Silkworm Hemocytes Research Articles

Effects of beauvericin on the blood cells of Bombyx mori

In this study, silkworms were treated by injection of the bioactive depsipeptide beauvericin (BEA) to explore its effect on the cellular immunity of larvae of the silkworm Bombyx mori. The results showed that: The LC50 of BEA for silkworms on the 3rd day of the 4th instar was 362.36 µM. The total count of circulating hemocytes in the silkworms decreased at 12 h after injection with 350 µM BEA, and reached the minimum value at 72 h post-treatment; at 48 h post-treatment, a large number of nodules formed by the aggregation of blood cells of the silkworms were observed under the light microscope. The survival rate of hemocytes in the larvae treated with BEA was significantly reduced in a dose-dependent manner in vivo and in vitro. The encapsulation of Q-Sepharose Fast Flow (QFF) gel particles by hemocytes in the treatment group was significantly higher than that in the control group at 1.5 h and 3 h post-treatment (P < 0.05). Moreover, the melanization ratio of QFF gel particles kept increasing with treatment time. The melanization rate at 24 h after treatment was significantly higher than that at other times (P < 0.05), reaching 55.33 %. Under the scanning electron microscope, BEA-treated larvae showed protrusions on the surface of their blood cells in vivo. Under the transmission electron microscope, it was observed that silkworm hemocytes were vacuolated. This study demonstrated that BEA had an effect on the blood cells of silkworms, and has thrown some light on the inhibitory effect and mechanism of BEA on insect cellular immunity.

KPI5 Is Involved in the Regulation of the Expression of Antibacterial Peptide Genes and Hemolymph Melanization in the Silkworm, Bombyx mori.

Kunitz-type protease inhibitors (KPIs) are ubiquitously found in many organisms, and participate in various physiological processes. However, their function in insects remains to be elucidated. In the present study, we characterized and functionally analyzed silkworm KPI5. Sequence analysis showed that KPI5 contains 85 amino acids with six conserved cysteine residues, and the P1 site is a phenylalanine residue. Inhibitory activity and stability analyses indicated that recombinant KPI5 protein significantly inhibited the activity of chymotrypsin and was highly tolerant to temperature and pH. The spatio-temporal expression profile analysis showed that KPI5 was synthesized in the fat body and secreted into the hemolymph. In vivo induction analysis showed that the expression of KPI5 in the fat body was significantly upregulated by pathogen-associated molecular patterns (PAMPs). Binding assays suggested that KPI5 can bind to pathogens and PAMPs. In vitro pathogen growth inhibition assay and encapsulation analysis indicated that KPI5 can neither kill pathogenic bacteria directly nor promote the encapsulation of agarose beads by silkworm hemocytes. Recombinant protein injection test and CRISPR/Cas9-mediated knockdown showed that KPI5 promotes the expression of antimicrobial peptides (AMPs) in the fat body. Moreover, the survival rate of individuals in the KPI5 knockdown group was significantly lower than that of the control group after pathogen infection. Phenoloxidase (PO) activity assays showed that KPI5 significantly inhibited the hemolymph PO activity and melanization induced by PAMPs. These findings suggested that KPI5 plays a dual regulatory role in innate immunity by promoting the expression of antimicrobial peptides in the fat body and inhibiting hemolymph melanization. Our study furthers the understanding of the function of insect KPIs and provides new insights into the regulatory mechanism of insect immune homeostasis.

Identification of Silkworm Hemocyte Subsets and Analysis of Their Response to Baculovirus Infection Based on Single-Cell RNA Sequencing.

A wide range of hemocyte types exist in insects but a full definition of the different subclasses is not yet established. The current knowledge of the classification of silkworm hemocytes mainly comes from morphology rather than specific markers, so our understanding of the detailed classification, hemocyte lineage and functions of silkworm hemocytes is very incomplete. Bombyx mori nucleopolyhedrovirus (BmNPV) is a representative member of the baculoviruses and a major pathogen that specifically infects silkworms (Bombyx mori) and causes serious losses in sericulture industry. Here, we performed single-cell RNA sequencing (scRNA-seq) of hemocytes in BmNPV and mock-infected larvae to comprehensively identify silkworm hemocyte subsets and determined specific molecular and cellular characteristics in each hemocyte subset before and after viral infectmadion. A total of 20 cell clusters and their potential marker genes were identified in silkworm hemocytes. All of the hemocyte clusters were infected by BmNPV at 3 days after inoculation. Interestingly, BmNPV infection can cause great changes in the distribution of hemocyte types. The cells appearing in the infection group mainly belong to prohemocytes (PR), while plasmatocytes (PL) and granulocytes (GR) are very much reduced. Furthermore, we found that BmNPV infection suppresses the RNA interference (RNAi) and immune response in the major hemocyte types. In summary, our results revealed the diversity of silkworm hemocytes and provided a rich resource of gene expression profiles for a systems-level understanding of their functions in the uninfected condition and as a response to BmNPV.

