of BmNOS were induced by HCl treatment. However, in the case of diapause and non-diapause eggs during embryogenesis, changes in the BmNOS activity and gene expressions did not coincide except 48-60 h after oviposition in diapause eggs. The results imply that changes in BmNOS activity during the embryogenesis of diapause and non-diapause eggs are regulated not only at the level of transcription but also post-transcription. The distribution and localization of BmNOS were also investigated with an immunohistochemical technique using antibodies against the universal NOS; the localization of BmNOS was observed mainly in the cytoplasm of yolk cells in diapause eggs and HCl-treated diapause eggs. These data suggest that BmNOS has an important role in the early embryonic development of the B. mori., (© 2016 Japanese Society of Developmental Biologists.)

Scavenger receptor class B is involved in various indispensable physiological processes, like the formation and inhibition of atherosclerosis or other cardiovascular diseases, innate immune defense and the removal of apoptotic cells. Here, we cloned BmSCRB8, a member of scavenger receptor class B in silkworm. We obtained the full-length cDNA sequence of BmSCRB8 by rapid amplification of cDNA ends (RACE), including 2 668 bp. The ORF of BmSCRB8 is 1 704 bp, encoding 567 amino acids. Online software prediction indicated that the molecular weight of BmSCRB8 is 63.87 kDa and the isoelectric point (pI) is 6.06. The space-time expression profile of BmSCRB8 was detected by reverse transcription PCR (RT-PCR), which implicated that BmSCRB8 is extensively expressed in each tissue and at each stage of blood. In addition, BmSCRB8 is highest expressed in fat body of silkworm, and is highly expressed in metamorphosis periods. Anti-BmSCRB8 polyclonal antibody was generated through prokaryotic expression, protein purification and mice immunization. Simultaneously, we constructed BmSCRB8 eukaryotic vector and then transfected embryonic cell line of silkworm. Immunofluorescence and overexpression showed that BmSCRB8 expressed specifically in membrane. Western blotting demonstrated that BmSCRB8 protein can be specifically recognized by anti-serum generated after mice immunization.

Basic helix loop helix (bHLH) transcription factor plays an important role in biological processes. Bmsage is a class of bHLH transcription factor highly expressed in the silk gland of Bombyx mori, which is not only involved in the developmental regulation of the silk gland cells at the embryonic period, but also plays a crucial regulatory role during the synthesis of silk protein. However, currently, much of the property and structure of Bmsage is still remained unknown. To study the property, structure and biological role of Bmsage, we constructed several prokaryotic expression vectors of Bmsage fused with NusA, MBP, SUMO, Trx and His tags, respectively, then screened and determined the best soluble expression vector and condition of Bmsage in Escherichia coli combining with the induction temperature and IPTG concentration, and further purified the recombinant Bmsage by Ni-column affinity chromatography according to the established expression condition and characterized its secondary structure using circular dichroism spectra. The results showed that NusA and MBP could significantly enhance the soluble expression of Bmsage in E. coli, but it was difficult to separate Bmsage from these tags. SUMO could not only increase the soluble expression of Bmsage in E. coli to a certain degree, but also be effectively separated from Bmsage. Other tags did not effectively promote the soluble expression of Bmsage in E. coli. Circular dichroism spectra showed that the purified Bmsage had well-defined α-helix structure in solution, indicating that SUMO may promote the correct folding of Bmsage into native-like structure. These work not only establish a foundation for further study of the property, structure and function of Bmsage, but also provide a reference for the expression and purification of other similar proteins.

In Bombyx mori (B. mori), Fem piRNA originates from the W chromosome and is responsible for femaleness. The Fem piRNA-PIWI complex targets and cleaves mRNAs transcribed from the Masc gene. Masc encodes a novel CCCH type zinc-finger protein and is required for male-specific splicing of B. mori doublesex (Bmdsx) transcripts. In the present study, several silkworm strains carrying a transgene, which encodes a Fem piRNA-resistant Masc mRNA (Masc-R), were generated. Forced expression of the Masc-R transgene caused female-specific lethality during the larval stages. One of the Masc-R strains weakly expressed Masc-R in various tissues. Females heterozygous for the transgene expressed male-specific isoform of the Bombyx homolog of insulin-like growth factor II mRNA-binding protein (ImpM) and Bmdsx. All examined females showed a lower inducibility of vitellogenin synthesis and exhibited abnormalities in the ovaries. Testis-like tissues were observed in abnormal ovaries and, notably, the tissues contained considerable numbers of sperm bundles. Homozygous expression of the transgene resulted in formation of the male-specific abdominal segment in adult females and caused partial male differentiation in female genitalia. These results strongly suggest that Masc is an important regulatory gene of maleness in B. mori., Competing Interests: The authors have declared that no competing interests exist.

