D‐3‐phosphoglycerate dehydrogenase from the silkworm Bombyx mori: Identification,functional characterization,and expression

D‐3‐phosphoglycerate dehydrogenase (PHGDH) is a key enzyme involved in the synthesis of l ‐serine. Despite the high serine content in silk proteins and the crucial role of PHGDH in serine biosynthesis, PHGDH has not been described in silkworms to date. Here, we identified PHGDH in the silkworm Bombyx mori and evaluated its biochemical properties. On the basis of the amino acid sequence and phylogenetic tree, this PHGDH has been categorized as a new type and designated as bmPHGDH. The recombinant bmPHGDH was overexpressed and purified to homogeneity. Kinetic studies revealed that PHGDH uses NADH as a coenzyme to reduce phosphohydroxypyruvate. High expression levels of bmphgdh messenger RNA (mRNA) were observed in the middle part of the silk gland and midgut in a standard strain of silkworm. Moreover, a sericin‐deficient silkworm strain displayed reduced expression of bmphgdh mRNA. These findings indicate that bmPHGDH might play a crucial role in the provision of l ‐serine in the larva of B. mori.     

Expression of the Japanese oak silkworm Antheraea yamamai fibroin gene in the domesticated silkworm Bombyx mori

Abstract To understand the evolutionary conservation of the gene expression mechanism and secretion machinery between Antheraea and Bombyx fibroins, we introduced the genomic A. yamamai fibroin gene into the domesticated silkworm, B. mori. The spliced A. yamamai fibroin mRNA appeared only in the posterior region of the silk gland of the transgenic silkworm, suggesting that the functions of the fibroin promoter region and the splicing machinery are conserved between these two species. The A. yamamai fibroin protein was detected in the lumen of the silk gland of the transgenic silkworm, albeit at lower levels compared with the B. mori‐type fibroin. We found a strong degeneration of the posterior region of the silk gland of the transgenic silkworm. As a result, the cocoon shell weight was much lower in the transgenic silkworm than in the non‐transgenic line. These results indicate that the promoter function and splicing machinery are well conserved between A. yamamai and B. mori but that the secretion mechanism of fibroin is diversified between the two.     

Expression pattern and tissue localization of the class B scavenger receptor BmSCRBQ4 in Bombyx mori

Class B scavenger receptors (SR‐Bs) are cell surface glycoproteins involved in various physiological processes in vivo, including the transport and metabolism of lipids, binding and phagocytosis of xenobiotics, and signaling. But little information is available about silkworm SR‐Bs; it is necessary to study these SR‐Bs for revealing their function. In this study, we cloned the full‐length coding sequence of BmSCRBQ4, a SR‐B gene from the silkworm Bombyx mori L. We found that the BmSCRBQ4 gene consists of nine exons and eight introns, with an open reading frame of 1371 bp encoding 456 amino acids. Gene expression studies determined that BmSCRBQ4 messenger RNA (mRNA) was expressed in unfertilized eggs, during embryonic development and throughout the majority of the larval period. Expression of mRNA was detected in the mid gut, middle silk gland, posterior silk gland, head, integumentum, fat body, testes and the ovaries of the larval B. mori Dazao strain, as well as in the silkworm cell lines BmN and BmE. Protein expression studies found BmSCRBQ4 protein was expressed only in the testes, fat body and middle silk gland of larvae, as well as in the silkworm cell lines BmN and BmE. The BmSCRBQ4 protein showed variability in banding patterns in different tissues and cells when analyzed by Western blotting. Immunohistochemical staining showed that the BmSCRBQ4 protein localizes to the constitutive membranes or cellular membranes of these tissues. These results indicated that BmSCRBQ4 gene may play some physiologically relevant roles at the cell surface in each tissue.     