Five Silkworm 30K Proteins Are Involved in the Cellular Immunity against Fungi.

Simple SummaryThe molecular mechanism of 30K proteins in anti-fungal immunity remains unclear. Here, we examined the mRNA levels of 30K proteins, including BmLP1, BmLP2, BmLP3, BmLP4, and BmLP7, and found that all of these proteins were significantly upregulated after injection of pathogen-associated molecular patterns to the fifth instar larvae, implying their involvement in immune response. The binding assay results showed that only BmLP1 and BmLP4 can bind to both fungal cells and silkworm hemocytes. In vitro, the encapsulation of hemocytes on day 5 of the fifth instar larval stage was promoted by the coating of agarose beads with recombinant BmLP1 and BmLP4. Therefore, these results demonstrate that 30K proteins are involved in the cellular immunity of silkworms by acting as pattern recognition molecules to directly recruit hemocytes to the fungal surface. We believe that our study makes a significant contribution to the literature because it provides insights into the 30K-mediated cellular immunity in silkworms.Background: 30K proteins are a major group of nutrient storage proteins in the silkworm hemolymph. Previous studies have shown that 30K proteins are involved in the anti-fungal immunity; however, the molecular mechanism involved in this immunity remains unclear. Methods: We investigated the transcriptional expression of five 30K proteins, including BmLP1, BmLP2, BmLP3, BmLP4, and BmLP7. The five recombinant 30K proteins were expressed in an Escherichia coli expression system, and used for binding assays with fungal cells and hemocytes. Results: The transcriptional expression showed that the five 30K proteins were significantly upregulated after injection of pathogen-associated molecular patterns to the fifth instar larvae, indicating the possibility of their involvement in immune response. The binding assay showed that only BmLP1 and BmLP4 can bind to both fungal cells and silkworm hemocytes. Furthermore, we found that BmLP1-coated and BmLP4-coated agarose beads promote encapsulation of hemocytes in vitro. The hemocyte encapsulation was blocked when the BmLP1-coated beads were preincubated with BmLP1 specific polyclonal antibodies. Conclusions: These results demonstrate that 30K proteins are involved in the cellular immunity of silkworms by acting as pattern recognition molecules to directly recruit hemocytes to the fungal surface.

Integrin β3 plays a novel role in innate immunity in silkworm, Bombyx mori

Integrins are transmembrane receptors that play essential roles in many physiological and pathological processes through cell-to-cell and cell-to-extracellular matrix (ECM) interactions. In the current study, a 2653-bp full-length cDNA of a novel integrin β subunit (designated Bmintegrin β3) was obtained from silkworm hemocytes. Bmintegrin β3 has the typical conserved structure of the integrin β family. The qRT-PCR results showed that Bmintegrin β3 was specifically expressed in the hematological system and that its expression was significantly increased after challenge with different types of PAMPs and bacteria. The recombinant Bmintegrin β3 protein displayed increased aggregation with S. aureus, suggesting that Bmintegrin β3 might directly bind to PAMPs. Interestingly, Bmintegrin β3 knockdown promoted PPO1, PPO2, BAEE, SPH78, SPH125, and SPH127 expression and accelerated the melanization process. Unexpectedly, the expression of genes related to phagocytosis, the Toll pathway, and the IMD pathway was also up-regulated after Bmintegrin β3 knockdown. Thus, Bmintegrin β3 might be a pattern recognition protein (PRP) for PAMPs and might directly bind to bacteria and enhance the phagocytosis activity of hemocytes. Moreover, Bmintegrin β3 and its ligand might negatively regulate the expression of immune-related genes through an unknown mechanism. In summary, our studies provide new insights into the immune functions of Bmintegrin β3 from the silkworm, Bombyx mori.

Cypermethrin Formulation (Ustad-10 EC) Induces Genotoxicity via Apoptosis, Affects Nutritional Physiology, and Modulates Immune Response in Silkworm Philosamia ricini (Lepidoptera: Saturniidae).