The temporal control mechanisms that precisely control animal development remain largely elusive. The timing of major developmental transitions in insects, including molting and metamorphosis, is coordinated by the steroid hormone 20-hydroxyecdysone (20E). 20E involves feedback loops to maintain pulses of ecdysteroid biosynthesis leading to its upsurge, whereas the underpinning molecular mechanisms are not well understood. Using the silkworm Bombyx mori as a model, we demonstrated that E75, the 20E primary response gene, mediates a regulatory loop between ecdysteroid biosynthesis and 20E signaling. E75 isoforms A and C directly bind to retinoic acid receptor-related response elements in Halloween gene promoter regions to induce gene expression thus promoting ecdysteroid biosynthesis and developmental transition, whereas isoform B antagonizes the transcriptional activity of isoform A/C through physical interaction. As the expression of E75 isoforms is differentially induced by 20E, the E75-mediated regulatory loop represents a fine autoregulation of steroidogenesis, which contributes to the precise control of developmental timing., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The silk gland in silkworm (Bombyx mori) is a highly specialized organ that specifically synthesizes silk proteins. A function shift to the synthesis of large quantities of 30K proteins occurs in the degenerating silk gland cells during larval-pupal metamorphosis. The posterior silk gland developmental mutant model of silkworm was used in this study and changes in the programmed cell death (PCD) regulatory signals and 30K protein synthesis during silk gland degeneration were investigated. The results showed that PCD induced by 20-hydroxyecdysone was initiated early during larval-pupal metamorphosis in the mutant, but PCD proceeded slowly, resulting in the degeneration process of the silk gland being extended and took almost twice the time compared with the wild type. Caspase-dependent pathway signals regulated by Dronc in the silk gland cells of the mutant were significantly reduced, while the PCD initiation signal regulated by the Atg family was not delayed or reduced, and PCD-related epigenetic modification such as lysine methylation, acetylation, and succinylation, and tyrosine phosphorylation changed significantly. During the degeneration process in the mutant, 30K proteins were efficiently synthesized in the silk gland cells in stage PP1 even when no caspase protein was detected. Degeneration of the silk gland is a PCD process in which autophagy and apoptosis may participate. The degeneration process was regulated by a caspase-dependent pathway, while the synthesis of 30K proteins along with silk gland degeneration may not be entirely dependent on caspase signals.

We aimed to understand the underlying mechanism that regulates successively expressed cuticular protein (CP) genes around pupation in Bombyx mori. Quantitative PCR was conducted to clarify the expression profile of CP genes and ecdysone-responsive transcription factor (ERTF) genes around pupation. Ecdysone pulse treatment was also conducted to compare the developmental profiles and the ecdysone induction of the CP and ERTF genes. Fifty-two CP genes (RR-1 13, RR-2 18, CPG 8, CPT 3, CPFL 2, CPH 8) in wing discs of B. mori were examined. Different expression profiles were found, which suggests the existence of a mechanism that regulates CP genes. We divided the genes into five groups according to their peak stages of expression. RR-2 genes were expressed until the day of pupation and RR-1 genes were expressed before and after pupation and for longer than RR-2 genes; this suggests different construction of exo- and endocuticular layers. CPG, CPT, CPFL and CPH genes were expressed before and after pupation, which implies their involvement in both cuticular layers. Expression profiles of ERTFs corresponded with previous reports. Ecdysone pulse treatment showed that the induction of CP and ERTF genes in vitro reflected developmental expression, from which we speculated that ERTFs regulate CP gene expression around pupation., (© 2016 The Royal Entomological Society.)

Heat shock proteins (Hsps) are involved in a variety of critical biological functions, including protein folding, degradation, and translocation and macromolecule assembly, act as molecular chaperones during periods of stress by binding to other proteins. Using expressed sequence tag (EST) and silkworm (Bombyx mori) transcriptome databases, we identified 27 cDNA sequences encoding the conserved J domain, which is found in DnaJ-type Hsps. Of the 27 J domain-containing sequences, 25 were complete cDNA sequences. We divided them into three types according to the number and presence of conserved domains. By analyzing the gene structures, intron numbers, and conserved domains and constructing a phylogenetic tree, we found that the DnaJ family had undergone convergent evolution, obtaining new domains to expand the diversity of its family members. The acquisition of the new DnaJ domains most likely occurred prior to the evolutionary divergence of prokaryotes and eukaryotes. The expression of DnaJ genes in the silkworm was generally higher in the fat body. The tissue distribution of DnaJ1 proteins was detected by western blotting, demonstrating that in the fifth-instar larvae, the DnaJ1 proteins were expressed at their highest levels in hemocytes, followed by the fat body and head. We also found that the DnaJ1 transcripts were likely differentially translated in different tissues. Using immunofluorescence cytochemistry, we revealed that in the blood cells, DnaJ1 was mainly localized in the cytoplasm., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Sericin was extracted from three strains of Thai Bombyx mori silk cocoons (white shell Chul1/1, greenish shell Chul3/2, and yellow shell Chul4/2) by a high-pressure and high-temperature technique. The characteristics of sericin extracted from different fractions (15, 45, and 60 min extraction process) were compared. No differences in amino acid composition were observed among the three fractions. For all silk strains, sericin extracted from a 15-min process presented the highest molecular weight. The biological potential of the different sericin samples as a bioadditive for 3T3 fibroblast cells was assessed. When comparing sericin extracted from three silk strains, sericin fractions extracted from Chul4/2 improved cell proliferation, while sericin from Chul 1/1 activated Type I collagen production to the highest extent. This study allows the natural variability of sericin obtained from different sources and extraction conditions to be addressed and provides clues for the selection of sericin sources.

Runt-related (RUNX) transcription factors are evolutionarily conserved either in vertebrate or invertebrate. Lozenge (Lz), a members of RUNX family as well as homologue of AML-1, functions as an important transcription factor regulating the hemocytes differentiation. In this paper, we identified and characterized RUNX family especially Lz in silkworm, which is a lepidopteran model insect. The gene expression analysis illustrated that BmLz was highly expressed in hemocytes throughout the whole development period, and reached a peak in glutonous stage. Over-expression of BmLz in silkworm accelerated the melanization process of hemolymph, and led to instantaneously up-regulation of prophenoloxidases (PPOs), which were key enzymes in the melanization process. Further down-regulation of BmLz expression by RNA interference resulted in the significant delay of melanization reaction of hemolymph. These findings suggested that BmLz regulated the melanization process of hemolymph by inducing PPOs expression, and played a critical role in innate immunity defense in silkworm., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The silk gland of Bombyx mori (BmSG) has gained significant attention by dint of superior synthesis and secretion of proteins. However, the application of BmSG bioreactor is still a controversial issue because of low yields of recombinant proteins. Here, a 3057 bp full-length coding sequence of Hpl was designed and transformed into the silkworm genome, and then the mutant (Hpl/Hpl) with specific expression of Hpl in posterior BmSG (BmPSG) was obtained. In the mutants, the transcription level of Fib-L and P25, and corresponding encoding proteins, did not decrease. However, the mRNA level of Fib-H was reduced by 71.1%, and Fib-H protein in the secreted fibroin was decreased from 91.86% to 71.01%. The mRNA level of Hpl was 0.73% and 0.74% of Fib-H and Fib-L, respectively, while HPL protein accounted for 18.85% of fibroin and 15.46% of the total amount of secreted silk protein. The exogenous protein was therefore very efficiently translated and secreted. Further analysis of differentially expressed gene (DEG) was carried out in the BmPSG cells and 891 DEGs were detected, of which 208 genes were related to protein metabolism. Reduced expression of endogenous silk proteins in the BmPSG could effectively improve the production efficiency of recombinant exogenous proteins.