Oxidative stress,DNA damage,and histological alterations in Bombyx mori exposed orally to pesticide dimethoate

Pesticides are an essential part of agricultural practices that ward off pathogens and diseases from the agricultural crop. However, apart from target organisms, these chemicals also have adverse effects on non-target organisms. Dimethoate is an insecticide used extensively in agriculture and horticulture practices worldwide. We used the silkworm Bombyx mori as a model organism to study the effect of commercial formulation of dimethoate (Dimethoate-30% EC) on the gut, silk gland, and fat body tissues. LD₅₀ of dimethoate-30% EC on silkworm (B. mori) was 997 ppm, as reported in a previous study. We used concentrations of 25, 50, and 100 ppm in our experiments. Our results showed that sub-lethal doses of dimethoate caused weight loss and induced damage at the histological level to the mid-gut, silk gland, and fat body of B. mori. It also caused a decrease in the level of antioxidants like CAT, SOD, GPx, GSH, and GST, indicating that dimethoate has produced a shift of ROS balance towards free radical generation and therefore resulted in overall damage to this organism. Sub-lethal doses of this pesticide also caused lipid peroxidation in the silk gland, gut, and fat body of B. mori, damaging these tissues. The disruption was also seen in the mid-gut and middle silk gland at the DNA level, where it caused single-strand breaks, as was revealed by single cell gel electrophoresis studies. Damage at histological, biochemical, and molecular levels was most extreme at a concentration of 100 ppm, the highest sub-lethal concentration given to B. mori.     

Fenoxycarb modulates ecdysone receptor B1 and programmed cell death of the anterior silk gland of silkworm Bombyx mori

Fenoxycarb, O‐ethyl N‐(2‐(4‐phenoxyphenoxy)‐ethyl) carbamate has been shown to be one of the most potent juvenile hormone analogues against a variety of insect species. In the present study, topical application of fenoxycarb to fifth‐instar larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) was performed immediately after the fourth ecdysis (on day 0), day 3 and day 6 of the instar and then its effects on the anterior silk glands (ASG) and ecdysone receptor B1 (EcR‐B1) protein were investigated during larval pupal development. Fenoxycarb application increased the instar length and prevented metamorphic events, depending on the application time. The ASGs of B. mori undergo programmed cell death during the larval–pupal metamorphosis and an insect steroid, 20‐hydroxyecdysone (20E), triggers this cell death. The exact mechanism by which 20E and juvenile hormone regulates programmed cell death in insect tissues is poorly understood. To gain insights into how juvenile hormone regulates metamorphic events like programmed cell death in the anterior silk glands, we analyzed the progression of programmed cell death with morphological observations and biochemical experiments like acid phosphatase activity and DNA electrophoresis. Then we examined the EcR‐B1 protein levels and their relationships with programmed cell death. Our results indicated that fenoxycarb modulates programmed cell death of the anterior silk glands and EcR‐B1 protein level, depending on the application time. Fenoxycarb may exhibit its effects in at least two different ways: (i) acting on prothoracic gland secretory activity; and/or (ii) regulation of EcR‐B1 expression in the anterior silk glands for programmed cell death process.     

The expression analysis of silk gland-enriched intermediate-size non-coding RNAs in silkworm Bombyx mori

Small non-protein coding RNAs （ncRNAs） play important roles in development, stress response and other cellular processes. Silkworm is an important model for studies on insect genetics and control of Lepidopterous pests. We have previously identified 189 novel intermediate-size ncRNAs in silkworm Bombyx mori, including 40 ncRNAs that showed altered expression in different developmental stages. Here we characterized the functions of these 40 ncRNAs by measuring their expressions in six tissues of the fifth instar larvae using Northern blot and real-time polymerase chain reaction assays. We identified nine ncRNAs （four small nucleolar RNAs and five unclassified ncRNAs） that were enriched in silk gland, including four ncRNAs that showed silk gland-specific expression. We further showed that three of nine silk gland-enriched ncRNAs were predominantly expressed in the anterior silk gland, whereas another three ncRNAs were highly accumulated in the posterior silk gland, suggesting that they may play different roles in fibroin synthesis. Furthermore, an unclassified ncRNA, Bm- 152, exhibited converse expression pattem with its antisense host gene gartenzwerg in diverse tissues, and might regulate the expression of gartenzwerg through RNA-protein complex. In addition, two silk gland-enriched ncRNAs Bm-102 and Bm-159 can be found in histone modification complex, which indicated that they might play roles through epigenetic modifications. Taken together, we provided the first expression and preliminary functional analysis of silk gland-enriched ncRNAs, which will help understand the molecular mechanism of silk gland-development and fibroin synthesis.     