Cypermethrin is a pyrethroid insecticide with high insecticidal activity, low mammalian toxicity, and biodegradability. The present study aimed to determine the acute toxicity and evaluate the secondary toxic effects of a commercial formulation of cypermethrin on silkworm Philosamia ricini Hutt of Northeast India. The potential genotoxicity of cypermethrin on silkworm hemocyte was examined by comet assay, caspase activation, and annexin V affinity assay. Alteration in nutritional physiology and histoarchitecture of the gut region was evaluated. Additionally, immunotoxicological effect of cypermethrin was studied by phenoloxidase (PO), lysozyme assay, and abundance of circulating hemocytes. The LC50 value at 24-, 48-, 72-, and 96-h exposure period was recorded as 185.96, 105.34, 72.42, and 58.41 µg/liter, respectively. Approximately sevenfold increase in mean comet tail length was observed at 24 h posttreatment with sublethal concentrations of cypermethrin. Cypermethrin also induced apoptosis and activated caspase reaction in silkworm hemocytes. Moreover, a significant decrease in digestive enzyme activity was observed at higher concentrations of cypermethrin. In cypermethrin-exposed groups, alteration in histoarchitecture was also observed in the form of ruptured microvilli and thin, deformed, fused mucous layer. The PO enzyme and lysozyme enzyme activity was also altered with sublethal concentration of cypermethrin. Total hemocyte count was reduced to 10587.10, 10052.30, 9234.30, and 8842.60 per mm3 with 10, 20, 30, and 40 µg/liter, respectively. The results offer new insights into the negative consequences of very low concentrations of cypermethrin formulations on nonmulberry silkworm of Northeast India.

Larval Exposure to Chlorpyrifos Affects Nutritional Physiology and Induces Genotoxicity in Silkworm Philosamia ricini (Lepidoptera: Saturniidae).

Chlorpyrifos is a most widely used organophosphate insecticide because of its cost effectiveness and degradable nature. However, this pesticide enters and contaminates the environment either by direct application, spray drifts or crop run off and shows adverse effect on the non-targeted organisms. Philosamia ricini (eri silkworm), one of the most exploited, domesticated and commercialized non mulberry silkworm is known for mass production of eri silk. The silkworm larvae get exposed to pesticide residues on the leaves of food plants. The present study investigates the effect of commercial formulation of chlorpyrifos (Pyrifos-20 EC) on eri silkworm. Initially the LC50 value of chlorpyrifos was determined at 24–96 h and further experiments were carried out with sub lethal concentrations of the chlorpyrifos after 24 h of exposure period. The potential toxicity of chlorpyrifos was evaluated as a fuction of metabolism and nutritional physiology in 3rd, 4th, and 5th instar larvae. Alteration in histoarchitecture of 5th instar eri silkworm gut exposed to sub lethal concentration of chlorpyrifos formulation was also studied. Chlorpyrifos induced genotoxicity in silkworm hemocytes was also investigated by single cell gel electrophoresis, micronuclei assay, and apoptosis assay. Herein, LC50 values of chlorpyrifos were calculated as 3.83, 3.35, 2.68, and 2.35 mg/L at 24, 48, 72, and 96h respectively. A significant decrease in trehalose activity along with digestive enzyme activity was observed in chlorpyrifos affected groups (P < 0.05). Further, genotoxicity study revealed higher tail percentage, tail length and tail moment of the damage DNA in chlorpyrifos exposed groups (P < 0.001). Moreover, at 2.0 mg/L concentration, ~10 fold increases in tail length was observed as compared to the control. Results showed activation of caspase activity following 24 h chlorpyrifos exposure (1.5 and 2.0 mg/L) in a dose-dependent manner. Moreover, in control group less number of apoptotic cells was detected, however in both chlorpyrifos exposed groups' numbers of apoptotic cells were statistically higher (P < 0.001). Taken together, this study provides evidence that chlorpyrifos pollution might have adverse effect on overall nutritional physiology and genotoxicity of eri silkworm that could lead to reduced survivability of this economically beneficial insect.

