Antioxidant Activity and Mechanism of Action of Amwaprin: A Protein in Honeybee (Apis mellifera) Venom.

Bee venom contains several bioactive components, including enzymatic and non-enzymatic proteins. There is increasing interest in the bioactive components of bee venom since they have exhibited various pharmacological effects. Recently, Apis mellifera waprin (Amwaprin) was identified as a novel protein in Apis mellifera (honeybee) venom and characterized as an antimicrobial agent. Herein, the novel biological function of Amwaprin as an antioxidant is described. In addition, the antioxidant effects of Amwaprin in mammalian cells were investigated. Amwaprin inhibited the growth of, oxidative stress-induced cytotoxicity, and inflammatory response in mammalian NIH-3T3 cells. Amwaprin decreased caspase-3 activity during oxidative stress and exhibited protective activity against oxidative stress-induced cell apoptosis in NIH-3T3 and insect Sf9 cells. The mechanism underlying the cell protective effect of Amwaprin against oxidative stress is due to its direct binding to the cell membrane. Furthermore, Amwaprin demonstrated radical-scavenging activity and protected against oxidative DNA damage. These results suggest that the antioxidant capacity of Amwaprin is attributed to the synergistic effects of its radical-scavenging action and cell shielding, indicating its novel role as an antioxidant agent.

Bombyx mori Ecdysone Receptor B1 May Inhibit BmNPV Infection by Triggering Apoptosis.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a serious threat to sericulture. Nevertheless, no effective control strategy is currently available. The innate immunity of silkworm is critical in the antiviral process. Exploring its molecular mechanism provides theoretical support for the prevention and treatment of BmNPV. Insect hormone receptors play an essential role in regulating host immunity. We found a correlation between Bombyx mori ecdysone receptor B1 (BmEcR-B1) and BmNPV infection, whereas the underlying mechanism remains unclear. In this study, the expression patterns and sequence characteristics of BmEcR-B1 and its isoform, BmEcR-A, were initially analyzed. BmEcR-B1 was found to be more critical than BmEcR-A in silkworm development and responses to BmNPV. Moreover, RNAi and an overexpression in BmN cells showed BmEcR-B1 had antiviral effects in the presence of 20-hydroxyecdysone (20E); Otherwise, it had no antiviral activity. Furthermore, BmEcR-B1 was required for 20E-induced apoptosis, which significantly suppressed virus infection. Finally, feeding 20E had no significant negative impacts on larval growth and the cocoon shell, suggesting the regulation of this pathway has practical value in controlling BmNPV in sericulture. The findings of this study provide important theoretical support for understanding the mechanism of the silkworm innate immune system in response to BmNPV infection.

Ingestion of heat-killed pathogens confers transgenerational immunity to the pathogens via the vitellogenin-hypopharyngeal gland axis in honeybees.

Honeybee diseases are a serious threat to beekeeping and pollination. Transgenerational immune priming (TGIP) has been attracting increasing attention as a promising strategy to protect honeybee colonies from infections. This study investigated whether feeding honeybees (Apis mellifera) with a heat-killed pathogen cocktail can provide them with transgenerational immunity to these pathogens. We found that vitellogenin (Vg) and defensin-1 were highly upregulated in nurse bees upon feeding them with a cocktail of heat-killed Ascosphaera apis and Paenibacillus larvae (A + P cocktail). Pathogen-pattern-recognition receptor genes in the Toll signaling pathway were upregulated in nurse bees upon ingestion of the A + P cocktail. In the nurse bees of the hives supplied with the A + P cocktail, Vg was upregulated in the fat body, and the defensin-1 expression and Vg uptake in the hypopharyngeal glands were induced. Consequently, the major proteins in royal jelly were upregulated. In addition, defensin-1 was upregulated in the queen larvae and young worker larvae in these hives. In correlation, the young worker larvae showed high pathogen resistance to P. larvae infection. Thus, our findings imply that introduction of a heat-killed pathogen cocktail into hives is an efficient strategy for conferring honeybees with social immunity through TGIP.

Identification of waprin and its microbicidal activity: A novel protein component of honeybee (Apis mellifera) venom.

