Identification and expression of the achaete-scute complex in the silkworm, Bombyx mori

Recently, the study of achaete-scute ( AS-C ) homologues has contributed enormously to understanding of gene duplication and function evolution, particularly in Diptera. We identified four AS-C homologue genes in the silkworm, Bombyx mori , referred to as BmASH , BmASH2 , BmASH3 , and Bmase . The complex displayed tandem array structure in the genome. Analysis of spatial expression profiles showed that they all were expressed in obviously higher levels in wing disc than in other tissues, suggesting that they might play important roles in the development of the wing. Furthermore, we found that their expression profiles in the wing discs were mostly correlated with the development of the scales, especially the BmASH gene. RNA interference results further indicated that BmASH was necessary for scale formation in silkworm wing.     

Lipopolysaccharide elicits expression of immune-related genes in the silkworm, Bombyx mori

Lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria, was found to be unable to activate immune-related genes in Drosophila melanogaster . In contrast, highly purified LPS elicited immune-related gene expression in the fat body of Bombyx mori . However, the level of activation by highly purified LPS was lower than crude LPS and peptidoglycan. Furthermore, synthetic lipid A also activated these genes, suggesting that B. mori possesses unknown signal pathways to activate immune-related genes by LPS. Up-regulation of antimicrobial peptide genes by highly purified LPS was not confirmed in the immune-responsive cell line, NIAS-Bm-aff3, suggesting that some factors necessary for signal transduction activated by LPS are deficient in this cell line.     

Two Tor genes in the silkworm Bombyx mori

Target of rapamycin (TOR), a member of the phosphatidylinositol kinase‐related kinase family, plays a critical role in the regulation of growth, metabolism, development and survival, at both the cellular and the organismal levels. Two paralogous Tor genes, BmTor1 and BmTor2, were identified as a pair of inverted repeats in the genome of the silkworm Bombyx mori. The synteny of BmTor1 and CG8360 indicates that BmTor1 is the orthologue while BmTor2 is a duplicate. Analyses of the two BmTor genes at both the nucleotide and amino acid levels reveal that they are evolutionally and structurally conserved. The two BmTor genes had similar expression patterns of tissue distribution with highest levels in the nervous system, and nearly identical developmental change profiles with maximal levels during the 4^th‐larval‐moulting and the larval–pupal transition stages. Furthermore, both BmTor genes were up‐regulated by either starvation or the moulting hormone 20‐hydroxyecdysone (20E), while BmTor2 was more sensitive to both treatments than BmTor1. For the first time, we have identified two copies of the Tor gene in a higher eukaryote, which are induced by starvation and 20E during the larval moulting and the larval–pupal transition stage.     

Characterization of an entomopathogenic fungi target integument protein,Bombyx mori single domain von Willebrand factor type C,in the silkworm,Bombyx mori

The insect cuticle works as the first line of defence to protect insects from pathogenic infections and water evaporation. However, the old cuticle must be shed in order to enter the next developmental stage. During each ecdysis, moulting fluids are produced and secreted into the area among the old and new cuticles. In a previous study, the protein Bombyx mori single domain von Willebrand factor type C (BmSVWC; BGIBMGA011399) was identified in the moulting fluids of Bo. mori and demonstrated to regulate ecdysis. In this study we show that in Bo. mori larvae, BmSVWC primarily locates to the integument (epidermal cells and cuticle), wing discs and head. During the moulting stage, BmSVWC is released into the moulting fluids, and is then produced again by epidermal cells after ecdysis. Fungal infection was shown to decrease the amount of BmSVWC in the cuticle, which indicates that BmSVWC is a target protein of entomopathogenic fungi. Thus, BmSVWC is mainly involved in maintaining the integrity of the integument structure, which serves to protect insects from physical damage and pathogenic infection.     

Bmmar6, a second mori subfamily mariner transposon from the silkworm moth Bombyx mori

A second member of the divergent mori subfamily of mariner transposons, Bmmar6, is described from the silkworm moth Bombyx mori genome. A confident consensus sequence for Bmmar6 was obtained from a single genomic copy, 17 EST sequences, and the direct sequencing of a 'family' sequence from an amplification of all full-length genomic copies. Bmmar6 is most similar to Bmmar1 in the mori subfamily, which now also includes several fly and nematode transposons. These might be viewed as a discrete family of transposons within the IS630-Tc1-mariner superfamily with a distinctive D,D37D catalytic motif, and another small divergent D,D41D clade is recognized as their sister group of transposons.     