Thermal induction of parthenogenesis (also known as thermal parthenogenesis) in silkworms is an important technique that has been used in artificial insemination, expansion of hybridization, transgenesis and sericultural production; however, the exact mechanisms of this induction remain unclear. This study aimed to investigate the gene expression profile in silkworms undergoing thermal parthenogenesis using RNA-seq analysis. The transcriptome profiles indicated that in non-induced and induced eggs, the numbers of differentially expressed genes (DEGs) for the parthenogenetic line (PL) and amphigenetic line (AL) were 538 and 545, respectively, as determined by fold-change ≥ 2. Gene ontology (GO) analysis showed that DEGs between two lines were mainly involved in reproduction, formation of chorion, female gamete generation and cell development pathways. Upregulation of many chorion genes in AL suggests that the maturation rate of AL eggs was slower than PL eggs. Some DEGs related to reactive oxygen species removal, DNA repair and heat shock response were differentially expressed between the two lines, such as MPV-17, REV1 and HSP68. These results supported the view that a large fraction of genes are differentially expressed between PL and AL, which offers a new approach to identifying the molecular mechanism of silkworm thermal parthenogenesis.

Lepidoptera is a large order of insects that have major impacts on humans as agriculture pests. The midgut is considered an important target for insect control. In the present study, 10 up-regulated, 18 down-regulated, and one newly emerged protein were identified in the transgenic Trichoderma-treated midgut proteome. Proteins related to stress response, biosynthetic process, and metabolism process were further characterized through quantitative real-time PCR (qPCR). Of all the identified proteins, the glutathione S-transferase sigma 2 (GSTs2) gene displayed enhanced expression when larvae were fed with Trichoderma wild-type or transgenic strains. Down regulation of GSTs2 expression by RNA interference (RNAi) resulted in inhibition of silkworm growth when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. Weight per larva decreased by 18.2%, 11.9%, and 10.7% in the untreated control, ddH2O, and GFP dsRNA groups, respectively, at 24h, while the weight decrease was higher at 42.4%, 28.8% and 32.4% at 72 h after treatment. Expression of glutathione S-transferase omega 2 (GSTo2) was also enhanced when larvae were fed with mulberry leaves treated with the transgenic Trichoderma strain. These results indicated that there was indeed correlation between enhanced expression of GSTs2 and the anti-stress response of silkworm larvae against Trichoderma. This study represents the first attempt at understanding the effects of transgenic organisms on the midgut proteomic changes in silkworm larvae. Our findings could not only broaden the biological control targets of insect at the molecular level, but also provide a theoretical foundation for biological safety evaluation of the transgenic Trichoderma strain., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The peptide-N (4)-(N-acetyl-β-D-glucosaminyl) asparagine amidase F (PNGase F) catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from glycoproteins. The PNGase F has broad substrate specificity and thus is extensively used for the structural and functional studies of the glycoproteins. In this study, we tried to produce active recombinant PNGase F as secreted and intracellular-expressed forms using baculovirus expression vector system (BEVS) through silkworm larvae or cultured cells. PNGase F itself contains potential N-linked glycosylation sites and we found that it was N-glycosylated when PNGase F secreted from silkworm cells. Intriguingly, the secreted recombinant PNGase F has the lower catalytic activity and self-digests its N-linked glycans and therefore this secreted form of this enzyme produced from BEVS is not appropriate for carbohydrate chain analysis. Instead, we successfully mass-produced (2.1 mg/20 silkworm larvae) and purified active recombinant PNGase F as an intracellular protein without N-glycosylations. Besides, we confirmed by directed mutagenesis that several amino acid residues are crucial for the function of PNGase F. Our results provide an alternative method for the mass production of active enzymes involved in the study of glycoproteins.

In the present study, we examined whether microorganisms collaterally ingested by insects with their food activate the innate immune system to confer systemic resistance against subsequent bacterial invasion. Silkworms orally administered heat-killed Pseudomonas aeruginosa cells showed resistance against intra-hemolymph infection by P. aeruginosa. Oral administration of peptidoglycans, cell wall components of P. aeruginosa, conferred protective effects against P. aeruginosa infection, whereas oral administration of lipopolysaccharides, bacterial surface components, did not. In silkworms orally administered heat-killed P. aeruginosa cells, P. aeruginosa growth was inhibited in the hemolymph, and mRNA amounts of the antimicrobial peptides cecropin A and moricin were increased in the hemocytes and fat body. Furthermore, the amount of paralytic peptide, an insect cytokine that activates innate immune reactions, was increased in the hemolymph of silkworms orally administered heat-killed P. aeruginosa cells. These findings suggest that insects sense bacteria present in their food by peptidoglycan recognition, which activates systemic immune reactions to defend the insects against a second round of infection.