Bee venom is a rich source of biologically and pharmacologically active proteins. Waprin is a protein component of venoms; however, waprin has yet to be identified in bee venom. Moreover, the biological functions of waprin in venoms remain poorly characterized. Thus, in this study, we have identified and characterized waprin: a novel protein component from the venom of honeybees (Apis mellifera). The waprin in A. mellifera venom (Amwaprin) was found to consist of an 80-amino acid mature peptide, in which the whey acidic protein domain contains four conserved disulfide bonds. We discovered the presence of the Amwaprin protein in secreted venom by using an antibody against recombinant Amwaprin produced in baculovirus-infected insect cells. Recombinant Amwaprin exhibited inhibitory activity against microbial serine proteases and elastases but not thrombin or plasmin. It recognized carbohydrates in the microbial cell wall molecules and bound to the live microbial surfaces. The binding action of Amwaprin produced its microbicidal activity by inducing structural damage to bacterial and fungal cell walls. In addition, recombinant Amwaprin is heat-stable and contains no hemolytic activity. These findings demonstrate that Amwaprin acts as a microbicidal and anti-elastolytic agent.

Fumarate mitigates disruption induced by fenpropathrin in the silkworm Bombyx mori (Lepidoptera): A metabolomics study.

The silkworm Bombyx mori L. is a model organism of the order Lepidoptera. Understanding the mechanism of pesticide resistance in silkworms is valuable for Lepidopteran pest control. In this study, comparative metabolomics was used to analyze the metabolites of 2 silkworm strains with different pesticide resistance levels at 6, 12, and 24 h after feeding with fenpropathrin. Twenty-six of 27 metabolites showed significant differences after fenpropathrin treatment and were classified into 6 metabolic pathways: glycerophospholipid metabolism, sulfur metabolism, glycolysis, amino acid metabolism, the urea cycle, and the tricarboxylic acid (TCA) cycle. After analyzing the percentage changes in the metabolic pathways at the 3 time points, sulfur metabolism, glycolysis, and the TCA cycle showed significant responses to fenpropathrin. Confirmatory experiments were performed by feeding silkworms with key metabolites of the 3 pathways. The combination of iron(II) fumarate + folic acid (IF-FA) enhanced fenpropathrin resistance in silkworms 6.38 fold, indicating that the TCA cycle is the core pathway associated with resistance. Furthermore, the disruption of several energy-related metabolic pathways caused by fenpropathrin was shown to be recovered by IF-FA in vitro. Therefore, IF-FA may have a role in boosting silkworm pesticide resistance by modulating the equilibrium between the TCA cycle and its related metabolic pathways.

Antimicrobial Activity of Apidermin 2 from the Honeybee Apis mellifera.

Apidermins (APDs) are known as structural cuticular proteins in insects, but their additional roles are poorly understood. In this study, we characterized the honeybee, Apis mellifera, APD 2 (AmAPD 2), which displays activity suggesting antimicrobial properties. In A. mellifera worker bees, the AmAPD 2 gene is transcribed in the epidermis, hypopharyngeal glands, and fat body, and induced upon microbial ingestion. Particularly in the epidermis of A. mellifera worker bees, the AmAPD 2 gene showed high expression and responded strongly to microbial challenge. Using a recombinant AmAPD 2 peptide, which was produced in baculovirus-infected insect cells, we showed that AmAPD 2 is heat-stable and binds to live bacteria and fungi as well as carbohydrates of microbial cell wall molecules. This binding action ultimately induced structural damage to microbial cell walls, which resulted in microbicidal activity. These findings demonstrate the antimicrobial role of AmAPD 2 in honeybees.

Bee Venom Induces Acute Inflammation through a H2O2-Mediated System That Utilizes Superoxide Dismutase.

