Specific binding of silkworm Bombyx mori 30-kDa lipoproteins to carbohydrates containing glucose

Insect lectins are important as part of nonspecific self-defense, but their antifungal mechanisms remain to be elucidated. Fungi contain glucans on the cell surface and insect glucan-binding proteins are considered to be essential for antifungal mechanisms. We purified glucose-binding proteins from hemolymph of pupae of the silkworm Bombyx mori, and the amino acid sequence analysis showed that their two proteins are 30-kDa lipoproteins, major components of B. mori hemolymph. These lipoproteins specifically bound to glucose and glucans, suggesting that they are involved in insect self-defense systems.     

Glucan-Binding Activity of Silkworm 30-kDa Apolipoprotein and Its Involvement in Defense against Fungal Infection

The silkworm Bombyx mori 30-kDa lipoproteins (6G1 and 19G1), major components of the hemolymph, were shown to bind to glucans. 6G1 apolipoprotein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant 6G1 apolipoprotein specifically bound to β-glucan, but not to chitin, mannan, peptidoglycan, or oligosaccharide chains on glycoproteins. The β-glucan binding of the recombinant 6G1 was inhibited by laminaribiose and laminarin, a soluble glucan, but not by lipopolysaccharide or insect blood sugar, trehalose at physiological concentration. Furthermore, the recombinant 6G1 was shown to participate in the activation of prophenoloxidase cascade and to interfere with hyphal growth of the entomopathogenic fungus Paecilomyces tenuipes, injected into pupae of B. mori. These results suggest that 6G1 lipoprotein plays a role in the protection of B. mori against invading fungi.     

Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites

In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine‐rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain‐like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level.     

Clathrin‐dependent endocytosis predominantly mediates protein absorption by fat body from the hemolymph in Bombyx mori

During insect larval–pupal metamorphosis, proteins in the hemolymph are absorbed by the fat body for the maintenance of intracellular homeostasis; however, the type of proteins and how these proteins are internalized into the fat body are unclear. In Bombyx mori, the developmental profiles of total proteins in the hemolymph and fat body showed that hemolymph‐decreased protein bands (55–100 kDa) were in accordance with those protein bands that increased in the fat body. Inhibition of clathrin‐dependent endocytosis predominantly blocked the transportation of 55–100 kDa proteins from the hemolymph into the fat body, which was further verified by RNA interference treatment of Bmclathrin. Six hexamerins were shown to comprise ～90% of the total identified proteins in both the hemolymph and fat body by mass spectrum (MS) analysis. In addition, hemolymph‐specific proteins were mainly involved in material transportation, while fat body‐specific proteins particularly participated in metabolism. In this paper, four hexamerins were found for the first time, and potential proteins absorbed by the fat body from the hemolymph through clathrin‐dependent endocytosis were identified. This study sheds light on the protein absorption mechanism during insect metamorphosis.     

Immunity of the greater wax moth Galleria mellonella

Investigation of insect immune mechanisms provides important information concerning innate immunity, which in many aspects is conserved in animals. This is one of the reasons why insects serve as model organisms to study virulence mechanisms of human pathogens. From the evolutionary point of view, we also learn a lot about host–pathogen interaction and adaptation of organisms to conditions of life. Additionally, insect‐derived antibacterial and antifungal peptides and proteins are considered for their potential to be applied as alternatives to antibiotics. While Drosophila melanogaster is used to study the genetic aspect of insect immunity, Galleria mellonella serves as a good model for biochemical research. Given the size of the insect, it is possible to obtain easily hemolymph and other tissues as a source of many immune‐relevant polypeptides. This review article summarizes our knowledge concerning G. mellonella immunity. The best‐characterized immune‐related proteins and peptides are recalled and their short characteristic is given. Some other proteins identified at the mRNA level are also mentioned. The infectious routes used by Galleria natural pathogens such as Bacillus thuringiensis and Beauveria bassiana are also described in the context of host–pathogen interaction. Finally, the plasticity of G. mellonella immune response influenced by abiotic and biotic factors is described.     

Localization and functional analysis of the insect‐specific RabX4 in the brain of Bombyx mori

Rab proteins are small monomeric GTPases/GTP‐binding proteins, which form the largest branch of the Ras superfamily. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane trafficking. RabX4 is an insect‐specific Rab protein that has no close homolog in vertebrates. There is little information about insect‐specific Rab proteins. RabX4 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX4 were produced in rabbits for western immunoblotting and immunohistochemistry. Western blotting of neural tissues revealed a single band, at approximately 26 kD. RabX4‐like immunohistochemical reactivity was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum in the brain. Further immunohistochemical analysis revealed that RabX4 colocalized with Rab6 and bombyxin in the corpus allatum, a neuronal organ that secretes neuropeptides synthesized in the brain into the hemolymph. RabX4 expression in the frontal ganglion, part of the insect stomatogastric nervous system that is found in most insect orders, was restricted to two neurons on the outer region and did not colocalize with allatotropin or Rab6. Furthermore, RNA interference of RabX4 decreased bombyxin expression levels in the brain. These findings suggest that RabX4 is involved in the neurosecretion of a secretory organ in Bombyx mori.     