Bombyx mori, a lepidopteran insect, is one of the earliest models for pattern recognition of Gram-negative bacteria, which may induce the IMD pathway for production of antibacterial peptides. So far, several recognition proteins have been reported in B. mori. However, the connection between pattern recognition of Gram negative bacteria and activation of BmRelish1, a transcription factor controlled by the IMD pathway remains largely unknown. In the present study, we identify BmCPT1, a cuticle protein bearing a Tweedle domain. Its gene expression is co-regulated by NF-kappaB and juvenile hormone signals. BmCPT1 is induced by Escherichia coli in fat bodies and hemocytes, but is constitutively expressed in the epidermis. In vitro binding assays indicate that BmCPT1 protein recognizes and binds to E. coli peptidoglycan. Post-transcriptionally modified BmCPT1 in the hemolymph binds to E. coli cells through interactions with peptidoglycan recognition protein-5 (BmPGRP5) and lipopolysaccharide binding protein (BmLBP). Transgenic overexpression of BmCPT1 causes the upregulated expression of BmRelish1 and clear induction of two gloverin genes. Therefore, BmCPT1 may work along with BmPGRP-S5 and BmLBP to recognize E. coli in the hemolymph and indirectly activate BmRelish1 to induce antimicrobial peptide synthesis., (Copyright © 2014. Published by Elsevier Ltd.)

Metabolic profiling of silkworm, especially the factors that affect silk synthesis at the metabolic level, is little known. Herein, metabolomic method based on gas chromatography-mass spectrometry was applied to identify key metabolic changes in silk synthesis deficient silkworms. Forty-six differential metabolites were identified in Nd group with the defect of silk synthesis. Significant changes in the levels of glycine and uric acid (up-regulation), carbohydrates and free fatty acids (down-regulation) were observed. The further metabolomics of silk synthesis deficient silkworms by decreasing silk proteins synthesis using knocking out fibroin heavy chain gene or extirpating silk glands operation showed that the changes of the metabolites were almost consistent with those of the Nd group. Furthermore, the increased silk yields by supplying more glycine or its related metabolite confirmed that glycine is a key metabolite to regulate silk synthesis. These findings provide important insights into the regulation between metabolic profiling and silk synthesis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Vitellogenin (Vg) is synthesized in the fat body of the female silkworm Bombyx mori and transported to the oocyte as a source of nutrition for embryo development. It is well known that ecdysone regulates physiological, developmental and behavioral events in silkworm. However, it is still not clear how the ecdysone regulates B. mori Vg (BmVg) transcription., Methods: Electrophoretic mobility shift assay (EMSA) and cell transfection assay were used to reveal whether BmBrC-Z2 is involved in regulating BmVg transcription. RNAi was employed to illustrate the function of BmBrC-Z2 in the silkworm egg formation and development., Results: (1) The transcription of BmVg can be induced by ecdysone in the female fat body. (2) Three putative BrC-Z2 cis-response elements were mapped to regions flanking the BmVg gene. (3) BmBrC-Z2 required direct binding to the cis-response elements on the BmVg promoter. (4) Over-expression of three BmBrC isoforms in the cell line showed that only BmBrC-Z2 could induce the BmVg promoter activity. (5) RNA interference (RNAi) of BmBrC-Z2 in female remarkably reduced BmVg synthesis and led to destructive affection on egg formation. The dsRNA of BmBrC-Z2 treated moths laid fewer and whiter eggs compared to the control., Conclusions: BmBrC-Z2 transported the ecdysone signal then regulated BmVg transcription directly to control vitellogenesis and egg formation in the silkworm., General Significance: The results of this study revealed that BmBrC-Z2 as a key factor to mediate ecdysone regulates reproduction in the silkworm., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The silkworm, Bombyx mori, is an important model of lepidoptera insect, and it has been used for several models of human diseases. In human being, long-term high-sugar diet can induce the occurrence of diabetes and other related diseases. Interestingly, our experiments revealed the high glucose diet also has a suppressive effect on the development of silkworms. To investigate the molecular mechanism by which high-glucose diet inhibited the midgut growth in silkworms, we employed comparative proteomic analysis to globally identify proteins differentially expressed in normal and high-glucose diet group silkworms. In all, 28 differently proteins were suppressed and 5 proteins induced in high-glucose diet group. Gene ontology analysis showed that most of these differently proteins are mainly involved in metabolic process, catalytic and cellular process. A development related protein, imaginal disk growth factor (IDGF), was further confirmed by western blot exclusively expressing in the normal diet group silkworms. Taken together, our data suggests that IDGF plays a critical role in impairing the development of silkworms by a high-glucose diet., Biological Significance: Glucose has been thought to play essential roles in growth and development of silkworm. In this paper, we certified firstly that high-glucose diet can suppress the growth of silkworm, and comparative proteomic was employed to reveal the inhibition mechanism. Moreover, an important regulation related protein (IDGF) was found to involve in this inhibition process. These results will help us get a deeper understanding of the relationship between diet and healthy. Furthermore, IDGF may be the critical protein for reducing the blood sugar in silkworm, and it may be used for screening human hypoglycemic drug. The work has not been submitted elsewhere for publication, in whole or in part, and all the authors have approved the manuscript., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In the silkworm Bombyx mori, pigment mutants with diverse body colors have been maintained throughout domestication for about 5000 years. The silkworm larval body color is formed through the mutual interaction of melanin, ommochromes, pteridines and uric acid. These pigments/compounds are synthesized by the cooperative action of various genes and enzymes. Previous reports showed that melanin, ommochrome and pteridine are increased in silkworm quail (q) mutants. To understand the pigment increase and alterations in pigment synthesis in q mutant, transcriptome profiles of the silkworm integument were investigated at 16 h after head capsule slippage in the fourth molt in q mutants and wild-type (Dazao). Compared to the wild-type, 1161 genes were differentially expressed in the q mutant. Of these modulated genes, 62.4% (725 genes) were upregulated and 37.6% (436 genes) were downregulated in the q mutant. The molecular function of differently expressed genes was analyzed by Blast2GO. The results showed that upregulated genes were mainly involved in protein binding, small molecule binding, transferase activity, nucleic acid binding, specific DNA-binding transcription factor activity and chromatin binding, while exclusively down-expressed genes functioned in oxidoreductase activity, cofactor binding, tetrapyrrole binding, peroxidase activity and pigment binding. We focused on genes related to melanin, pteridine and ommochrome biosynthesis; transport of uric acid; and juvenile hormone metabolism because of their importance in integument coloration during molting. This study identified differently expressed genes implicated in silkworm integument formation and pigmentation using silkworm q mutant. The results estimated the number and types of genes that drive new integument formation.