Venoms from venomous arthropods, including bees, typically induce an immediate local inflammatory response; however, how venoms acutely elicit inflammatory response and which components induce an inflammatory response remain unknown. Moreover, the presence of superoxide dismutase (SOD3) in venom and its functional link to the acute inflammatory response has not been determined to date. Here, we confirmed that SOD3 in bee venom (bvSOD3) acts as an inducer of H2O2 production to promote acute inflammatory responses. In mouse models, exogenous bvSOD3 rapidly induced H2O2 overproduction through superoxides that are endogenously produced by melittin and phospholipase A2, which then upregulated caspase-1 activation and proinflammatory molecule secretion and promoted an acute inflammatory response. We also showed that the relatively severe noxious effect of bvSOD3 elevated a type 2 immune response and bvSOD3 immunization protected against venom-induced inflammation. Our findings provide a novel view of the mechanism underlying bee venom-induced acute inflammation and offer a new approach to therapeutic treatments for bee envenoming and bee venom preparations for venom therapy/immunotherapy.

Dual function of a bumblebee (Bombus ignitus) serine protease inhibitor that acts as a microbicidal peptide and anti-fibrinolytic venom toxin.

In bee venoms, low-molecular-weight peptides, including serine protease inhibitors (SPIs), exhibit multifunctional activities. Although SPIs in bee venoms are relatively well known, those that function in both the body and secreted venom of bees are not well-characterized. In this study, we identified a bumblebee (Bombus ignitus) SPI (BiSPI) that displays microbicidal and anti-fibrinolytic activities. BiSPI was found to consist of a trypsin inhibitor-like domain containing a P1 site and ten cysteine residues. We observed that the BiSPI gene was ubiquitously transcribed in the body, including the venom glands. In correlation, the BiSPI protein was detected both in the body and secreted venom by using an antibody against a recombinant BiSPI peptide produced in baculovirus-infected insect cells. Recombinant BiSPI exhibited inhibitory activity against trypsin but not chymotrypsin and inhibited microbial serine proteases and plasmin but not elastase or thrombin. Moreover, recombinant BiSPI recognized carbohydrates and bound to fungi and gram-negative and gram-positive bacteria. Consistent with these properties, recombinant BiSPI exhibited microbicidal activities against bacteria and fungi through induction of structural damage by binding to the microbial surfaces. Additionally, recombinant BiSPI inhibited the plasmin-mediated degradation of human fibrin and was thus concluded to exhibit anti-fibrinolytic activity. Moreover, the peptide showed no effect on hemolysis. These findings demonstrate the dual function of BiSPI, which acts as a microbicidal peptide and anti-fibrinolytic venom toxin.

Differential Expression of Major Royal Jelly Proteins in the Hypopharyngeal Glands of the Honeybee Apis mellifera upon Bacterial Ingestion.

Honeybee vitellogenin (Vg) transports pathogen fragments from the gut to the hypopharyngeal glands and is also used by nurse bees to synthesize royal jelly (RJ), which serves as a vehicle for transferring pathogen fragments to the queen and young larvae. The proteomic profile of RJ from bacterial-challenged and control colonies was compared using mass spectrometry; however, the expression changes of major royal jelly proteins (MRJPs) in hypopharyngeal glands of the honeybee Apis mellifera in response to bacterial ingestion is not well-characterized. In this study, we investigated the expression patterns of Vg in the fat body and MRJPs 1-7 in the hypopharyngeal glands of nurse bees after feeding them live or heat-killed Paenibacillus larvae. The expression levels of MRJPs and defensin-1 in the hypopharyngeal glands were upregulated along with Vg in the fat body of nurse bees fed with live or heat-killed P. larvae over 12 h or 24 h. We observed that the expression patterns of MRJPs and defensin-1 in the hypopharyngeal glands and Vg in the fat body of nurse bees upon bacterial ingestion were differentially expressed depending on the bacterial status and the time since bacterial ingestion. In addition, the AMP genes had increased expression in young larvae fed heat-killed P. larvae. Thus, our findings indicate that bacterial ingestion upregulates the transcriptional expression of MRJPs in the hypopharyngeal glands as well as Vg in the fat body of A. mellifera nurse bees.

Upregulation of Transferrin and Major Royal Jelly Proteins in the Spermathecal Fluid of Mated Honeybee (Apis mellifera) Queens.

