Silkworm Hemolymph Research Articles

A novel silkworm infection model with fluorescence imaging using transgenic Trichosporon asahii expressing eGFP

Trichosporon asahii is a pathogenic fungus that causes deep mycosis in patients with neutropenia. Establishing an experimental animal model for quantitatively evaluating pathogenicity and developing a genetic recombination technology will help to elucidate the infection mechanism of T. asahii and promote the development of antifungal drugs. Here we established a silkworm infection model with a transgenic T. asahii strain expressing eGFP. Injecting T. asahii into silkworms eventually killed the silkworms. Moreover, the administration of antifungal agents, such as amphotericin B, fluconazole, and voriconazole, prolonged the survival time of silkworms infected with T. asahii. A transgenic T. asahii strain expressing eGFP was obtained using a gene recombination method with Agrobacterium tumefaciens. The T. asahii strain expressing eGFP showed hyphal formation in the silkworm hemolymph. Both hyphal growth and the inhibition of hyphal growth by the administration of antifungal agents were quantitatively estimated by monitoring fluorescence. Our findings suggest that a silkworm infection model using T. asahii expressing eGFP is useful for evaluating both the pathogenicity of T. asahii and the efficacy of antifungal drugs.

Differential Count of Haemocyte from the Haemolymph of Muga Silkworm (Antheraea assamensis H.) reared on the host plant

Five types of circulating haemocytes have been identified from the haemolymph of 5th instar larvae of the Muga Silkworm (Antheraea assamensis) reared on Som leaves (Machilus bombycina) at Govt. Basic Muga Seed Farm, Khanapara, Assam, India. Haemocytes were identified by means of light microscopy as prohaemocytes, plasmatocytes, granulocytes, spherulocytes and oenocytoids. Differential count of haemocyte from the haemolymph of fifth-instar larvae of Muga Silkworm were also studied and calculated. Among them, prohaemocytes were found to be the most abundant cell types in the haemolymph, whereas spherulocytes the rarest.

Responses of detoxification enzymes in the midgut of Bombyx mori after exposure to low-dose of acetamiprid

Bombyx mori is an important economic insect. However, the environmental pollution caused by the widespread use of neonicotinoid insecticides has significantly affected the safe production of sericulture. In this paper, we determined the LC50 of acetamiprid, a kind of neonicotinoid insecticides, to 5th instar silkworm larvae, examined its residues in hemolymph and midgut of silkworm after continuous exposure to low-dose of acetamiprid, and investigated the transcription level of detoxifying-related genes and the activity of detoxifying enzymes. The results showed that acetamiprid was highly toxic (24-h LC50, 1.50 mg/L) to silkworm larvae. After continuous exposure to low-dose of acetamiprid (0.15 mg/L), the acetamiprid residue concentrations in hemolymph and midgut were 0.90 and 0.58 μg/mg, respectively, at 48 h, but all decreased at 96 h. At 24 h of acetamiprid exposure, the transcription levels of CYP4M5 and CYP6AB4 and the P450 enzyme activity were significantly enhanced. However, the transcription levels of CarE and CarE-11 and the activity of CarE enzymes were both inhibited by acetamiprid exposure. After 24 h–72 h of acetamiprid exposure, the transcription levels of GSTe3 and GSTd1 were significantly up-regulated, and the GST enzyme activity was also significantly elevated from 48 h to 96 h. Furthermore, the expression levels of FoxO, CncC and Keap1, the key upstream genes of detoxification enzymes, showed a similar trend as the GST genes. These results indicated that acetamiprid was reduced in midgut and the expression of GSTs was upregulated may via FoxO/CncC/Keap1 signaling pathway, which plays a key role in detoxification responses.