Many biological phenotypes of male and female silkworms (Bombyx mori) are quite different, and one of the major differences is the growth rate at various larval stages. Nutrient utilization by midgut varies with sexes. However, the molecular basis of this variation is not clear. To understand the molecular mechanism, comparative proteomic approach was employed to investigate the variation of midgut proteomes between male and female silkworms. Totally, 32 proteins that were grouped into four categories were differentially expressed and subsequently identified by mass spectrometry. Gene ontology analysis revealed that these proteins were attributed with biological functions such as binding, catalytic, and transporter, and these proteins were involved in biological process such as cellular process, localization, and metabolic process. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that these proteins were involved in pathways such as glycolysis, gluconeogenesis, oxidative phosphorylation, and purine metabolism. At transcription level, the expressional variation was confirmed for six identified proteins including muscle glycogen phosphorylase, uridine 5'-monophosphate synthase, cone cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha, ATP synthase, thiol peroxiredoxin, and serpin-2. This study provides useful information for understanding the mechanisms of nutrient absorption and the protein-protein interaction in the silkworm., (© The Author 2014. Published by Oxford University Press on behalf of the Entomological Society of America.)

A scaleless wing mutant of silkworm, Bombyx mori, has much fewer scales than wild type (WT). The scaleless phenotype was associated with tracheal system developmental deficiency and excessive apoptosis of scale cells. In this study, the wing discs proteins of WT and scaleless during pupation were studied using 2-DE and mass spectrometry. Of the 99 identified protein spots, four critical differentially expressed proteins between WT and scaleless were further verified using Q-PCR. At the first day of pupation (P0) in WT, imaginal disk growth factor (IDGF) was upregulated, whereas actin-depolymerizing factor 1 (ADF1) and profilin (PFN), which associated with cellular motility and cytoplasmic extension, were downregulated. We speculated their coaction counteracts the correct organization of the tracheal system in wing disc. Thiol peroxiredoxin (TPx) was upregulated in scaleless at P0, but its mRNA higher expression occurred in the day before pupation (S4). TPx could inhibit the formation of hydrogen peroxide, preventing the release of cytochrome C and activation of the caspase family protease. Its higher expression in scaleless was responsible for the apoptosis of scale cells delayed. The results provide further evidence that the scaleless phenotype was related to the tracheal system developmental deficiency and excessive apoptosis of scale cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The present study was conducted to clarify the regulatory mechanism of cuticular protein genes of Bombyx mori expressed in wing discs in the prepupal stage. BHR3, BHR4, E74A, and ßFTZ-F1 were successively expressed in wing discs at the pre-pupal stage. BHR3 showed different ecdysone responsiveness from other ecdysone-responsive transcription factors (ERTFs) and was induced by ecdysone addition but showed decrease by ecdysone removal after treatment (ecdysone pulse). BHR4 and E74A were induced by the ecdysone addition and by the ecdysone pulse. ßFTZ-F1 was not induced by the ecdysone addition but was induced by the ecdysone pulse. Thus, ERTFs showed different hormone responsiveness, resulting in the different expression timing. We selected cuticular protein genes that showed the same expression stage with each transcription factor and examined their ecdysone responsiveness. The developmental expression and ecdysone responsiveness of BmorCPH5, BmorCPR34, BmorCPR23, and BmorCPR99 resembled those of BHR3, BHR4, E74A, and ßFTZ-F1, respectively. The results of the transient reporter assay strongly suggested the regulation of each cuticular protein promoter by ERTF. These ERTFs regulated different cuticular protein genes and determined their expression timing and probably the nature of the cuticle layer of insects., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The vacuolar protein sorting 4 (Vps4) protein is essential for the multivesicular body (MVB) pathway, virus budding process and cytokinesis. Vps4 has been identified and characterized from many species, but not from silkworm Bombyx mori. In this study, we firstly identified and cloned the silkworm homologous gene for VPS4, expressed it in Escherichia coli, purified and characterized the protein designated as BmVps4. The BmVps4 cDNA contains an open reading frame of 1,314 bp, and encodes a protein of 438 amino acid residues. BmVps4 is of high sequence-similarity to Vps4 proteins from other species. The recombinant BmVps4 shows ATPase activity, which can be stimulated by Mg(2+) and inhibited by dominant mutations. Together, our data suggest BmVps4 is the genuine silkworm homologue of Vps4. To our knowledge, this is the first-time characterization of any silkworm MVB proteins. This study will facilitate further investigation of silkworm MVB pathway and its possible roles in the infection and budding of B. mori nuclear polyhedrosis virus (BmNPV), which is one of the most common and severe pathogens for silkworms. The cloned BmVps4 sequence is deposited in GenBank (Accession number GQ995504).

