Expressional analysis of the silkworm storage protein 1 and identification of its interacting proteins

Storage proteins are haemolymph‐specific proteins in insects, mainly synthesized in the fat body, released into the haemolymph, and then selectively reabsorbed by the fat body before pupation. These storage proteins play an important role in insect metamorphosis and egg development. Some of these storage proteins are responsive to pathogen infection and can even suppress pathogen multiplication. However, the mechanisms of the physiological, biochemical and immune‐responsive functions of storage proteins remain unclear. In this study, the expression patterns of Bombyx mori storage protein 1 (BmSP1) during the larval stage were analysed. Then, BmSP1 protein fused with enhanced green fluorescent protein (EGFP) was successfully expressed in a B. mori baculovirus vector expression system. Quantitative real‐time PCR showed that the expression level of BmSP1 increased with the advance of instars and reached the highest level in the fifth instar, especially in the fat body. Recombinant BmSP1 expressed in silkworm larvae inhibited haemolymph melanization. Then, proteins that interact with BmSP1 were identified with EGFP used as an antigenic determinant by co‐immunoprecipitation. A 30 kDa low molecular weight lipoprotein PBMHP‐6 precursor (BmLP6) was shown to interact with BmSP1. Yeast two‐hybrid experiments confirmed the interaction between BmSP1 and BmLP6. The results obtained in this study will be helpful for further study of the functions of BmSP1 and BmLP6 in the regulatory network of silkworm development and innate immunity.     

Function of a TGF‐β inducible nuclear protein in the silk gland in Bombyx mori

A TGF‐β inducible nuclear protein 1 (BmTINP1) was cloned from silkworm, Bombyx mori. Polyclonal antibodies against BmTINP1 were produced and subsequently used in immunoblotting and immunohistochemistry analyses. The immunoblotting analyses demonstrated that BmTINP1 was specifically expressed in the anterior silk gland (ASG) and the middle silk gland (MSG) but not in the posterior silk gland (PSG). There were two bands that suggested the existence of an isoform of BmTINP1. The expression profiles of BmTINP1 in ASGs and MSGs were similar, and they manifested a high level of expression throughout the period during which silk gland grew exponentially. Immunohistochemistry results revealed that BmTINP1 was translocated from the nucleus into the cytoplasm when larvae developed from the 4th‐HCS into the 5th instar. 20‐hydroxyecdysone (20E) promotes the translocation, while the methoprene [a juvenile hormone (JH) analog] restrains the process. Our findings indicate that BmTINP1 is involved in silk produce along with the rapid growth of ASGs and MSGs during the last instar larvae, and the process could be regulated by hormones via control of BmTINP1 translocation from the nucleus to the cytoplasm.     

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.     

Ribosomal protein L5 of Bombyx mori: cDNA sequence and tissue differences in the L5 mRNA content

A gene encoding the ribosomal protein L5, which assembles with the 5S rRNA into one of the components of the 60S ribosomal subunit, was identified in insects for the first time. We report on the isolation of Bombyx mori cDNA, whose putative translation product is 60–76% identical with the L5 sequences known from other animals and about 50% identical with the L5 of rice and yeasts. Bombyx contains a single copy of the L5 gene, which is constitutively expressed as a 1.1 kb mRNA in all tissues. The ratio of L5 mRNA to total RNA appears to reflect the proteosynthetic tissue capacity. A very high level of L5 mRNA is maintained in functional silk glands. A rapid decline of the ratio of L5 mRNA to the rRNA, which occurs in the silk glands after cocoon spinning, indicates differences in the stability of these two RNA classes.     

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.     

Antimicrobial peptides from Bombyx mori: a splendid immune defense response in silkworms

Bombyx mori L., a primary producer of silk, is the main tool in the sericulture industry and provides the means of livelihood to a large number of people. Silk cocoon crop losses due to bacterial infection pose a major threat to the sericulture industry. Bombyx mori L., a silkworm of the mulberry type, has a sophisticated inherent innate immune mechanism to combat such invasive pathogens. Among all the components in this defense system, antimicrobial peptides (AMPs) are notable due to their specificity towards the invading pathogens without harming the normal host cells. Bombyx mori L. so far has had AMPs identified that belong to six different families, namely cecropin, defensin, moricin, gloverin, attacin and lebocin, which are produced by the Toll and immune deficiency (IMD) pathways. Their diverse modes of action depend on microbial pathogens and are still under investigation. This review examines the recent progress in understanding the immune defense mechanism of Bombyx mori based on AMPs.

AMPs produced by B. mori induced by microbial challenge in the fat body.     

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.     

Modular cis‐regulatory logic of yellow gene expression in silkmoth larvae

Colour patterns in butterflies and moths are crucial traits for adaptation. Previous investigations have highlighted genes responsible for pigmentation (ie yellow and ebony). However, the mechanisms by which these genes are regulated in lepidopteran insects remain poorly understood. To elucidate this, molecular studies involving dipterans have largely analysed the cis‐regulatory regions of pigmentation genes and have revealed cis‐regulatory modularity. Here, we used well‐developed transgenic techniques in Bombyx mori and demonstrated that cis‐regulatory modularity controls tissue‐specific expression of the yellow gene. We first identified which body parts are regulated by the yellow gene via black pigmentation. We then isolated three discrete regulatory elements driving tissue‐specific gene expression in three regions of B. mori larvae. Finally, we found that there is no apparent sequence conservation of cis‐regulatory regions between B. mori and Drosophila melanogaster, and no expression driven by the regulatory regions of one species when introduced into the other species. Therefore, the trans‐regulatory landscapes of the yellow gene differ significantly between the two taxa. The results of this study confirm that lepidopteran species use cis‐regulatory modules to control gene expression related to pigmentation, and represent a powerful cadre of transgenic tools for studying evolutionary developmental mechanisms.     

Identification and characterization of a Masculinizer homologue in the diamondback moth,Plutella xylostella

Recently, a novel sex‐determination system was identified in the silkworm (Bombyx mori) in which a piwi‐interacting RNA (piRNA) encoded on the female‐specific W chromosome silences a Z‐linked gene (Masculinizer) that would otherwise initiate male sex‐determination and dosage compensation. Masculinizer provides various opportunities for developing improved genetic pest management tools. A pest lepidopteran in which a genetic pest management system has been developed, but which would benefit greatly from such improved designs, is the diamondback moth, Plutella xylostella. However, Masculinizer has not yet been identified in this species. Here, focusing on the previously described ‘masculinizing’ domain of B. mori Masculinizer, we identify P. xylostella Masculinizer (PxyMasc). We show that PxyMasc is Z‐linked, regulates sex‐specific alternative splicing of doublesex and is necessary for male survival. Similar results in B. mori suggest this survival effect is possibly through failure to initiate male dosage compensation. The highly conserved function and location of this gene between these two distantly related lepidopterans suggests a deep role for Masculinizer in the sex‐determination systems of the Lepidoptera.     

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.