Functional characterization of the vitellogenin promoter in the silkworm,Bombyx mori

Genetic transformation and genome editing technologies have been successfully established in the lepidopteran insect model, the domesticated silkworm, Bombyx mori, providing great potential for functional genomics and practical applications. However, the current lack of cis‐regulatory elements in B. mori gene manipulation research limits further exploitation in functional gene analysis. In the present study, we characterized a B. mori endogenous promoter, Bmvgp, which is a 798‐bp DNA sequence adjacent to the 5′‐end of the vitellogenin gene (Bmvg). PiggyBac‐based transgenic analysis shows that Bmvgp precisely directs expression of a reporter gene, enhanced green fluorescent protein (EGFP), in a sex‐, tissue‐ and stage‐specific manner. In transgenic animals, EGFP expression can be detected in the female fat body from larval?pupal ecdysis to the following pupal and adult stage. Furthermore, in vitro and in vivo experiments revealed that EGFP expression can be activated by 20‐hydroxyecdysone, which is consistent with endogenous Bmvg expression. These data indicate that Bmvgp is an effective endogenous cis‐regulatory element in B. mori.     

Membrane-penetrating trehalase from silkworm Bombyx mori. Molecular cloning and localization in larval midgut

The main blood sugar in insects, trehalose, differs from glucose in mammals. To incorporate trehalose into cells and utilize it, tissue cells possess the enzyme trehalase (EC3.2.1.28), which catalyses trehalose into glucose, in the organellar membrane or in the cytoplasm. Soluble and membrane-bound trehalase proteins have been isolated from insects. To date, however, only genes encoding the soluble trehalase have been reported in insects. Soluble trehalase is therefore believed to become localized on the cell surface via modification. In contrast, cDNAs encoding trehalase localized on the apical cell surface via the glycosylphosphatidylinositol-anchor have been isolated from mammalian small intestines. The amino acid sequence contains a specific hydrophobic region and an upstream omega site, which is cleaved for glycosylphosphatidylinositol-attachment, at the C-terminus. Here, we describe a cDNA from the silkworm Bombyx mori that encodes a novel trehalase (type-2) with one transmembrane domain and lacking the omega site. Immunoblotting and immunohistochemical analyses demonstrated that in the midgut tissue of Bombyx larvae, soluble trehalase-1 is present mainly in goblet cell cavities, but membrane-bound trehalase-2 is predominantly seen on the visceral muscle surrounding the midgut. To our knowledge, this is the first report of a cDNA encoding trehalase that penetrates the cell membrane in insects and its cellular localization.     

Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori

Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern‐recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N‐acetylmuramoyl‐L‐alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP‐S4, a short‐form PGRP from the domesticated silkworm, Bombyx mori. The PGRP‐S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP‐S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP‐S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn²⁺. Scanning electron microscopy showed that PGRP‐S4 disrupted the bacterial cell surface. PGRP‐S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP‐S4 has multiple functions in immunity.     

Immunoglobulin superfamily is conserved but evolved rapidly and is active in the silkworm, Bombyx mori

Immunoglobulin superfamily (IgSF) proteins are known for their abilities to specifically recognize and adhere to cells. In this paper, we predicted the presence of 133 IgSF proteins in the silkworm ( Bombyx mori ) genome. Comparison with similar proteins in other model organisms ( Caenorhabditis elegans , Drosophila melanogaster , Anopheles gambiae , Apis mellifera and Homo sapiens ) indicated that IgSF proteins are conserved but have rapidly evolved from worms to human beings. However, these proteins are well conserved amongst insects. Silkworm microarray-based expression data showed tissue expression of 57 IgSF genes and microbe-induced differential expression of 37 genes. Based on the expression data, we can conclude that the silkworm IgSF is active.     

Induction of gene expression of antibacterial proteins by chitin oligomers in the silkworm, Bombyx mori

Activation of antibacterial protein genes by various chitin oligomers (from dimer to hexamer) was investigated by Northern blot analysis using cDNAs encoding cecropin B, attacin and lebocin from Bombyx mori as probes. All chitin oligomers tested were found to strongly trigger expression of cecropin B, attacin and lebocin genes. Furthermore, gene expression triggered by chitin oligomers was confirmed to occur specifically in the fat body and haemocytes. These results suggest that chitin oligomers have the same characteristics as those of lipopolysaccharide (LPS) and peptidoglycan in triggering gene expression of insect antibacterial proteins.     