The 30K proteins comprise about 35% of the total embryo yolk proteins and function as storage proteins during embryonic development of the domesticated silkworm Bombyx mori. The most abundant components of hemolymph are 30K proteins in the early and middle pupal stages. In the present study, the 30K protein BmLP7 was purified from larval hemolymph by chromatography. We prepared the antibody of this protein and found that it could bind to both BmLP3 and BmLP7. We used western blotting to analyze the dynamic change of BmLP3 and BmLP7 proteins in the hemolymph during development and found their concentration decreased dramatically from day 4 pupae, which appears to be linked to their accumulation in the oocyte for forming yolk granule since then. We found BmLP3 and BmLP7 proteins reduced significantly in day 10 eggs (the day before hatching). The crude extract of the newly hatched larvae showed proteolytic activity against BmLP3 and BmLP7 and immunohistochemistry showed BmLP3 and BmLP7 were degraded in the embryonic gut lumen in day 10 eggs. These systematic studies of BmLP3 and BmLP7 reveal their synthesis, transportation and degradation, which could represent the experience of all 30K proteins.

Dynein light chains function as motor acceptor to recruit cargos, which play vital roles in many cellular processes such as intracellular transport and mitosis. In this study, we cloned and expressed the dynein light chain LC7 gene BmRobl in silkworm. The full-length cDNA of the dynein light chain LC7 gene BmRobl is 757 bp and encoded 97 aa polypeptide. Its molecular weight was ~11 kDa confirmed by western blotting. The tissue and stage expression profile of BmRobl drafted by real time PCR revealed that presence of BmRobl transcript was examined in all tissue but prominent expression level was found in brain, wing disc, ovary and testis. In metamorphosis wing disc, BmRobl reached to peak during the prepupae stage compared with the larval and pupal stages. This indicated BmRobl might involve in wing discs development during metamorphosis. Besides, in vitro wing discs 20E cultivation was performed and BmRobl expression profile was detected. The results demonstrated that the BmRobl gene was significantly up-regulated with increase of 20E concentration; the mRNA level peaked at 2 μg/ml of 20E. However, the BmRobl expression nearly has no change cultivated by 20 μg/ml 20E compared with 0.02 μg/ml 20E. These indicated that BmRobl expression might directly or indirectly induced by 20E, besides, high concentration 20E was far too inducible, suggesting that low concentrations of ecdysteroid induce cell proliferation, whereas high concentrations inhibit cell proliferation. Moreover, the transport role of BmRobl was clarified by UV challenge and vanadate cultivation. Both the real time PCR and western blotting results showed that the BmRobl gene was degraded with increase in the concentration of sodium vanadate combined with elongation in the time of UV challenge. Interestingly, compared with the single treatment group and non-treatment group, the group treated by both sodium vanadate and UV have severe degradation. This indicated that UV and vanadate might down-regulate BmRobl synergetically. It was further speculated that BmRobl may function as a positive regulator of the dynein complex during cellular transport.

This study demonstrates that a 30K protein was gradually synthesized in primary-cultured motoneurons from the accessory planta retractor (APR) of the 6th abdominal ganglion (APR6) in silkworm ventral ganglia through stimulation of hemolymph. An increase in 30K protein synthesis resulted in an inhibition of programmed cell death (PCD) of APR6 motoneurons. The 30K protein was gradually synthesized from the 30Kc6 gene of identified APR6s in day-6 4th instars to day-9 5th instar larvae, but synthesis of the 30K protein ceased in isolated APR6s of day-1 pupa, which normally begin to undergo PCD. When pupal APR6s were treated with larval hemolymph, however, the 30K protein was synthesized suggesting the existence of an anti-PCD factor in the larval hemolymph. An increase of 30K protein within the APR6s was confirmed by antiserum made against the recombinant 30K protein that originated from the APR 30Kc6 gene. Larval APR6, in which PCD was induced with 20-hydroxyecdysone (20E) added to the primary culture, exhibited some PCD characteristics of shrinkage of cell bodies, axonal fragmentation and loss of mitochondrial function. These results provide new insights on the survival or PCD of insect motoneurons through stimulation of hemolymph., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme for vitamin B(6) metabolism in animals. It catalyzes the ATP-dependent phosphorylation of pyridoxal, generating pyridoxal 5'-phosphate, an important cofactor for many enzymatic reactions. Bombyx mori PLK (BmPLK) is 10 or more residues shorter than mammalian PLKs, and some amino acid residues conserved in the PLKs from mammals are not maintained in the protein. Multiple sequence alignment suggested that amino acid residues Thr(47), Ile(54), Arg(88), Asn(121) and Glu(230) might play important roles in BmPLK. In this study, we used a site-directed specific mutagenesis approach to determine the functional significance of these particular amino acid residues in BmPLK. Our results demonstrated that the mutation of Asn(121) to Glu did not affect the catalytic function of BmPLK. The corresponding site-directed mutants of Thr(47) to Asn, Ile(54) to Phe, and Arg(88) to Ile displayed a decreased catalytic efficiency and an elevated Km value for substrate relative to the wild-type value, and no enzyme activity could be detected in mutant of Trp(230) to Glu. Circular dichroism analysis revealed that the mutation of Trp(230) to Glu resulted in mis-folding of the protein. Our results provided direct evidence that residue Trp(230) is crucial to maintain the structural and functional integrity of BmPLK. This study will add to the existing understanding of the characteristic of structure and function of BmPLK., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The hycu-ep32 gene of Hyphantria cunea NPV can inhibit Bombyx mori nucleopolyhedrovirus (BmNPV) multiplication in co-infected cells, but it is not known whether the overexpression of the hycu-ep32 gene has an antiviral effect in the silkworm, Bombyx mori. Thus, we constructed four transgenic vectors, which were under the control of the 39 K promoter of BmNPV (39 KP), Bombyx mori A4 promoter (A4P), hr3 enhancer of BmNPV combined with 39 KP, and hr3 combined with A4P. Transgenic lines were created via embryo microinjection using practical diapause silkworm. qPCR revealed that the expression level of hycu-ep32 could be induced effectively after BmNPV infection in transgenic lines where hycu-ep32 was controlled by hr3 combined with 39 KP (i.e., HEKG). After oral inoculation of BmNPV with 3 × 10(5) occlusion bodies per third instar, the mortality with HEKG-B was approximately 30% lower compared with the non-transgenic line. The economic characteristics of the transgenic lines remained unchanged. These results suggest that overexpression of an exogenous antiviral gene controlled by an inducible promoter and enhancer is a feasible method for breeding silkworms with a high antiviral capacity.