Gene Expression Profile of Bombyx mori Hemocyte under the Stress of Destruxin A

Destruxin A (DA) is a cyclo-peptidic mycotoxin from the entomopathogenic fungus Metarhizium anisopliae. To uncover potential genes associated with its molecular mechanisms, a digital gene expression (DGE) profiling analysis was used to compare differentially expressed genes in the hemocytes of silkworm larvae treated with DA. Ten DGE libraries were constructed, sequenced, and assembled, and the unigenes with least 2.0-fold difference were further analyzed. The numbers of up-regulated genes were 10, 20, 18, 74 and 8, as well as the numbers of down-regulated genes were 0, 1, 8, 13 and 3 at 1, 4, 8, 12 and 24 h post treatment, respectively. Totally, the expression of 132 genes were significantly changed, among them, 1, 3 and 12 genes were continually up-regulated at 4, 3 and 2 different time points, respectively, while 1 gene was either up or down-regulated continually at 2 different time points. Furthermore, 68 genes were assigned to one or multiple gene ontology (GO) terms and 89 genes were assigned to specific Kyoto Encyclopedia of Genes and Genomes (KEGG) Orthology. In-depth analysis identified that these genes putatively involved in insecticide resistance, cell apoptosis, and innate immune defense. Finally, twenty differentially expressed genes were randomly chosen and validated by quantitative real-time PCR (qRT-PCR). Our studies provide insights into the toxic effect of this microbial insecticide on silkworm's hemocytes, and are helpful to better understanding of the molecular mechanisms of DA as a biological insecticide.

Serratia marcescens Suppresses Host Cellular Immunity via the Production of an Adhesion-inhibitory Factor against Immunosurveillance Cells

Injection of a culture supernatant of Serratia marcescens into the bloodstream of the silkworm Bombyx mori increased the number of freely circulating immunosurveillance cells (hemocytes). Using a bioassay with live silkworms, serralysin metalloprotease was purified from the culture supernatant and identified as the factor responsible for this activity. Serralysin inhibited the in vitro attachment of both silkworm hemocytes and murine peritoneal macrophages. Incubation of silkworm hemocytes or murine macrophages with serralysin resulted in degradation of the cellular immune factor BmSPH-1 or calreticulin, respectively. Furthermore, serralysin suppressed in vitro phagocytosis of bacteria by hemocytes and in vivo bacterial clearance in silkworms. Disruption of the ser gene in S. marcescens attenuated its host killing ability in silkworms and mice. These findings suggest that serralysin metalloprotease secreted by S. marcescens suppresses cellular immunity by decreasing the adhesive properties of immunosurveillance cells, thereby contributing to bacterial pathogenesis.

Serratia marcescens Induces Apoptotic Cell Death in Host Immune Cells via a Lipopolysaccharide- and Flagella-dependent Mechanism

Injection of Serratia marcescens into the blood (hemolymph) of the silkworm, Bombyx mori, induced the activation of c-Jun NH(2)-terminal kinase (JNK), followed by caspase activation and apoptosis of blood cells (hemocytes). This process impaired the innate immune response in which pathogen cell wall components, such as glucan, stimulate hemocytes, leading to the activation of insect cytokine paralytic peptide. S. marcescens induced apoptotic cell death of silkworm hemocytes and mouse peritoneal macrophages in vitro. We searched for S. marcescens transposon mutants with attenuated ability to induce apoptosis of silkworm hemocytes. Among the genes identified, disruption mutants of wecA (a gene involved in lipopolysaccharide O-antigen synthesis), and flhD and fliR (essential genes in flagella synthesis) showed reduced motility and impaired induction of mouse macrophage cell death. These findings suggest that S. marcescens induces apoptosis of host immune cells via lipopolysaccharide- and flagella-dependent motility, leading to the suppression of host innate immunity.

Purification and characterization of silkworm hemocytes by flow cytometry

Hemocyte functions are well-investigated in the silkworm, Bombyx mori, however, detailed analysis of each hemocyte subset has been hampered by the lack of appropriate separation method. Here we use an array of flow cytometric analyses to characterize silkworm hemocytes with various molecular probes, such as propidium iodide, green fluorescence protein, monoclonal antibodies, and fluorescent lectins. Of these, separation using propidium iodide was the simplest and provided most reliable results for the isolation of the hemocyte subsets. cDNAs were then synthesized from these sorted populations and subset-specific gene expression was examined by RT-PCR. Granulocytes, plasmatocytes, and oenocytoids expressed different classes of immune genes, suggesting that they have multiple roles in silkworm immunity. In contrast, a contribution of spherulocytes to immunity was not documented in that they failed to express most of the genes. The functions of spherulocytes are thus likely to be distinct from those of the other three hemocyte subsets.