Proteomic profiling of the hemolymph at the fifth instar of the silkworm Bombyx mori

Abstract Two‐dimensional gel electrophoresis (2‐DE) followed by matrix‐assisted laser desorption ionization – time‐of‐flight/time‐of‐flight mass spectrometry (MS) analysis were used to charaterize the hemolymph proteomic profiles of the silkworm, Bombyx mori. At days 4 (V4) and 5 (V5) of the fifth (final) instar, when the larvae were at the fast‐growing stage, we found dramatic changes in spots representing proteins having an approximate molecular weight (MW) of 30 kDa. Of these spots, four 30K proteins were highly up‐regulated, implying a close association with the growth and development of B. mori larvae. To understand the molecular basis and underlying mechanisms involved in development and metamorphosis, the proteome of whole hemolymph at V5 was analyzed using shotgun liquid chromatography tandem mass spectrometry with an LTQ‐Orbitrap. A total of 108 proteins were identified without any false discovery hits. These proteins were involved in a variety of cellular functions, including metabolism, development, nutrient transport and reserve, and defense response. Gene ontology analysis showed that 3.4% of these proteins had nutrient reservoir activities and 5.7% were involved in the response to stimulus. Pathway analysis revealed that 22 proteins with common targets were involved in various cellular processes such as immunity, differentiation, proliferation and metamorphosis. These results suggested that some key factors such as the 30K proteins in hemolymph play important roles in B. mori growth and development. Moreover, the multiple functions of hemolymph may be operated by a complex biological network.     

High-level expression of orange fluorescent protein in the silkworm larvae by the Bac-to-Bac system

This novel orange fluorescent protein (OFP) emits brilliant orange fluorescent light. OFP has high fluorescence quantum yield, fast maturation rate, and stability, which imply this protein should be the most favorable biotechnological tools used to investigate the function of target gene by visualizing, monitoring, and quantifying in living cells. B. mori, silkworm has been used as an important bioreactor for the production of recombinant proteins through baculovirus expression system (BES). In this paper, we used infection technique which introduced the baculovirus DNA into silkworms using a cationic lipofectin reagent instead of directly injecting the virus, and demonstrated a high-level expression of the orange fluorescent protein (OFP) gene in the Bombyx mori, silkworm larvae. When recombinant rBacmid/BmNPV/OFP DNA ranging from 50–100 ng/larval was injected, a sufficient OFP expression in hemolymph was harvested. The recombinant viruses could be obtained from the hemolymph of infected larvae and stored as seed which could be used for the large-scale expression. This procedure omitted the costly and labor-consumed insect cell culture. Further investigation of OFP should provide us with more insight in unlocking the mystery of the mechanisms of autocatalytic bioluminescence and its utilization in biotechnology.     

Comparison of Hemolymph and Holotissues of Different Species of Insects as Diet Components forin VitroRearing ofTrichogramma galloiZucchi andT. pretiosumRiley

The aim of this work was to compare pupal holotissues (Diatraea saccharalis, Anticarsia gemmatalis,andBombyx mori) and larval hemolymph of several moth species (Helicoverpa zeaandBombyx mori) commonly produced in Brazilian laboratories as components of artificial diets forin vitrorearing ofTrichogramma galloiandTrichogramma pretiosum.Diets based on larval hemolymph ofH. zeaproduced the best development of both natural enemies, although a diet with pupal holotissues ofB. moriallowed full development ofT. pretiosum.Amino acid analysis showed qualitative differences among the insect derivates and eggs of the natural and alternative hosts, but with a quantitative similarity. Electrophoretic analysis resulting in a diverse protein-stained bands evidenced the qualitative differences among these components.     

Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins

The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography–tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49 %) were predicted to function as binding proteins and 46 % were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.     

Two Crystal Structures of Bombyx mori Lipoprotein 3 - Structural Characterization of a New 30-kDa Lipoprotein Family Member

The 30-kDa family of lipoproteins from insect hemolymph has been the focus of a number of studies over the last few years. Recently, four crystal structures of Bombyx mori lipoprotein 7 have been determined. Here we report two crystal structures of another member of the 30-kDa lipoprotein family, Bombyx mori lipoprotein 3 (Bmlp3). The protein was isolated from its natural source, mulberry silkworm hemolymph. It crystallized in two different crystal forms, Bmlp3-p21 (space group P2₁) and Bmlp3-c2 (space group C2). The crystal structures were solved by molecular replacement using the coordinates of Bmlp7 as a starting model. The crystals of Bmlp3-p21 diffracted X-rays to 2.4 Å resolution and of Bmlp3-c2 to 2.1 Å resolution. Bmlp3 has an overall fold characteristic of 30-kDa lipoproteins, with a VHS-type N-terminal domain and β-trefoil C-terminal domain. Structural comparison of Bmlp3 and Bmlp7 shows that the loops present in the C-terminal domain are flexible and participate in dimer formation. Additionally, new putative binding sites of Bmlp3 have been analyzed in detail and the electrostatic potential of the protein surface at physiological pH 7.4 conditions has been calculated. The results of these calculations are the starting point for an explanation of the recently reported cell-penetrating properties of the 30-kDa lipoproteins.     