INDUCED PHENOLOXIDASE PROFILES IN SILKWORM Bombyx mori (L.) UNDER BIOTIC STRESS

Phenol oxidase (PO) is one of the stress enzyme protein in living organism. The conversion of Pro-PO into an activation form of PO required a stress protein. In the present study has emerged with the novel finding of induced phenoloxidase was identified under bacterial endotoxin viz., Lipopolysaccharide (LPS) activity using silkworm Bombyx mori as an animal model. The PO enzyme plays an important role for insect survival during pathophysiological conditions. The enzyme activity was analyzed from ten different silkworm races with two phenolic substrates viz., L-Dopa and Dopamine by Native-PAGE. The bacterially induced PO was found in hemolymph and midgut of silkworm, PO3 were induced by LPS injection. In control PO1 & PO2 are non-bacterially induced protein having the molecular weight of 72 and 71. The results shown that there is no substrate specificity and similar functional activity was found in hemolymph and midgut under pathogenic condition. It was observed that bacterially inducible PO clearly differed from non-inducible PO (control). At final observation of induced isozymes of PO in the haemolymph and midgut system of tolerant silkworm races points out the existence of biochemical immunity against biotic stress of LPS. This is the first report to document the silkworm immunity under the LPS toxin in different silkworm races to identify the tolerant and susceptibility against a biotic stress.

Virus induced global protein synthesis shutdown in Antheraea mylitta infected with Bombyx mori nuclear polyhedrosis virus

An abortive infection system was studied involving Antheraea mylitta and Bombyx mori nuclear polyhedrosis virus (BmNPV). SDS-PAGE of healthy and infected A. mylitta silkworm's hemolymph proteins was analysed and mortality rate was used as index to verify infection. In A. mylitta cells, BmNPV infection resulted in a severe global protein synthesis shutdown during early post infection i.e. within 24 hrs. These results indicate that the restriction of BmNPV multiplication in A. mylitta is caused by a global protein synthesis shutdown resulting in non-productive infection evident from our observation that there was no significant mortality of A. mylitta silkworms administered with BmNPV.

A systematic and methodical approach for the efficient purification of recombinant protein from silkworm larval hemolymph

The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.

Silk Protein as a Fetal Bovine Serum Substitute for Animal Cell Culture

Fetal Bovine Serum (FBS) is an essential substance added to animal cell culture medium. However, its composition is unclear causing problems such as development of an immune response when cultured cells are transplanted into the human body. In this study, silk sericin, silk fibroin, and hemolymph obtained from silkworms were added to the cell culture medium in order to determine if it can replace FBS. After establishment of the cell culture, cell proliferation and expression levels of cell growth-related genes were compared with those of control cells (cells cultured in the medium with 10% FBS). Results showed that the test group treated with silk fibroin extracted from a Korean silkworm variety, Kumokjam could replace 10% FBS. In addition, expression levels of cell growth related genes such as Fibronectin and TGF-β1 increased significantly in cells cultured using silk fibroin, depending on the concentration used in cell adhesion and cell proliferation [24]. To date, no studies have been conducted to find a replacement for FBS. Thus, this study was carried out to develop a substitute for FBS by using silkworm-derived alternatives such as silkworm hemolymph, silk sericin, and silk fibroin, which are cheap and have various physiological effects, cell promoting effects, and can be mass produced.

Identification of secretion domain of Neospora caninum profilin

Profilin (PROF) is a small actin-binding protein presented in apicomplexan protozoa. It was previously reported that Neospora caninum profilin (NcPROF) is secreted into the hemolymph of silkworm larvae regardless of the lack of an identified regular secretion signal peptide. To date, which domain is required for its secretion still remains unknown. To this end, we express a fluorescent protein (mCherry) fused with NcPROF at its N-terminus or C-terminus. Both fusion proteins were expressed and secreted into the culture supernatant from Bm5 cells or hemolymph from silkworm larvae, respectively. To further narrow down the C-terminal minimal domain required for its secretion, we constructed three truncated C-terminal domain constructions, ΔN (aa41–163), ΔN1 (aa50–163), and ΔN2 (aa144–163) respectively. All three fusion proteins were detected in the culture supernatant of Bm5 cells and silkworm hemolymph. Surprisingly, a 20-aa C-terminal α-helix domain facilitates the secretion of mCherry, allowing purification of ΔN2-mCherry from silkworm larval hemolymph by affinity chromatography. Taken together, the secretion domain from NcPROF was identified, indicating that can be utilized for the secretory expression of recombinant proteins in the future.

Bmserpin2 Is Involved in BmNPV Infection by Suppressing Melanization in Bombyx mori.

