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 effect of acetylation on the protein stability of BmApoLp‐III in the silkworm,Bombyx mori

Acetylation is an important, reversible posttranslational modification to a protein. In a previous study, we found that there were a large number of acetylated sites in various nutrient storage proteins of the silkworm haemolymph. In this study, we confirmed that acetylation can affect the stability of nutrient storage protein Bombyx mori apolipophorin‐III (BmApoLp‐III). First, the expression of BmApoLp‐III could be upregulated when BmN cells were treated with the deacetylase inhibitor panobinostat (LBH589); similarly, the expression was downregulated when the cells were treated with the acetylase inhibitor C646. Furthermore, the increase in acetylation by LBH589 could inhibit the degradation and improve the accumulation of BmApoLp‐III in BmN cells treated with cycloheximide and MG132 respectively. Moreover, we found that an increase in acetylation could decrease the ubiquitination of BmApoLp‐III and vice versa; therefore, we predicted that acetylation could improve the stability of BmApoLp‐III by competing for ubiquitination and inhibiting the protein degradation pathway mediated by ubiquitin. Additionally, BmApoLp‐III had an antiapoptosis function that increased after LBH589 treatment, which might have been due to the improved protein stability after acetylation. These results have laid the foundation for further study on the mechanism of acetylation in regulating the storage and utilization of silkworm nutrition.     

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.     

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.     

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.     

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.     

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.