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.     

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.     

Transgenic characterization of two testis‐specific promoters in the silkworm,Bombyx mori

Sex‐specific regulatory elements are key components for developing insect genetic sexing systems. The current insect genetic sexing system mainly uses a female‐specific modification system whereas little success was reported on male‐specific genetic modification. In the silkworm Bombyx mori, a lepidopteran model insect with economic importance, a transgene‐based, female‐specific lethality system has been established based on sex‐specific alternative splicing factors and a female‐specific promoter BmVgp (vitellogenin promoter) has been identified. However, no male‐specific regulatory elements have yet been identified. Here we report the transgenic identification of two promoters that drive reporter gene expression in a testis‐specific manner in B. mori. Putative promoter sequences from the B. mori Radial spoke head 1 gene (BmR1) and beta‐tubulin 4 gene (Bmβ4) were introduced using piggybac‐based germline transformation. In transgenic silkworms, expression of the reporter gene enhanced green fluorescent protein (EGFP) directed by either BmR1 promoter (BmR1p) or Bmβ4p showed precisely testis‐specific manners from the larval to adult stage. Furthermore, EGFP expression of these two transgenic lines showed different localization in the testis, indicating that BmR1p or Bmβ4p might be used as distinct regulatory elements in directing testis‐specific gene expression. Identification of these testis‐specific promoters not only contributes to a better understanding of testis‐specific gene function in insects, but also has potential applications in sterile insect techniques for pest management.     

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.     

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.     

Functional characterization of an aldose reductase (bmALD1) obtained from the silkworm Bombyx mori

We describe a new member of the aldo‐keto reductase (AKR) superfamily in the silkworm Bombyx mori. On the basis of its amino acid sequence and phylogenetic tree, this AKR belongs to the AKR1B family and has been designated as bmALD1. In the current study, recombinant bmALD1 was overexpressed, purified to homogeneity and kinetically characterized. We discovered that bmALD1 uses NADPH as a coenzyme to reduce carbonyl compounds such as DL‐glyceraldehyde, glucose and 2‐nonenal. No NADH‐dependent activity was detected. To the best of our knowledge, bmALD1 is only the third AKR characterized in silkworm which, given its substrate specificity, could play a major role in glucose metabolism and antioxidant reactions. Our data provide an increased understanding of insect AKR function.     

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.     

The gustatory receptor family in the silkworm moth Bombyx mori is characterized by a large expansion of a single lineage of putative bitter receptors

The gustatory receptor (Gr) family of insect chemoreceptors includes receptors for sugars and bitter compounds, as well as cuticular hydrocarbons and odorants such as carbon dioxide. We have annotated a total of 65 Gr genes from the silkworm Bombyx mori genome. The Gr family in the silkworm moth includes putative carbon dioxide receptors and sugar receptors, as well as duplicated orthologues of the orphan DmGr43a receptor. Most prominent in this 65‐gene family, however, is a single large expansion of 55 Grs that we propose are predominantly ‘bitter’ receptors involved in perception of the large variety of secondary plant chemicals that caterpillars and moths encounter. These Grs might therefore mediate food choice and avoidance as well as oviposition site preference.     

Structure and expression of tandemly duplicated xanthine dehydrogenase genes of the silkworm (Bombyx mori)

Xanthine dehydrogenase (XDH) is a molybdoenzyme which catalyses oxidation of xanthine and hypoxanthine to uric acid. We isolated genomic clones of silkworm (Bombyx mori) XDH genes (BmXDH1 and BmXDH2). The BmXDH2 The BmXDH2 gene is located upstream from the BmXDH1 gene and they show a tandemly duplicated structure. Both BmXDH genes were expressed in the fat body and Malpighian tubules, whereas only the BmXDH1 gene was expressed in the midgut. Phylogenetic analysis indicates that BmXDH gene duplication occurred after the divergence of the silkworm and dipteran species. Intron insertion site comparison shows that some introns were lost during insect XDH gene evolution.     

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.     

Utilization of Bombyx mori larvae as a surrogate animal model for evaluation of the anti-infective potential of oxazolidinones

Silkworm (Bombyx mori) larvae were investigated as an alternative animal model for the efficacy testing of novel oxazolidinones. The minimal lethal dose (MLD) for Staphylococcus aureus was 1–5 × 10⁷ CFU per larva, exhibiting more than 90% mortality within 2 to 4 days post-infection. Treatment with vancomycin, linezolid, and novel oxazolidinones (RBx 7644, RBx 8700, and RBx 2006171) showed survival in a dose-dependent manner. The antibacterial potential of RBx 7644 and RBx 8700 was compared with that of linezolid and that of vancomycin and the effective doses (ED)₅₀ obtained in the silkworm model were 37.89, 24.96, 13.38, and 30.72 mg/kg body weight, respectively. The ED₅₀ values showed a similar trend in a murine model of infection. Owing to the small size of the larvae, very small amounts of new chemical entities (NCEs) are required to test their in vivo efficacy in this model. Thus, the silkworm model may be used in the early stage of new discovery research.     

Bombyx mori Ras proteins BmRas1, BmRas2 and BmRas3 are neither farnesylated nor palmitoylated but are geranylgeranylated

The lipid modifications which occur on Bombyx mori Ras proteins BmRas1, BmRas2 and BmRas3 were studied by metabolic labelling in an insect cell‐free protein synthesis system and in a baculovirus expression system, using specific inhibitors of protein prenylation and protein palmitoylation. In addition, the subcellular localization of BmRas proteins was examined using EGFP fusion proteins of constitutively active forms of BmRas proteins transiently expressed in Sf9 cells. As a result, it was revealed that the three B. mori Ras proteins BmRas1, BmRas2 and BmRas3 are neither farnesylated nor palmitoylated but are geranylgeranylated for localization to the plasma membrane of insect cells. Thus, the mechanism of membrane binding of insect Ras proteins is quite different from that reported for mammalian Ras proteins.     

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.