Transgenic protein production in silkworm silk glands requires cathepsin and chitinase of Autographa californica multicapsid nucleopolyhedrovirus

The silkworm Bombyx mori represents an established in vivo system for the production of recombinant proteins. Baculoviruses have been extensively investigated and optimised for the expression of high protein levels inside the haemolymph of larvae and pupae of this lepidopteran insect. Current technology includes deletion of genes responsible for the activity of virus-borne proteases, which in wild-type viruses, cause liquefaction of the host insect and enhance horizontal transmission of newly synthesised virus particles. Besides the haemolymph, the silk gland of B. mori provides an additional expression system for recombinant proteins. In this paper, we investigated how silk gland can be efficiently infected by a Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV). We demonstrated that the viral chitinase and the cysteine protease cathepsin are necessary to permit viral entry into the silk gland cells of intrahaemocoelically infected B. mori larvae. Moreover, for the first time, we showed AcMNPV crossing the basal lamina of silk glands in B. mori larvae, and we assessed a new path of infection of silk gland cells that can be exploited for protein production.     

Carbonic anhydrase generates a pH gradient in Bombyx mori silk glands

Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown.In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient.B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.     

Preventive effects of N‐acetyl‐l‐cysteine against imidacloprid intoxication on Bombyx mori larvae

Imidacloprid, a widely used neonicotinoid insecticide, is toxic to silkworm (Bombyx mori). To explore whether N‐acetyl‐l ‐cysteine (NAC) has an effect on preventing silkworm (B. mori) from toxification caused by imidacloprid, we fed the fifth‐instar larvae with mulberry leaves dipped in 200 mg/L NAC solution before exposing in imidacloprid, and investigated the silkworm growth, survival rate, feed efficiency, cocoon quality, and the activities of antioxidant enzymes in midgut. The results showed that addition of NAC could significantly increase body weight, survival rate, and feed efficiency of imidacloprid poisoned silkworm larvae (P < 0.05), as well as cocoon mass, cocoon shell mass, and the ratio of cocoon shell (P < 0.05). Furthermore, it could significantly promote the activities of the antioxidant enzymes including superoxide dismutase, catalase, and glutathione peroxide in the midgut of fifth‐instar larvae under imidacloprid exposure at the late stage of treatment. In addition, it also could downregulate the malondialdehyde content. The results of our findings proved that the added NAC may have some beneficial effects on protection or restoration of antioxidant balance in imidacloprid exposed larvae.     

Identification and characterization of the diuretic hormone 31 receptor in the silkworm Bombyx mori

The receptor for diuretic hormone 31 (DH31R) was identified in the silkworm Bombyx mori. A heterologous expression system revealed that an orphan G-protein coupled receptor, BNGR-B1, responded to DH31 and upregulated the intracellular cAMP level. DH31R (BNGR-B1) was predominantly expressed in the anterior silk gland, midgut, and ovary, whereas DH31 was predominantly expressed in the central nervous system and midgut.     

