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.     

Evaluating the evolution and function of the dynamic Venom Y protein in ectoparasitoid wasps

Venom of the parasitoid wasp Nasonia vitripennis changes the metabolism and gene expression in its fly host Sarcophaga bullata to induce developmental arrest, suppression of the immune response and various other venom effects. Yet, the venom of ectoparasitoid wasps has not been fully characterized. A major component of N. vitripennis venom is an uncharacterized, high‐expressing protein referred to as Venom Y. Here we describe the evolutionary history and possible functions of this venom protein. We found that Venom Y is a relatively young gene that has duplicated to form two distinct paralogue groups. A copy of Venom Y has been recruited as a venom protein in at least five wasp species. Functional analysis found that Venom Y affects detoxification and immunity genes in envenomated fly hosts. Many of these genes are fat‐body specific, suggesting that Venom Y may have a targeted effect on fat body tissue. We also show that Venom Y may mitigate negative effects of other venom proteins. Finally, protein sequencing indicates that Venom Y is post‐translationally modified. This study contributes to elucidating parasitoid venom by using RNA interference knockdown to investigate venom protein function in the context of the whole venom cocktail.     

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.     

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.     

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.     

Silencing cathepsin L expression reduces Myzus persicae protein content and the nutritional value as prey for Coccinella septempunctata

Gut‐expressed aphid genes, which may be more easily inhibited by RNA interference (RNAi) constructs, are attractive targets for pest control efforts involving transgenic plants. Here we show that expression of cathepsin L, which encodes a cysteine protease that functions in aphid guts, can be reduced by expression of an RNAi construct in transgenic tobacco. The effectiveness of this approach is demonstrated by up to 80% adult mortality, reduced fecundity, and delayed nymph production of Myzus persicae (green peach aphids) when cathepsin L expression was reduced by plant‐mediated RNAi. Consistent with the function of cathepsin L as a gut protease, M. persicae fed on the RNAi plants had a lower protein content in their bodies and excreted more protein and/or free amino acids in their honeydew. Larvae of Coccinella septempunctata (seven‐spotted ladybugs) grew more slowly on aphids having reduced cathepsin L expression, suggesting that prey insect nutritive value, and not just direct negative effects of the RNAi construct, needs to be considered when producing transgenic plants for RNAi‐mediated pest control.     

Evolution of the neural sex‐determination system in insects: does fruitless homologue regulate neural sexual dimorphism in basal insects?

In the brain of holometabolous insects such as the fruit fly Drosophila melanogaster, the fruitless gene produces sex‐specific gene products under the control of the sex‐specific splicing cascade and contributes to the formation of the sexually dimorphic circuits. Similar sex‐specific gene products of fruitless homologues have been identified in other holometabolous insects such as mosquitoes and a parasitic wasp, suggesting the fruitless‐dependent neural sex‐determination system is widely conserved amongst holometabolous insects. However, it remains obscure whether the fruitless‐dependent neural sex‐determination system is present in basal hemimetabolous insects. To address this issue, identification, characterization, and expression analyses of the fruitless homologue were conducted in the two‐spotted cricket, Gryllus bimaculatus, as a model hemimetabolous insect. The Gryllus fruitless gene encodes multiple isoforms with a unique zinc finger domain, and does not encode a sex‐specific gene product. The Gryllus Fruitless protein is broadly expressed in the neurones and glial cells in the brain, and there was no prominent sex‐related difference in the expression levels of Gryllus fruitless isoforms. The results suggest that the Gryllus fruitless gene is not involved in the neural sex‐determination in the cricket brain.     

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.     

MicroRNA miR‐252 targets mbt to control the developmental growth of Drosophila

Developmental growth is an intricate process involving the coordinated regulation of the expression of various genes, and microRNAs (miRNAs) play crucial roles in diverse processes throughout animal development. The ecdysone‐responsive miRNA, miR‐252, is normally upregulated during the pupal and adult stages of Drosophila development. Here, we found that overexpression of miR‐252 in the larval fat body decreased total tissue mass through a reduction in both cell size and cell number, causing a concomitant decrease in larval size. Furthermore, miR‐252 overexpression led to a delayed larval‐to‐pupal transition with defective anterior spiracle eversion, as well as a decrease in adult size and mass. Conversely, adult flies lacking miR‐252 showed an increase in mass compared with control flies. We found that miR‐252 directly targeted mbt, encoding a p21‐activated kinase, to repress its expression. Notably, co‐overexpression of mbt rescued the developmental and growth defects associated with miR‐252 overexpression, indicating that mbt is a biologically relevant target of miR‐252. Overall, our data support a role for the ecdysone/miR‐252/mbt regulatory axis in growth control during Drosophila development.     

Establishment of a specific cell death induction system in Bombyx mori by a transgene with the conserved apoptotic regulator,mouse Bcl‐2‐associated X protein (mouse Bax)

The induction of apoptosis in vivo is a useful tool for investigating the functions and importance of particular tissues. B‐cell leukaemia/lymphoma 2‐associated X protein (Bax) functions as a pro‐apoptotic factor and induces apoptosis in several organisms. The Bax‐mediated apoptotic system is widely conserved from Caenorhabditis elegans to humans. In order to establish a tissue‐specific cell death system in the domestic silkworm, Bombyx mori, we constructed a transgenic silkworm that overexpressed mouse Bax (mBax) in particular tissues by the Gal4‐upstream activation sequence system. We found that the expression of mBax induced specific cell death in the silk gland, fat body and sensory cells. Fragmentation of genomic DNA was observed in the fat body, which expressed mBax, thereby supporting apoptotic cell death in this tissue. Using this system, we also demonstrated that specific cell death in sensory cells attenuated the response to the sex pheromone bombykol. These results show that we successfully established a tissue‐specific cell death system in vivo that enabled specific deficiencies in particular tissues. The inducible cell death system may provide useful means for industrial applications of the silkworm and possible utilization for other species.     

Novel insights into the regulation of coagulation by factor V isoforms,tissue factor pathway inhibitorα, and protein S

Factor V (FV) is a regulator of both pro‐ and anticoagulant pathways. It circulates as a single‐chain procofactor, which is activated by thrombin or FXa to FVa that serves as cofactor for FXa in prothrombin activation. The cofactor function of FVa is regulated by activated protein C (APC) and protein S. FV can also function as an anticoagulant APC cofactor in the inhibition of FVIIIa in the membrane‐bound tenase complex (FIXa/FVIIIa). In recent years, it has become clear that FV also functions in multiple ways in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. Of particular importance is a FV splice variant (FV‐Short) that serves as a carrier and cofactor to TFPIα in the inhibition of FXa. FV‐Short is generated through alternative splicing of exon 13 that encodes the large activation B domain. A highly negatively charged binding site for TFPIα is exposed in the C‐terminus of the FV‐Short B domain, which binds the positively charged C‐terminus of TFPIα, thus keeping TFPIα in circulation. The binding of TFPIα to FV‐Short is also instrumental in localizing the inhibitor to the surface of negatively charged phospholipids, where TFPIα inhibits FXa in process that is stimulated by protein S. Plasma FV activation intermediates and partially proteolyzed platelet FV similarly bind TFPIα with high affinity and regulate formation of prothrombinase. The novel insights gained into the interaction between FV isoforms, TFPIα, and protein S have opened a new avenue for research about the mechanisms of coagulation regulation and also for future development of therapeutics aimed at modulating coagulation.     

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.