The posterior silk gland (PSG) is the most important suborgan responsible for the synthesis and secretion of silk core fibroin proteins in silkworm. Here, we performed genome-scale expression profiling analysis of silkworm PSG at the fourth molting (M4) and at day 1 (V1), day 3 (V3), day 5 (V5), and wandering stage (W) of the fifth instar by microarray analysis with 22 987 probes. We found that the five genes of silk proteins secreted from PSG including fibroin heavy (H) and light (L) chains, P25, seroin 1, and seroin 2 basically showed obvious up-regulation at V3 which lasted to V5, while slight down-regulation at W. The expression of translation-related genes including ribosomal proteins and translation initiation factors generally remained stable from M4 to V5, whereas it showed clear down-regulation at W. Clustering analysis of the 643 significantly differentially expressed transcripts revealed that 43 of the important genes including seroin 1 and sugar transporter protein had co-expression patterns which were consistent with the rate changes of fibroin synthesis and PSG growth. Pathway analysis disclosed that the genes in different clusters might have co-regulations and direct interactions. These genes were supposed to be involved in the fibroin synthesis and secretion. The differential expression of several hormone-related genes also suggested their functions on the regulation of PSG development and fibroin synthesis. 2D gel-based proteomics and phosphoproteomics profiling revealed that the phosphorylated proteins accounted for no more than one-sixth of the total proteins at each stage, which was much lower than the level in normal eukaryotic cells. Changes in the phosphorylation status and levels of several proteins such as actin-depolymerizing factor 1 and enolase might be deeply involved in fibroin secretion and tissue development. Shotgun proteomic profiling combined with label-free quantification analysis on the PSG at V3, V5, and W revealed that many small heat shock proteins (sHSP) were specially expressed at W, which was substantially consistent with the results from 2-DE analysis, and implied the close correlations of sHSP with the physiological states of PSG at W. A majority of significantly up-regulated proteins at V5 were related to ribosome pathway, which was different from the microarray results, implying that the translation-level regulation of ribosomal proteins might be critical for fibroin synthesis. In contrast, the ubiquitin-proteasome pathway related proteins appeared obviously up-regulated at W, suggesting that the programmed cell death process of PSG cells might be started before cocooning.

Sericulture has been greatly advanced by applying hybrid breeding techniques to the domesticated silkworm, Bombyx mori, but has reached a plateau during the last decades. For the first time, we report improved silk yield in a GAL4/UAS transgenic silkworm. Overexpression of the Ras1(CA) oncogene specifically in the posterior silk gland improved fibroin production and silk yield by 60%, while increasing food consumption by only 20%. Ras activation by Ras1(CA) overexpression in the posterior silk gland enhanced phosphorylation levels of Ras downstream effector proteins, up-regulated fibroin mRNA levels, increased total DNA content, and stimulated endoreplication. Moreover, Ras1 activation increased cell and nuclei sizes, enriched subcellular organelles related to protein synthesis, and stimulated ribosome biogenesis for mRNA translation. We conclude that Ras1 activation increases cell size and protein synthesis in the posterior silk gland, leading to silk yield improvement.

Yellow proteins form a large family in insects. In Drosophila melanogaster, there are 14 yellow genes in the genome. Previous studies have shown that the yellow gene is necessary for normal pigmentation; however, the roles of other yellow genes in body coloration are not known. Here, we provide the first evidence that yellow-e is required for normal body color pattern in insect larvae. In two mutant strains, bts and its allele bts2, of the silkworm Bombyx mori, the larval head cuticle and anal plates are reddish brown instead of the white color found in the wild type. Positional cloning revealed that deletions in the Bombyx homolog of the Drosophila yellow-e gene (Bmyellow-e) were responsible for the bts/bts2 phenotype. Bmyellow-e mRNA was strongly expressed in the trachea, testis, and integument, and expression markedly increased at the molting stages. This profile is quite similar to that of Bmyellow, a regulator of neonatal body color and body markings in Bombyx. Quantitative reverse transcription-PCR analysis showed that Bmyellow-e mRNA was heavily expressed in the integument of the head and tail in which the bts phenotype is observed. The present results suggest that Yellow-e plays a crucial role in the pigmentation process of lepidopteran larvae.

MicroRNAs (miRNAs) are pivotal regulators involved in various physiological and pathological processes via their post-transcriptional regulation of gene expressions. We sequenced 14 libraries of small RNAs constructed from samples spanning the life cycle of silkworms, and discovered 50 novel miRNAs previously not known in animals and verified 43 of them using stem-loop RT-PCR. Our genome-wide analyses of 27 species-specific miRNAs suggest they arise from transposable elements, protein-coding genes duplication/transposition and random foldback sequences; which is consistent with the idea that novel animal miRNAs may evolve from incomplete self-complementary transcripts and become fixed in the process of co-adaptation with their targets. Computational prediction suggests that the silkworm-specific miRNAs may have a preference of regulating genes that are related to life-cycle-associated traits, and these genes can serve as potential targets for subsequent studies of the modulating networks in the development of Bombyx mori.