Cloning and characterization of a pyridoxine 5'-phosphate oxidase from silkworm, Bombyx mori

A cDNA encoding Pyridoxine 5'-phosphate oxidase (PNPO) from Bombyx mori was cloned and characterized (G en B ank accession number: DQ452398). The cDNA encodes a polypeptide of 257 amino acid residues. The recombinant enzyme purified from Escherichia coli exhibited maximal activity at pH 9.0, and the K _m values for the substrates of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate were determined as 0.65 and 1.15 µmol/l. It was found that B. mori PNPO shares 51.44% homology with humans, but several function-related, key amino acid residues in B. mori PNPO are different from the human and E. Coli gene. B. mori has a single copy of the PNPO gene, which spans a 3.5 kb region and contains five exons and four introns. B. mori PNPO is a homodimer, with each monomer containing nine antiparallel β-strands and five α-helical segments. The secondary structure was deduced from computational study.     

cDNA structure and characterization of a kinesin-like protein from the silkworm Bombyx mori

We have isolated a 1224 bp cDNA clone from a Bombyx mori embryonic cDNA library which contains sequences homologous to the kinesin-like protein gene, ncd , which is required for distribution of chromosomes at meiosis in Drosophila melanogaster females. This clone includes both a microtubule motor and the ATP-binding domains found in kinesin-like proteins. The motor domain is classified in the group of the BimC and cut7 , which have a role in spindle formation during mitosis of Aspergillus nidulans and Schizosaccharomyces pombe , respectively. However, the location of the domain at the carboxy terminus is not common in this family, except for ncd and KAR3.     

Female-biased expression of odourant receptor genes in the adult antennae of the silkworm, Bombyx mori

Olfaction plays an important role in the life history of insects, including key behaviours such as host selection, oviposition and mate recognition. Odour perception by insects is primarily mediated by the large diverse family of odourant receptors (Ors) that are expressed on the dendrites of olfactory neurones housed within chemosensilla. However, few Or sequences have been identified from the Lepidoptera, an insect order that includes some of the most important pest species worldwide. We have identified 41 Or gene sequences from the silkworm (Bombyx mori) genome, more than double the number of published Or sequences from the Lepidoptera. Many silkworm Ors appear to be orthologs of the 17 published tobacco budworm (Heliothis virescens) Ors indicating that many Or lineages may be conserved within the Lepidoptera. The majority of the Or genes are expressed in adult female and male antennae (determined by quantitative real-time PCR analysis), supporting their probable roles in adult olfaction. Several Or genes are expressed at high levels in both male and female antennae, suggesting they mediate the perception of common host or conspecific volatiles important to both sexes. BmOrs 45-47 group together in the same phylogenetic branch and all three are expressed at moderate female-biased ratios, six to eight times higher in female compared to male moth antennae. Interestingly, BmOrs19 and 30 appear to be expressed predominantly in female antennae, opposite to that of the published silkworm pheromone receptors BmOrs 1 and 3 that are specific to male antennae. These results suggest that BmOr19 and 30 may detect odours critical to female behaviour, such as oviposition cues or male-produced courtship pheromones.     

cDNA cloning,characterization and gene expression of nitric oxide synthase from the silkworm,Bombyx mori

Molecular cloning and nucleotide sequencing of cDNA encoding Bombyx mori nitric oxide synthase (BmNOS) was conducted to analyse its possible role in insect immunity. The amino acid sequence deduced from the BmNOS cDNA showed 84%, 54% and 53% identity with those of NOSs from Manduca sexta, Drosophila melanogaster and Rhodonius prolixus. Recombinant BmNOS produced in insect cells using baculovirus was found to require NADPH, Ca2+, calmodulin and tetrahydrobiopterin (BH4) for its activity. The BmNOS gene was constitutively expressed at a low level in the larval fat body, haemocyte, Malpighian tubule and midgut, and adult antenna, and induced strongly in the fat body by lipopolysaccharide (LPS), suggesting that the BmNOS gene plays different physiological roles in different tissues. Injection of NO donors that produce NO in vivo induced the gene expression of an antibacterial peptide, cecropin B, strongly suggesting that NO produced by BmNOS following LPS stimulation is involved in signal transduction as a signalling molecule for immune gene expression.