Transgenic analysis of the BmBLOS2 gene that governs the translucency of the larval integument of the silkworm,Bombyx mori

The larval integument of the silkworm, Bombyx mori, is opaque because urate granules accumulate in the epidermis. Although the biosynthetic pathway of uric acid is well studied, little is known about how uric acid accumulates as urate granules in epidermal cells. In the distinct oily (od) mutant silkworm, the larval integument is translucent because of the inability to construct urate granules. Recently, we have found that the od mutant has a genomic deletion in the B. mori homologue of the human biogenesis of lysosome‐related organelles complex1, subunit 2 (BLOS2) gene (BmBLOS2). Here, we performed a molecular and functional characterization of BmBLOS2. Northern blot analysis showed that BmBLOS2 was ubiquitously expressed in various tissues. We analysed the structure of a newly isolated mutant (od^B) allelic to od and found a premature stop codon in the coding sequence of BmBLOS2 in this new mutation. Moreover, the translucent phenotype was rescued by the germ‐line transformation of the wild‐type BmBLOS2 allele into the od mutant. Our results suggest that BmBLOS2 is responsible for the od mutant phenotype and plays a crucial role in biogenesis of urate granules in the larval epidermis of the silkworm. The relationships amongst Hermansky–Pudlak syndrome (HPS) genes in mammals, granule group genes in Drosophila and translucent mutant genes in B. mori are discussed.     

Transgenic characterization of two silkworm tissue‐specific promoters in the haemocyte plasmatocyte cells

Haemocytes play crucial roles in insect metabolism, metamorphosis, and innate immunity. As a model of lepidopteran insects, the silkworm is a useful model to study the functions of both haematopoiesis and haemocytes. Tissue‐specific promoters are excellent tools for genetic manipulation and are widely used in fundamental biological research. Herein, two haemocyte‐specific genes, Integrin β2 and Integrin β3, were confirmed. Promoter activities of Integrin β2 and Integrin β3 were evaluated by genetic manipulation. Quantitative real‐time PCR and western blotting suggested that both promoters can drive enhanced green fluorescent protein (EGFP) specifically expressed in haemocytes. Further evidence clearly demonstrated that the transgenic silkworm exhibited a high level of EGFP signal in plasmatocytes, but not in other detected haemocyte types. Moreover, EGFP fluorescence signals were observed in the haematopoietic organ of both transgenic strains. Thus, two promoters that enable plasmatocytes to express genes of interest were confirmed in our study. It is expected that the results of this study will facilitate advances in our understanding of insect haematopoiesis and immunity in the silkworm, Bombyx mori.     

Comparative analysis of Bombyx mori nucleopolyhedrovirus responsive genes in fat body and haemocyte of B. mori resistant and susceptible strains

The infection profiles of the Bombyx mori nucleopolyhedrovirus (BmNPV) in B. mori larvae revealed that the virus invaded the fat body and haemocyte of both KN and 306 strains, which are highly resistant and susceptible, respectively, to BmNPV infection. However, viral proliferation was notably slowed in the resistant B. mori strain. Using suppression subtractive hybridization, two fat body cDNA libraries were constructed to compare BmNPV responsive gene expression levels between the two silkworm lines. In total, 96 differentially expressed genes were obtained. Real‐time quantitative PCR (qPCR) analysis confirmed that eight genes were significantly up‐regulated in the fat body and haemocyte of the KN strain following BmNPV injection. Our results suggest that these genes may have potential roles in B. mori antiviral infection mechanisms.     

Systemic disruption of the homeostasis of transfer RNA isopentenyltransferase causes growth and development abnormalities in Bombyx mori

Isopentenylation at A37 (i⁶A37) of some transfer RNAs (tRNAs) plays a vital role in regulating the efficiency and fidelity of protein synthesis. However, whether insects, which are well known for their highly efficient protein synthesis machinery, employ this regulatory mechanism remains uninvestigated. In the current study, a candidate tRNA isopentenyltransferase (IPT) gene with three alternative splicing isoforms (BmIPT1–BmIPT3) was identified in Bombyx mori (silkworm). Only BmIPT1 could complement a yeast mutant lacking tRNA IPT. Phylogenetic analysis showed that silkworm tRNA IPT is conserved in the Lepidoptera. BmIPT was expressed in all B. mori tissues and organs that were investigated, but was expressed at a significantly higher level in silk glands of the fourth instar compared to the first day of the fifth instar. Interestingly, BmIPT was expressed at a significantly higher level in the domesticated silkworm, B. mori, than in wild Bombyx mandarina in multiple tissues and organs. Knock‐down of BmIPT by RNA interference caused severe abnormalities in silk spinning and metamorphosis. Constitutive overexpression of BmIPT1 using a cytoplasmic actin 4 promoter in B. mori raised its messenger RNA level more than sixfold compared with nontransgenic insects and led to significant decreases in the body weight and cocoon shell ratio. Together, these results confirm the first functional tRNA IPT in insects and show that a suitable expression level of tRNA IPT is vital for silk spinning, normal growth, and metamorphosis. Thus, i⁶A modification at position A37 in tRNA probably plays an important role in B. mori protein synthesis.     

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.     