Comparative analysis of proteome maps of silkworm hemolymph during different developmental stages

Background  

The silkworm Bombyx mori is a lepidopteran insect with four developmental stages: egg, larva (caterpillar), pupa, and adult. The hemolymph of the silkworm is in an open system that circulates among all organs, and functions in nutrient and hormone transport, injury, and immunity. To understand the intricate developmental mechanisms of metamorphosis, silkworm hemolymph from different developmental stages, including the 3^rd day of fifth instar, the 6^th day of fifth instar, the 3^rd day of pupation, the 8^th day of pupal stage and the first day of the moth stage, was investigated by two-dimensional electrophoresis and mass spectrometry.     

Serum-free culture of an embryonic cell line from <Emphasis Type="Italic">Bombyx mori</Emphasis> and reinforcement of susceptibility of a recombinant BmNPV by cooling

We established the first continuous cell line that uses a serum-free culture from the embryo of Bombyx mori (Lepidoptera: Bombycidae), designated as NIAS-Bm-Ke17. This cell line was serially subcultured in the SH-Ke-117 medium. The cells adhere weakly to the culture flask, and most cells have an oval shape. The cell line was subcultured 154 times, and the population doubling time is 83.67 ± 5.22 h. Random amplification of polymorphic DNA-polymerase chain reaction with a tenmar single primer for discrimination of insect cell lines recognized the NIAS-Bm-Ke1 cell line as B. mori. This cell line does not support the growth of the B. mori nuclear polyhedrosis virus (BmNPV) in the absence of the heat-inactivated hemolymph of B. mori. However, the heat-inactivated hemolymph in 1% volume of the medium supported a high level of susceptibility to BmNPV. In addition, the cooling treatment of the cells at 2.5°C also enhanced the susceptibility. We report a new serum-free culture system of the B. mori cell line for the baculovirus expression vector system.     

NMR assignments of juvenile hormone binding protein in complex with JH III

A hemolymph juvenile hormone binding protein (JHBP) shuttles hydrophobic JH, a key hormone in regulation of the insect life cycle, from the site of the JH biosynthesis to the cells of target organs. We report complete NMR chemical shift assignments of Bombyx mori JHBP in the JH III-bound state.     

The dipteran parasitoid Exorista bombycis induces pro‐ and anti‐oxidative reactions in the silkworm Bombyx mori: Enzymatic and genetic analysis

Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro‐oxidative reactions and activation of anti‐oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione‐S‐transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro‐ and anti‐oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology.     

Effect of metamorphosis on the major hemolymph proteins of the silkworm

During the metamorphosis of the silkworm, Bombyx mori, three major hemolymph proteins (MHPs) (molecular weights 17,000, 25,000, 27,000) were detected and found to be distributed in the hemolymph and in the tissues of several organs, such as the fat body, midgut, ovary, testis, and even eggs. The MHPs in eggs gradually decreased and disappeared during embryogenesis. The formation, distribution, and utilization of MHPs in tissues other than the gonad, however, were not affected by sex. Radioisotope experiments in vivo revealed that the MHPs were synthesized at an early period of the fifth larval instar. The synthesis of at least two of them occurred in the fat body. MHPs in the hemolymph entered the tissues at the onset of the larval-pupal transformation. On the basis of their appearance, distribution, and depletion, the MHPs may be classified as reserve proteins which are synthesized in the larval stage and utilized later in the developmental stages.     

In vitro lipid transfer between lipoproteins and midgut-diverticula in the spider Polybetes pythagoricus

It has been already reported that most hemolymphatic lipids in the spider Polybetes pythagoricus are transported by HDL1 and VHDL lipoproteins. We studied in vitro the lipid transfer among midgut-diverticula (M-diverticula), and either hemolymph or purified lipoproteins as well as between hemolymphatic lipoproteins. M-diverticula and hemolymph were labeled by in vivo ¹⁴C-palmitic acid injection. In vitro incubations were performed between M-diverticula and either hemolymph or isolated lipoproteins. Hemolymph lipid uptake was associated to HDL1 (67%) and VHDL (32%). Release from hemolymph towards M-diverticula showed the opposite trend, VHDL 75% and HDL1 45%. Isolated lipoproteins showed a similar behavior to that observed with whole hemolymph. Lipid transfer between lipoproteins showed that HDL1 transfer more ¹⁴C-lipids to VHDL than vice versa. Only 38% FFA and 18% TAG were transferred from M-diverticula to lipoproteins, while on the contrary 75% and 73% of these lipids, respectively, were taken up from hemolymph. A similar trend was observed regarding lipoprotein phospholipids. This study supports the hypothesis that HDL1 and hemocyanin-containing VHDL are involved in the uptake and release of FFA, phospholipids and triacylglycerols in the spider P. pythagoricus. The data support a directional flow of lipids from HDL1 and VHDL suggesting a mode of lipid transport between lipoproteins and M-diverticula.     

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host–pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection‐driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six‐plex Tandem mass tag (TMT) labeling‐based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post‐infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self‐regulation and response to virus infection. This study provides new insights for understanding the host–virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.