Activation of the Silkworm Cytokine by Bacterial and Fungal Cell Wall Components via a Reactive Oxygen Species-triggered Mechanism

The insect cytokine paralytic peptide (PP) induces muscle contraction in silkworm larvae. Here we demonstrate that bacterial and fungal cell wall components peptidoglycan and glucan stimulate muscle contraction via activation of PP in the hemolymph. Anti-PP antibody suppressed the muscle contraction induced by PP, peptidoglycan, or glucan. The contraction was also inhibited by free radical scavengers and serine protease inhibitors. Moreover, injecting live silkworms with peptidoglycan or glucan generated the active form of PP. The active form of PP was also produced in vitro when peptidoglycan or glucan was incubated with hemolymph containing the PP precursor. Generation of the active form of PP was suppressed by free radical scavengers and serine protease inhibitors. Furthermore, PP activation in isolated hemolymph was inhibited by potassium cyanide, suggesting that cellular activity is involved. Stimulation by peptidoglycan promoted the generation of reactive oxygen species by silkworm hemocytes. The addition of either the active form of PP or anti-PP antibody to Staphylococcus aureus injected into silkworm larvae delayed or enhanced, respectively, the killing effect of S. aureus, suggesting that activated PP contributes to host resistance to infectious pathogens. These findings suggest that immunologic stimulants such as peptidoglycan or glucan induce reactive oxygen species production from larval hemocytes, followed by the activation of serine protease, which mediates the PP processing reaction and leads to defensive responses.

Silkworm (Bombyx mori) hemocytes do not produce reactive oxygen metabolites as a part of defense mechanisms.

To investigate whether hemocytes of Bombyx mori (Lepidoptera) larvae produce reactive oxygen species (ROS) as part of the oxidative killing of invading pathogens, the production of ROS was measured as a luminol- and lucigenin-enhanced chemiluminescence of unstimulated or stimulated (zymosan particles, phorbol myristate acetate, calcium ionophore, rice starch or Xenorhabdus nematophila) hemolymph. No detectable ROS production was found. The spontaneous and activated ROS production measured with hemocytes, i.e. under the conditions when the antioxidative potential of hemolymph plasma was eliminated, was again undetectable. Likewise, ROS production by isolated hemocytes was observed by spectrophotometric (NBT test, cytochrome c assay) and fluorimetric (using dihydrorhodamine and hydroethidine probes) methods. Hence none of the experimental approaches used indicated the production of ROS by hemocytes of B. mori larvae as part of their immune response.

Immunolocalization of prophenoloxidase among hemocytes of the silkworm, Bombyx mori

Silkworm ( Bombyx mori) hemocytes were fixed immediately after collection. Thin sections of the hemocytes were stained by an indirect immunogold staining method using rabbit anti-prophenoloxidase/IgG as a primary antibody and colloidal gold coated with goat anti-rabbit/IgG as a secondary antibody. Electron micrographs of the sections revealed that only plasmatocytes and oenocytoids have prophenoloxidase both in cytoplasm and nucleus whereas granulocytes, spherulocytes and prohemocytes do not have appreciable amounts of the proenzyme. Cytoplasmic inclusions of oenocytoids also contain the proenzyme. A wide variety of concentrations of prophenoloxidase was observed among oenocytoids. Plasmatocytes appeared to have less prophenoloxidase than any oenocytoids. Once materials in the granules of granulocyte were discharged into the plasma and formed coagula, they cross-reacted with antiprophenoloxidase/IgG, suggesting that prophenoloxidase was trapped in the coagula by unknown mechanisms. This observation is discussed in relation to the dispute concerning the presence of prophenoloxidase or phenoloxidase in the granulocyte.

The separation and the cytotoxic test of silkworm hemocytes

The separation and the cytotoxic test of silkworm hemocytes

On the Heart in Relation to Circulation of Hemocytes in Insects

Verson (1908) found that silkworm hemocytes did not enter the heart, and stated that this was because of the narrowness of the ostial slits. Absence of hemocytes from the silkworm heart was noted by Masera (1933) in his study on pulsation rates. According to Weber (1935) the hemocytes of adult Aleurodes are too large to enter the heart and only plasma circulates through it. Wiggles worth (1950) found that the hemocytes in Rhodnius fail to enter the heart lumen and on the basis of this observation and those of the previously mentioned workers has concluded that in insects “… only cell-free hemolymph can be said to truly circulate.” He goes on to say that “… it is obvious, therefore, that the numbers of circulating cells must vary enormously from time to time.”