A palmitoyltransferase Approximated gene Bm-app regulates wing development in Bombyx mori

The silkworm Bombyx mori is an important lepidopteran model insect in which many kinds of natural mutants have been identified.However,molecular mechanisms of most of these mutants remain to be explored.Here we report the identification of a gene Bm-app is responsible for the silkworm minute wing(mw)mutation which exhibits exceedingly small wings during pupal and adult stages.Compared with the wild type silkworm,relative messenger RNA expression of Bm-app is significantly decreased in the ul 1 mutant strain which shows mw phenotype.A 10 bp insertion in the putative promoter region of the Bm-app gene in mw mutant strain was identified and the dual luciferase assay revealed that this insertion decreased Bm-app promoter activity.Furthermore,clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases-mediated depletion of the Bm-app induced similar wing defects which appeared in the mw mutant,demonstrating that Bm-app controls wing development in B.mori.Bm-app encodes a palmitoyltransferase and is responsible for the palmitoylation of selected cytoplasmic proteins,indicating that it is required for cell mitosis and growth during wing development.We also discuss the possibility that Bm-app regulates wing development through the Hippo signaling pathway in B.mori.     

Photoreception in decapitated larvae of silkworm Bombyx mori

To investigate the photoreception that controls daily oscillations at the periphery in insects, we decapitated larvae of the silkworm Bombyx mori (Lepidoptera: Bombycidae) by ligature, and observed rhythms in their peripheral tissues under several light conditions. We measured the mRNA expression of period (per) and timeless (tim), which are homologues of Drosophila clock genes that function in the core oscillator of the circadian clock system. The expression of both per and tim significantly changed in the midgut, Malpighian tubules and silk glands of decapitated larvae exposed to photophase and scotophase that were reversed from the original daily light–dark cycle under which the larvae were housed. Under constant darkness, the daily expression of tim mRNA persisted for at least one cycle in the midgut and silk gland. In addition, an appropriate light stimulus under constant darkness induced a significant phase shift in the endogenous timing system (probably a circadian clock) that determined peak levels of tim mRNA expression in the midgut and silk glands of decapitated larvae. Since light regulated the gene expression rhythm in peripheral tissues of decapitated silkworm larvae, neither the brain nor eyes were essential for photoreception to control daily oscillations in these tissues. Thus, peripheral tissues in insects might directly use light even at the larval stage.     

Shotgun proteomic analysis of the Bombyx mori anterior silk gland: An insight into the biosynthetic fiber spinning process

The Bombyx mori anterior silk gland (ASG) is a natural fiber manipulator for the material provided by the middle and posterior silk glands. In view of the significant role of the ASG in the liquid–crystal spinning process, a shotgun proteomics approach was taken to study the relationship between the function of proteins in the silkworm ASG and the spinning mechanism. A total of 1132 proteins with 7647 unique peptides were identified in the ASG dataset including some involved in the cuticle, ion transportation, energy metabolism, and apoptosis. Two putative cuticle‐specific proteins were highly and specifically expressed in the ASG; therefore, the ASG dataset could provide clues for comprehensive understanding of the natural silk spinning mechanism in the silkworm. All MS data have been deposited in the ProteomeXchange with identifier PXD000090.     

Gene expression analysis in the larval silk gland of the eri silkworm Samia ricini

Insects produce silk for a range of purposes. In the Lepidoptera, silk is utilized as a material for cocoon production and serves to protect larvae from adverse environmental conditions or predators. Species in the Saturniidae family produce an especially wide variety of cocoons, for example, large, golden colored cocoons and those with many small holes. Although gene expression in the silk gland of the domestic silkworm (Bombyx mori L.) has been extensively studied, considerably fewer investigations have focused on members of the saturniid family. Here, we established expression sequence tags from the silk gland of the eri silkworm (Samia ricini), a saturniid species, and used these to analyze gene expression. Although we identified the fibroin heavy chain gene in the established library, genes for other major silk proteins, such as fibroin light chain and fibrohexamerin, were absent. This finding is consistent with previous reports that these latter proteins are lacking in saturniid silk. Recently, a series of fibrohexamerin‐like genes were identified in the Bombyx genome. We used this information to conduct a detailed analysis of the library established here. This analysis identified putative homologues of these genes. We also found several genes encoding small silk protein molecules that are also present in the silk of other Lepidoptera. Gene expression patterns were compared between eri and domestic silkworm, and both conserved and nonconserved expression patterns were identified for the tested genes. Such differential gene expression might be one of the major causes of the differences in silk properties between these species. We believe that our study can be of value as a basic catalogue for silk gland gene expression, which will yield to the further understanding of silk evolution.     