Melanization, an important defense response, plays a vital role in arthropod immunity. It is mediated by serine proteases (SPs) that convert the inactive prophenoloxidase (PPO) to active phenoloxidase (PO) and is tightly regulated by serine protease inhibitors (serpins) which belong to a well distributed superfamily in invertebrates, participating in immune mechanisms and other important physiological processes. Here, we investigated the Bmserpin2 gene which was identified from a transcriptome database in response to Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that Bmserpin2 was expressed in all tissues, with maximum expression in fat body. Upon BmNPV infection, the expression of Bmserpin2 was up-regulated in P50 (susceptible strain) and BC9 (resistant strain) in haemocytes, fat body and the midgut. However, up-regulation was delayed in BC9 (48 or 72 h), in contrast to P50 (24 h), after BmNPV infection. Meanwhile, Bmserpin2 could delay or inhibit melanization in silkworm haemolymph. Significant increased PO activity can be observed in Bmserpin2-depleted haemolymph under NPV infection. Furthermore, the viral genomic DNA copy number was decreased in Bmserpin2-depleted haemolymph. We conclude that Bmserpin2 is an inducible gene which might be involved in the regulation of PPO activation and suppressed melanization, and have a potential role in the innate immune system of B. mori.

Nosema bombycis suppresses host hemolymph melanization through secreted serpin 6 inhibiting the prophenoloxidase activation cascade

Nosema bombycis is a pathogen of the silkworm that belongs to the microsporidia, a group of obligate intracellular parasites related to fungi. N. bombycis infection causes the disease pébrine in silkworms. Insects utilize hemolymph melanization as part of the innate immune response to fight against pathogens, and melanization relies on a serine protease-mediated prophenoloxidase (PPO) activation cascade that is tightly regulated by serine protease inhibitors (serpins). Previous studies showed that N. bombycis infection suppressed silkworm hemolymph melanization, however the mechanism has not been elucidated. We hypothesize that N. bombycis can secret serpins (NbSPNs) to inhibit host serine proteases in the PPO activation cascade, thus suppressing phenoloxidase (PO) activity and the consequent melanization. We demonstrated in this study that N. bombycis infection suppressed silkworm PO activity and melanization and we identified the expression of N. bombycis serpin 6 (NbSPN6) in the hemolymph of the infected host. When recombinant NbSPN6 was added to normal hemolymph, PO activity was inhibited in a dose-dependent manner. Moreover, in vivo analysis by RNA interference technology showed that when NbSPN6 expression is blocked, the inhibitory effects on PO activity can be reversed and the proliferation of N. bombycis within host can be suppressed. These results demonstrated the indispensable role of NbSPN6 in successful pathogen infection. To further elucidate the molecular basis of NbSPN6 suppressing host defense, we determined that the host serine protease prophenoloxidase-activating enzyme (PPAE) is the direct target of NbSPN6 inhibition. Taken together, our novel study is the first to elucidate the molecular mechanism of pathogen-derived serpin inhibiting hemolymph melanization and, thus, regulating host innate immune responses. This study may also provide novel strategies for preventing microsporidia infection.

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.

Label-free LC-MS/MS proteomic analysis of the hemolymph of silkworm larvae infected with Beauveria bassiana

Beauveria bassiana, a pathogen of the economically important silkworm (Bombyx mori), causes serious losses in the sericulture industry; however, the mechanisms underlying B. bassiana infection and the silkworm response are not fully understood. To obtain new insights into the interaction between B. bassiana and its host, hemolymph samples from fifth instar silkworm larvae infected with B. bassiana were analyzed at 36-h post-inoculation using a label-free LC-MS/MS proteomic technique. In total, 671 proteins were identified in the hemolymph, including 87 differentially expressed proteins, 42 up-regulated and 45 down-regulated in infected larvae. Six were detected only in infected larvae, and five were detected only in uninfected larvae. Based on GO annotations, 48 of the differentially expressed proteins were involved in molecular functions, 42 were involved in biological processes, and 39 were involved in cell components. A KEGG pathway analysis indicated that these differentially expressed proteins participate in 85 signal transduction pathways, including the amoebiasis, MAPK signaling, Hippo signaling, Toll and Imd signaling, and lysosome pathways. The silkworm hemolymph is the main site for B. bassiana replication. We identified differentially expressed proteins involved in the regulation of the host response to B. bassiana infection, providing important experimental data for the identification of key factors contributing to the interaction between the pathogenic fungus and its host.