To identify Bombyx mori genes involved in the early response to injury and microbial challenge, we performed genome-wide gene expression-profiling experiments using oligonucleotide DNA microarrays. Of approximately 23,000 genes examined, 465 displayed changes in mRNA expression levels. Of these, 306 were induced and 159 were repressed in response to injury (injection with phosphate buffer saline) or challenges by Gram-negative (Serratia marcescens), Gram-positive bacteria (Staphylococcus aureus), or fungus (Beauveria bassiana). Many of these differentially expressed genes can be assigned to specific functional groups of the innate immune response, including recognition, signaling, melanization and coagulation, and antimicrobial peptides. Seventeen percent of differentially expressed genes encode proteins with no obvious similarity to known functional domains. Of particular interest is a member of the juvenile hormone-binding protein family, which was highly induced by both injury and microbial challenges. The possible role of juvenile hormone in innate immunity is discussed.

Based on the character of strong promoter of the fibroin gene and high level secretion of fibroin of Bombyx mori, we amplified the promoter of heavy chain gene (Fib-H) and its downstream signal peptide sequence (FibHS) by PCR. After that, we cloned the PCR product in pBluescriptII SK (+) to form the vector pSK-FibHS and analyzed its sequence. The sequence identity was 99% comparable to that of the reported sequence by Blast on line. Then we digested pSk-Ser-DsRed-PolyA with Sal IKpn I to get DsRed-PolyA DNA fragment and subcloned it into vector pSK-FibHS to generate a transitorily secretory expression vector pSK-FibHS-DsRed-PolyA. After identified the recombinant plasmid by restriction enzyme digestion, we transfected pSK-FibHS-DsRed-PolyA into BmN cells by liposome. From the cells transfected with the recombinant vector, what the red fluorescence could be detected verified that the recombinant vector could express DsRed in BmN cells transiently. Furthermore, when silkworm had been injected with the recombinant vector pSK-FibHS-DsRed-PolyA, red fluorescence could be observed in the lumen of silk gland of silkworm. The result indicated that DsRed expressed transiently and was secreted into lumen of the silk gland. Therefore, we supposed that the cloned sequence (FibHS) possessed signal peptide bio-function. Moreover, this study would lay a foundation for the research on secretory expression of exogenous gene by silk gland bioreactor.

Spiders and silkworms employ the complex flow of highly concentrated silk solution as part of silk fiber spinning. To understand the role of fluidic forces in this process, the flow of silk solution in the spider major ampullate and silkworm silk glands was investigated using numerical simulation. Our simulations demonstrate significant differences between flow in the spider and silkworm silk glands. In particular, shear flow effects are shown to be much greater in the spider than the silkworm, the silkworm gland exhibits a much different flow extension profile than the spider gland, and the residence time within the spider gland is eight times greater than in the silkworm gland. Lastly, simulations on the effect of spinning speed on the flow of silk solution suggest that a critical extension rate is the initiating factor for fiber formation from silk solution. These results provide new insight into silk spinning processes and will guide the future development of novel fiber spinning technologies.

Mulberry latex contains extremely high concentrations of alkaloidal sugar mimic glycosidase inhibitors, such as 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1) and 1-deoxynojirimycin (DNJ). Although these compounds do not harm the silkworm, Bombyx mori, a mulberry specialist, they are highly toxic to insects that do not normally feed on mulberry leaves. D-AB1 and DNJ are strong inhibitors of alpha-glucosidases (EC 3.2.1.20); however, they do not affect the activity of beta-fructofuranosidases (EC 3.2.1.26). Although alpha-glucosidase genes are found in a wide range of organisms, beta-fructofuranosidase genes have not been identified in any animals so far. In this study, we report the identification and characterization of beta-fructofuranosidase genes (BmSuc1 and BmSuc2) from B. mori. The BmSuc1 gene was highly expressed in the midgut and silk gland, whereas the expression of BmSuc2 gene was not detected. BmSuc1 encodes a functional beta-fructofuranosidase, whose enzymatic activity was not inhibited by DNJ or D-AB1. We also showed that BmSUC1 protein localized within the midgut goblet cell cavities. Collectively, our data clearly demonstrated that BmSuc1 serves as a sugar-digesting enzyme in the silkworm physiology. This anomalous presence of the beta-fructofuranosidase gene in the B. mori genome may partly explain why the silkworm can circumvent the mulberry's defense system.

A period-timeless (per-tim) based feedback loop is considered to be essential in generating circadian rhythms in Drosophila melanogaster. In addition to transcriptional regulation, the post-transcriptional modification is essential to the circadian oscillation of core clock proteins in the circadian system. Here we present expression profiles of the catalytic subunit of casein kinase 2alpha (ck2alpha) and casein kinase 2beta (ck2beta) in Bombyx mori. Southern blot analyses showed that ck2alpha and ck2beta of B. mori were single copy genes. Northern blot analyses demonstrated that both subunits were expressed in eggs, larval heads, adult heads, testes and ovaries. In situ hybridization analyses indicated that subunits were expressed in brain neurons expressing PER-like protein. Surprisingly, antisense RNAs of ck2alpha and ck2beta were also detected in the putative clock neurons. Temporal expressions of ck2alpha and ck2beta mRNAs were constant in adult heads under LD12:12. The core clock genes per and tim showed daily fluctuations of mRNA abundance in the embryonic stage that is photoperiod sensitive period to determine egg diapause in the next generation whereas the expression of ck2alpha and ck2beta was constant. No evidence supports that ck2alpha and ck2beta of B. mori were transcriptionally regulated by circadian oscillation, but histological data show a close association of ck2alpha and ck2beta with circadian system in B. mori.