Evolutionarily Conserved Repulsive Guidance Role of Slit in the Silkworm Bombyx mori

Axon guidance molecule Slit is critical for the axon repulsion in neural tissues, which is evolutionarily conserved from planarians to humans. However, the function of Slit in the silkworm Bombyx mori was unknown. Here we showed that the structure of Bombyx mori Slit (BmSlit) was different from that in most other species in its C-terminal sequence. BmSlit was localized in the midline glial cell, the neuropil, the tendon cell, the muscle and the silk gland and colocalized with BmRobo1 in the neuropil, the muscle and the silk gland. Knock-down of Bmslit by RNA interference (RNAi) resulted in abnormal development of axons and muscles. Our results suggest that BmSlit has a repulsive role in axon guidance and muscle migration. Moreover, the localization of BmSlit in the silk gland argues for its important function in the development of the silk gland.     

Some autophagic and apoptotic features of programmed cell death in the anterior silk glands of the silkworm,Bombyx mori

Programmed cell death has been subdivided into two major groups: apoptosis and autophagic cell death. The anterior silk gland of Bombyx mori degenerates during larval-pupal metamorphosis. Our findings indicate that two types of programmed cell death features are observed during this physiological process. During the prepupal period, pyknosis of the nucleus, cell detachment and membrane blebbing occur and they are the first signs of programmed cell death in the anterior silk glands. According to previous studies, all of these morphological appearences are common for both cell death types. Autophagy features are also exhibited during the prepupal period. One of the lysosomal marker enzymes, acid phosphatase, levels are high during this period then decrease gradually. Vacuole formation begins to appear first at the basal surface of the cell, then expands to the apical surface just before the larval pupal ecdysis. After larval-pupal ecdysis, DNA fragmentation, which is the obvious biochemical marker of apoptosis, is detected in agarose gel electrophoresis which also shows that caspase-like enzyme activities occur during the programmed cell death process of the anterior silk glands. Apoptosis and autophagic cell death interact with each other during the degeneration process of the anterior silk gland in Bombyx mori and this interaction occurs at a late phase of cell death. We suggest that only apoptotic cell death not enough for whole gland degeneration and that more effective degeneration occurs with this cooperation.

Addendum to: Goncu E, Parlak O. Morphological changes and patterns of ecdysone receptor B1 immunolocalization in the anterior silk gland undergoing programmed cell death in the silkworm, Bombyx mori. Acta Histochem 2008; In press.     

Discovering genes responsible for silk synthesis in Bombyx mori by piggyBac‐based random insertional mutagenesis

Silkworm mutants are valuable resources for both transgenic breeding and gene discovery. PiggyBac-based random insertional mutagenesis has been widely used in gene functional studies. In order to discover genes involved in silk synthesis, a piggyBac-based random insertional library was constructed using Bombyx mori, and the mutants with abnormal cocoon were particularly screened. By this means, a “thin cocoon” mutant was identified. This mutant revealed thinner cocoon shell and shorter posterior silk gland (PSG) compared with the wild type. The messenger RNA (mRNA) levels of all the three fibroin genes, including Fib-H, Fib-L and P25, were significantly down-regulated in the PSG of mutants. Four piggyBac insertion sites were identified in Aquaporin (AQP), Longitudinals lacking protein-like {Lola), Glutamyl aminopeptidase-like (GluAP) and Loc101744460. The mRNA levels of all the four genes were significantly altered in the silk gland of mutants. In particular, the mRNA amount of AQP, a gene responsible for the regulation of osmotic pressure, decreased dramatically immediately prior to the spinning stage in the anterior silk gland of mutants. The identification of the genes disrupted in the “thin cocoon” mutant in this study provided useful information for understanding silk production and transgenic breeding of silkworms in the future.