Sperm storage in the spermathecae of honeybee (Apis mellifera) queens is vital for reproduction of honeybees. However, the molecular mechanisms whereby queens store sperm in a viable state over prolonged periods in the spermatheca are not fully understood. Here, we conducted RNA sequencing analysis of the spermathecae in both virgin and mated A. mellifera queens 24 h after mating and observed that the genes encoding transferrin (Tf) and major royal jelly proteins (MRJPs) were differentially expressed in the spermathecae of mated queens. The concentrations of Tf and antioxidant proteins such as superoxide dismutase 1, catalase, and glutathione peroxidase as well as the levels of reactive oxygen species, H2O2, and iron were higher in the spermathecal fluid of the mated queens than in virgin queens. Tf upregulation is likely to perform a protective role against the Fenton reaction occurring between iron and H2O2 in the antioxidant pathway in the mated queen's spermathecal fluid. Furthermore, MRJPs-especially MRJP1, MRJP4, and MRJP6-were upregulated in the mated queen's spermathecal fluid, indicating that they may serve as antimicrobial and antioxidant agents as well as an energy source for stored sperm in the spermathecal fluid of honeybee queens. Together, our findings show that Tf and MRJPs are upregulated in the spermatheca and spermathecal fluid of mated honeybee queens.

Dual-Targeting Antiproliferation Hybrids Derived from 1-Deoxynojirimycin and Kaempferol Induce MCF-7 Cell Apoptosis through the Mitochondria-Mediated Pathway.

1-Deoxynojirimycin, an α-glucosidase inhibitor, possesses various biological activities such as antitumor, antidiabetic, and antiviral effects. However, the application of 1-deoxynojirimycin is restricted by its poor lipophilicity and low bioavailability. In this study, three 1-deoxynojirimycin derivatives (8-10) comprising 1-deoxynojirimycin and kaempferol were designed and synthesized to modify their pharmacokinetics and improve their antitumor efficacy. Among them, compound 10, a conjugate of 1-deoxynojirimycin and kaempferol linked through an undecane chain, exhibited excellent lipophilicity, antiproliferative effects, and α-glucosidase inhibitory activity. Compared with 1-deoxynojirimycin, kaempferol, and their combination, compound 10 downregulated cyclooxygenase-2 (COX-2) expression, arrested the cell cycle at the S phase, induced cellular apoptosis, and inhibited the migration of MCF-7 cells. Moreover, further investigation indicated that compound 10 induced MCF-7 cell apoptosis through a mitochondrial-mediated pathway via the loss of mitochondrial membrane potential. This led to increasing intracellular levels of reactive oxygen species (ROS) and Ca2+, the downregulation of Bcl-2 expression, and the upregulation of Bax levels.

Activator protein-1 mediated CYP6ER1 overexpression in the clothianidin resistance of Nilaparvata lugens (Stål).

BACKGROUND: Nilaparvata lugens, a destructive rice pest in Asia, has developed resistance to many insecticides, including the neonicotinoid clothianidin. CYP6ER1 plays an important role in N. lugens resistant to clothianidin, but only limited information on the transcriptional regulation of CYP6ER1 overexpression in clothianidin resistance is available. RESULTS: In this study, the transcription factor activator protein 1 (AP-1) was found to be overexpressed in a clothianidin-resistant strain of N. lugens and several field resistant populations. RNA interference-mediated silencing of NlAP-1 significantly decreased CYP6ER1 expression and increased the susceptibility of N. lugens to clothianidin. Additionally, NlAP-1 was highly expressed in egg and adult stages, and in midguts, and NlAP-1 was upregulated and induced to a greater extent in the clothianidin-resistant strain after exposure to clothianidin. Finally, dual-luciferase reporter assays confirmed the interaction between NlAP-1 and the two predicted binding sites in the CYP6ER1 promoter. CONCLUSION: NlAP-1 bound the -1388 to -1208-bp region of the CYP6ER1 promoter, enhancing its activity and then regulate the expression of CYP6ER1. These findings enhance our knowledge of the transcriptional regulation of the P450 genes that mediate insecticide resistance in insect pests. © 2021 Society of Chemical Industry.