Pharmacokinetics of anti-infectious reagents in silkworms

Silkworm microorganism infection models are useful for screening novel therapeutically effective antimicrobial agents. In this study, we used silkworms to investigate the pharmacokinetics and metabolism of antimicrobial agents, in which cytochrome P450 plays a major role. The pharmacokinetic parameters of the antimicrobial agents were determined based on their concentrations in the hemolymph after administration. The parameters, such as half-lives and distribution volumes, in silkworm were consistent with those in mammalian models. In addition, antifungal agents with reduced therapeutic effectiveness due to high protein-binding capacities in mammalian serum exhibited similar features in silkworm hemolymph. Cytochrome P450 enzymes, which metabolize exogenous compounds in mammalian liver, were distributed mainly in the silkworm midgut. Most of the compounds metabolized by cytochrome P450 in humans are also metabolized in the silkworm midgut. These findings suggest that the pharmacokinetics of antimicrobial agents are fundamentally similar between silkworms and mammals, and that therapeutic effects in the silkworm infection model reflect the pharmacokinetics of the test samples.

Antibacterial and anti‐inflammatory activities of the immune‐challenged silkworm (Bombyx mori) hemolymph with Lactobacillus cell wall extracts

AbstractInsects have been used by humans as food and traditional medicines for many years. Their antimicrobial and anti‐inflammatory activities are a main focus of medicinal insect research. In a previous study, hemocyte extracts prepared from Bombyx mori larvae were found to have anti‐inflammatory effects. Therefore, we injected Lactobacillus cell wall extracts dorsolaterally into the hemocoel to increase antimicrobial peptides produced by silkworm larva. The best overall antimicrobial activities were observed against E. coli after injection of 10 μL hemolymph from immune‐challenged larvae, although the tested extract did not affect the cell proliferation rate. The effects of the immune‐challenged B. mori hemolymph on anti‐inflammatory pathways were determined using reverse transcription quantitative polymerase chain reaction and western blotting to assess the expression of pro‐inflammatory molecules in macrophage‐like human monocytic leukemia THP‐1 cells following treatment with phorbol myristate acetate. The results demonstrated that the hemolymph of immune‐challenged silkworms had antibacterial and anti‐inflammatory effects. The effects of silkworms with immune‐challenged hemolymph could facilitate the development of new types of functional foods, feed additives, and biomaterials with antibacterial and anti‐inflammatory properties.

Overexpression of BmFoxO inhibited larval growth and promoted glucose synthesis and lipolysis in silkworm.

Forkhead box O (FoxO) is a downstream transcription factor of the insulin-signaling pathway, which plays vital roles in the growth and metabolism of organisms. In this study, BmFoxO was overexpressed in BmE cells, in which proliferation was inhibited and apoptosis was increased. The transgenic vector overexpressing BmFoxO was constructed, and the transgenic silkworm line A4FoxO was generated via embryonic microinjection. The body size of A4FoxO silkworm was smaller than that of non-transgenic silkworm (WT). The quantitative polymerase chain reaction results revealed that the insulin pathway was enhanced and the growth-related TOR pathway was suppressed. Furthermore, the translation of proteins in the fat body of A4FoxO silkworm was inhibited. The expression level of genes involved in the glucose synthesis and lipolysis pathways was increased, whereas that of genes involved in fat synthesis was decreased. Oil red O staining revealed that the amount of lipid droplets was reduced in A4FoxO silkworms compared with WT. Further analysis showed that the content of triglyceride and glycogen was significantly decreased in fat body, but the content of glucose and trehalose was increased in the hemolymph of A4FoxO silkworms. These results suggest that the enhanced expression of BmFoxO disturbs glycolipid metabolism and affects silkworm growth.

Staphylococcus aureus aggregation in the plasma fraction of silkworm hemolymph.

Staphylococcus aureus formed bacterial aggregates in the plasma fraction of the hemolymph of silkworm, the larva of Bombyx mori, in a growth-dependent manner. The addition of arabinose or galactose inhibited the formation of S. aureus aggregates in the silkworm plasma. Formation of the bacterial aggregates depended on S. aureus genes required for the synthesis of bacterial surface polysaccharides–ypfP and ltaA, which are involved in lipoteichoic acid synthesis, and the tagO gene, which is involved in wall teichoic acid synthesis. These findings suggest that S. aureus forms bacterial aggregates in the silkworm plasma via bacterial surface teichoic acids.

Secretory Nanoparticles of Neospora caninum Profilin-Fused with the Transmembrane Domain of GP64 from Silkworm Hemolymph.