Molecular cloning and characterization of peroxiredoxin 4 involved in protection against oxidative stress in the silkworm Bombyx mori

Peroxiredoxins (Prxs) are a ubiquitous family of proteins that play important roles in insects in protection against oxidative stress through the detoxification of cellular peroxides. Here, we describe the cloning and characterization of a Prx4 cDNA of the silkworm Bombyx mori (BmPrx4). The BmPrx4 gene has an open reading frame of 744 bp encoding 248 amino acids and a conserved motif, VCP, involved in its presumed redox functions. The heterologously expressed proteins of the gene in Escherichia coli showed antioxidant activity, removed hydrogen peroxide and protected DnA. Western blotting analysis showed the presence of BmPrx4 in the haemolymph, suggesting that the protein is secretable. Moreover, BmPrx4 was expressed at all developmental stages. The expression level of BmPrx4 was relatively low during the feeding stage but high at the wandering stage. BmPrx4 was induced by quercetin or temperature stress. Immunohistochemical analysis revealed that BmPrx4 is present in the brain, neurones and olfactory organ of the head in silkworms. Overall, our results indicate that the expression profile of BmPrx4 correlates well with protection from oxidative damage. Our data provide clues for the development of control technology for agricultural and forestry pests as the silkworm is a representative of lepidopteran pests.     

Subchronic toxicity of magnesium oxide nanoparticles to Bombyx mori silkworm

Despite many research efforts devoted to the study of the effects of magnesium oxide nanoparticles (MgO NPs) on cells or animals in recent years, data related to the potential long-term effects of this nanomaterial are still scarce. The aim of this study is to explore the subchronic effects of MgO NPs on Bombyx mori silkworm, a complete metamorphosis insect with four development stages (egg, larva, pupa, month). With this end in view, silkworm larvae were exposed to MgO NPs at different mass concentrations (1%, 2%, 3% and 4%) throughout their fifth instar larva. Their development, survival rate, cell morphology, gene expressions, and especially silk properties were compared with a control. The results demonstrate that MgO NPs have no significant negative impact on the growth or tissues. The cocooning rate and silk quality also display normal results. However, a total of 806 genes are differentially expressed in the silk gland (a vital organ for producing silk). GO (Gene Ontology) results show that the expression of many genes related to transporter activity are significantly changed, revealing that active transport is the main mechanism for the penetration of MgO NPs, which also proves that MgO NPs are adsorbed by cells. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis demonstrates that the longevity regulating pathway-worm, peroxisome and MAPK signaling pathway are closely involved in the biological effects of MgO NPs. Overall, subchronic exposure to MgO NPs induced no apparent negative impact on silkworm growth or silks but changed the expressions of some genes.

The subchronic toxicity of MgO NPs was studied by silkworm model, from the levels of animal entirety, tissues, and genes.     

A MC motif in silkworm Argonaute 1 is indispensible for translation repression

Small RNA‐mediated gene silencing is a fundamental gene regulatory mechanism, which is conserved in many organisms. Argonaute (Ago) family proteins in the RNA‐induced silencing complex (RISC) play crucial roles in RNA interference (RNAi) pathways. In the silkworm Bombyx mori, four Ago proteins have been identified, named as Ago1, Ago2, Ago3 and Siwi. Ago2 participates in double‐stranded RNA (dsRNA)‐induced RNAi, whereas Ago3 and Siwi are involved in the Piwi‐interacting RNA (piRNA) pathway. However, there is no experimental evidence concerning silkworm Ago1 (BmAgo1) in the RNAi mechanism. In the present study, we analysed the function of BmAgo1 in the microRNA (miRNA)‐mediated RNAi pathway using tethering and miRNA sensor reporter assays. These results clearly demonstrate that BmAgo1 plays an indispensable role in translation repression in silkworm. Moreover, coimmunoprecipitation data indicated that BmAgo1 interacts with BmDcp2, an orthologue of mRNA‐decapping enzyme 2 (Dcp2) protein in the Drosophila processing‐bodies (P‐bodies). Substitutions of two conserved phenylalanines (F522 and F557) by valines in the MC motif strongly impaired the function of BmAgo1 in translation repression and its localization in P‐bodies, suggesting that these two amino acid residues in the MC motif of BmAgo1 are prerequisites for mRNA translation repression in B. mori.     

Radiation-induced changes in gene expression in the silkworm revealed by serial analysis of gene expression (SAGE)

Serial analysis of gene expression (SAGE) was used to examine the profile of expressed genes during embryonic development in the domesticated silkworm, Bombyx mori, after irradiation with Cobalt-60. A comparison of the SAGE sequence tags derived from irradiated embryos with those from normal embryos revealed 673 differentially expressed genes (P < 0.01 and at least three folds change). Of these, 292 genes were highly expressed in normal embryos and 381 genes were highly expressed in irradiated embryos. These results provide valuable information for understanding the mechanisms of radiation-induced changes in gene expression. In addition, it was noted that the generation of longer cDNA fragments from SAGE tags is an efficient way to identify genes, thereby facilitating the analysis of large numbers of unknown genes.     

Establishment of transgenic silkworms expressing GAL4 specifically in the haemocyte oenocytoid cells

Insect haemocytes play significant roles in innate immunity. The silkworm, a lepidopteran species, is often selected as the model for studies into the functions of haemocytes in immunity; however, our understanding of the role of haemocytes remains limited because the lack of haemocyte promoters for transgene expression makes genetic manipulations difficult. In the present study, we aimed to establish transgenic silkworm strains expressing GAL4 in their haemocytes. First, we identified three genes with strong expression in haemocytes, namely, lp44, Haemocyte Protease 1 (HP1) and hemocytin. Transgenic silkworms expressing GAL4 under the control of the putative promoters of these genes were then established and expression was examined. Although GAL4 expression was not detected in haemocytes of HP1‐GAL4 or hemocytin‐GAL4 strains, lp44‐GAL4 exhibited a high level of GAL4 expression, particularly in oenocytoids. GAL4 expression was also detected in the midgut but in no other tissues, indicating that GAL4 expression in this strain is mostly oenocytoid‐specific. Thus, we have identified a promoter that enables oenocytoid expression of genes of interest. Additionally, the lp44‐GAL4 strain could also be used for other types of research, such as the functional analysis of genes in oenocytoids, which would facilitate advances in our understanding of insect immunity.