Effect of the silkworm pupa protein-glucose conjugate on the thermal stability and antioxidant activity of anthocyanins.

Anthocyanin (cyanidin-3-O-glucose) is a natural water-soluble pigment with a robust antioxidant capacity. However, its poor stability and bioavailability limits its application as a functional food ingredient. This study explored the ability of the silkworm pupa protein-glucose (Spp-Glu) conjugate, developed under wet-heating conditions, to improve the thermal stability and antioxidant activity of cyanidin-3-O-glucose (C3G) at pH 3.0 and 6.8. The characterization experiments suggested that C3G complexed with the Spp-Glu conjugate could modify the protein's microenvironment and cause unfolding of the protein's secondary structures under varied pH conditions. Spectroscopic techniques further revealed the formation of complexes via hydrophobic interactions and static quenching processes when C3G was bound to Spp or Spp-Glu. The formation of these complexes effectively attenuated C3G degradation, thereby enhancing its stability under heat treatment over a range of pH values, and the experiments measuring antioxidant activity suggested that the Spp-Glu conjugate formed does not affect the efficacy of C3G after complexation. Therefore, our study suggests that Spp-Glu has the potential to effectively protect and deliver anthocyanins during industrial application for functional food formulation.

Lipolytic Activity of a Carboxylesterase from Bumblebee (Bombus ignitus) Venom.

Bee venom is a complex mixture composed of peptides, proteins with enzymatic properties, and low-molecular-weight compounds. Although the carboxylesterase in bee venom has been identified as an allergen, the enzyme's role as a venom component has not been previously elucidated. Here, we show the lipolytic activity of a bumblebee (Bombus ignitus) venom carboxylesterase (BivCaE). The presence of BivCaE in the venom secreted by B. ignitus worker bees was confirmed using an anti-BivCaE antibody raised against a recombinant BivCaE protein produced in baculovirus-infected insect cells. The enzymatic activity of the recombinant BivCaE protein was optimal at 40 °C and pH 8.5. Recombinant BivCaE protein degrades triglycerides and exhibits high lipolytic activity toward long-chain triglycerides, defining the role of BivCaE as a lipolytic agent. Bee venom phospholipase A2 binds to mammalian cells and induces apoptosis, whereas BivCaE does not affect mammalian cells. Collectively, our data demonstrate that BivCaE functions as a lipolytic agent in bee venom, suggesting that BivCaE will be involved in distributing the venom via degradation of blood triglycerides.

Anti-fibrinolytic activity of a metalloprotease inhibitor from bumblebee (Bombus ignitus) venom.

Bee venom is a mixture of bioactive components that include proteases and protease inhibitors. A metalloprotease inhibitor has been predicted to be a bumblebee-specific toxin in the venom proteome of Bombus terrestris; however, the identification and functional roles of bee venom metalloprotease inhibitors have not been previously determined. In this study, we identified a bumblebee (B. ignitus) venom metalloprotease inhibitor (BiVMPI) that exhibits anti-fibrinolytic activity. BiVMPI contains a trypsin inhibitor-like cysteine-rich domain that exhibits similarity to inducible metalloprotease inhibitor. Using an anti-BiVMPI antibody raised against a recombinant BiVMPI protein produced in baculovirus-infected insect cells, the presence of BiVMPI in the venom gland and secreted venom of B. ignitus worker bees was confirmed. The recombinant BiVMPI protein demonstrated inhibitory activity against a metalloprotease, trypsin, chymotrypsin, protease K, and plasmin, but not subtilisin A, elastase, or thrombin. Additionally, the recombinant BiVMPI bound to plasmin and inhibited the plasmin-mediated degradation of fibrin, demonstrating an anti-fibrinolytic role for BiVMPI as a bee venom metalloprotease inhibitor. Our results provide the first evidence for the identification and anti-fibrinolytic activity of a metalloprotease inhibitor from bee venom.

Carboxylesterase genes in nitenpyram-resistant brown planthoppers, Nilaparvata lugens.