Neosporosis, which is caused by Neospora caninum, is a well-known disease in the veterinary field. Infections in pregnant cattle lead to abortion via transplacental (congenitally from mother to fetus) transmission. In this study, a N. caninum profilin (NcPROF), was expressed in silkworm larvae by recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid and was purified from the hemolymph. Three NcPROF constructs were investigated, native NcPROF fused with an N-terminal PA tag (PA-NcPROF), PA-NcPROF fused with the signal sequence of bombyxin from B. mori (bx-PA-NcPROF), and bx-PA-NcPROF with additional C-terminal transmembrane and cytoplasmic domains of GP64 from BmNPV (bx-PA-NcPROF-GP64TM). All recombinant proteins were observed extra- and intracellularly in cultured Bm5 cells and silkworm larvae. The bx-PA-NcPROF-GP64TM was partly abnormally secreted, even though it has the transmembrane domain, and only it was pelleted by ultracentrifugation, but PA-NcPROF and bx-PA-NcPROF were not. Additionally, bx-PA-NcPROF-GP64TM was successfully purified from silkworm hemolymph by anti-PA agarose beads while PA-NcPROF and bx-PA-NcPROF were not. The purified bx-PA-NcPROF-GP64TM protein bound to its receptor, mouse Toll-like receptor 11 (TLR-11), and formed unique nanoparticles. These results suggest that profilin fused with GP64TM was secreted as a nanoparticle with binding affinity to its receptor and this nanoparticle formation is advantageous for the development of vaccines to N. caninum.

Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system

Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost.

Manipulation of the silkworm immune system by a metalloprotease from the pathogenic bacterium Pseudomonas aeruginosa

Antimicrobial peptide (AMP) production and melanization are two key humoral immune responses in insects. Induced synthesis of AMPs results from Toll and IMD signal transduction whereas melanization depends on prophenoloxidase (PPO) activation system. During invasion, pathogens produce toxins and other virulent factors to counteract host immune responses. Here we show that the pathways leading to PPO activation and AMP synthesis in the silkworm Bombyx mori are affected by a metalloprotease, named elastase B, secreted by Pseudomonas aeruginosa (PAO1). The metalloprotease gene (lasB) was expressed shortly after PAO1 cells had been injected into the larval silkworm hemocoel, leading to an increase of elastase activity. Injection of the purified PAO1 elastase B into silkworm hemolymph compromised PPO activation. In contrast, the protease caused a level increase of gloverin, an AMP in the hemolymph. To verify our results obtained using the purified elastase B, we infected B. mori with PAO1 ΔlasB mutant and found that PO activity in hemolymph of the PAO1 ΔlasB-infected larvae was significantly higher than that in the wild type-infected. The mutant-inhabited hemolymph had lower levels of gloverin and antimicrobial activity. PAO1 ΔlasB showed a decreased viability in the silkworm hemolymph whereas the host had a lower mortality. In addition, the effects caused by the ΔlasB mutant were restored by a complementary strain. These data collectively indicated that the elastase B produced by PAO1 is an important virulent factor that manipulates the silkworm immune system during infection.

In vitro rearing of Ooencyrtus nezarae (Hymenoptera: Encyrtidae), an egg parasitoid of Riptortus pedestris (Hemiptera: Alydidae)

ABSTRACTOoencyrtus nezarae Ishii (Hymenoptera: Encyrtidae) is a polyphagous egg parasitoid of various true bugs, including Riptortus pedestris (Fabricius) (Hemiptera: Alydidae), a major pest of soybean and fruit trees in northeastern Asia. This study was conducted to develop artificial host eggs containing insect haemolymph for mass rearing of O. nezarae. The haemolymph of Chinese oak silkworm (Antheraea pernyi Guérin-Méneville) pupae was found to be most suitable for artificial eggs for the complete development of O. nezarae. Among 764 parasitoid eggs laid in artificial eggs, 49.2% successfully developed to adult parasitoids. The developmental time in artificial eggs was delayed by 3–5 days relative to that observed in natural host eggs; however there was no significant difference in the length of hind tibia between adults that emerged from artificial and natural host eggs. Moreover, new generations that emerged from artificial eggs reproduced on all egg types offered (either natural or artificial eggs). We propose that the artificial eggs developed herein can therefore be an effective means for mass rearing O. nezarae.