Carboxylesterases (CarEs) represent one of the major detoxification enzyme families involved in insecticide resistance. However, the function of specific CarE genes in insecticide resistance is still unclear in the insect Nilaparvata lugens (Stål), a notorious rice crop pest in Asia. In this study, a total of 29 putative CarE genes in N. lugens were identified, and they were divided into seven clades; further, the ß-esterase clade was significantly expanded. Tissue-specific expression analysis found that 17 CarE genes were abundantly distributed in the midgut and fat body, while 12 CarE genes were highly expressed in the head. The expression of most CarE genes was significantly induced in response to the challenge of nitenpyram, triflumezopyrim, chlorpyrifos, isoprocarb and etofenprox. Among these, the expression levels of NlCarE2, NlCarE4, NlCarE9, NlCarE17 and NlCarE24 were increased by each insecticide. Real-time quantitative polymerase chain reaction and RNA interference assays revealed the NlCarE1 gene to be a candidate gene mainly involved in nitenpyram resistance, while simultaneously silencing NlCarE1 and NlCarE19 produced a stronger effect than silencing either one individually, suggesting a cooperative relationship in resistance formation. These findings lay the foundation for further clarification of insecticide resistance mediated by CarE in N. lugens.

Chemical synthesis, inhibitory activity and molecular mechanism of 1-deoxynojirimycin-chrysin as a potent α-glucosidase inhibitor.

Hyperglycemia can be efficaciously regulated by inhibiting α-glucosidase activity and this is regarded as an effective strategy to treat type 2 diabetes. 1-Deoxynojimycin, an α-glucosidase inhibitor, can penetrate cells rapidly to potently inhibit α-glucosidase in a competitive manner. However, the application of 1-deoxynojimycin is limited by its poor lipophilicity and low bioavailability. Herein, three 1-deoxynojimycin derivatives 4-6 were designed and synthesized by linking 1-deoxynojimycin and chrysin to ameliorate the limitations of 1-deoxynojimycin. Among them, compound 6, a conjugate of 1-deoxynojimycin and chrysin linked by an undecane chain, could better bind to the α-glucosidase catalytic site, thereby exhibiting excellent α-glucosidase inhibitory activity (IC50 = 0.51 ± 0.02 µM). Kinetics analyses revealed that compound 6 inhibited the activity of α-glucosidase in a reversible and mixed competitive manner. Fluorescence quenching and UV-Vis spectra showed that compound 6 changed the conformation of the α-glucosidase via complex formation, which triggered a static fluorescence quenching of the enzyme protein.

Spider Silk Fibroin Protein Heterologously Produced in Rice Seeds Reduce Diabetes and Hypercholesterolemia in Mice.

Silk fibroin proteins are biomaterials with diverse applications. These spider and silkworm proteins have specific biological effects when consumed by mammals; in addition to reducing blood pressure and blood glucose and cholesterol levels, they have anti-human immunodeficiency virus activity. In the present study, rice (Oryza sativa) was engineered to produce the C-terminus of the major ampullate spidroin protein from the spider Araneus ventricosus under the control of a Prolamin promoter. Homozygous transgenic rice lines were identified, and the therapeutic effect of this spider silk fibroin protein on the lipid and glucose metabolism was analyzed in a mouse model. Feeding fat-fed mice, the transgenic rice seeds for four weeks reduced serum concentrations of triglycerides, total cholesterol, low-density lipoprotein cholesterol, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase, and lowered blood glucose levels. This is the first study to investigate the effects of consumption of rice seeds heterologously expressing spider silk fibroin protein in a mammalian model. Our findings suggest that functional foods containing spider silk fibroin protein might be useful as potential pharmaceutical materials for preventing and treating diabetes, hyperlipidemia, and hypercholesterolemia.

Honeybee (Apis cerana) vitellogenin acts as an antimicrobial and antioxidant agent in the body and venom.

Honeybee (Apis mellifera) egg-yolk protein vitellogenin (Vg) plays roles in immunity, antioxidation, and life span beyond reproduction, but it also acts as an allergen Api m 12 in venom. Here we established antimicrobial and antioxidant roles of honeybee Vg in the body and venom. Using the cDNA encoding Vg identified from Asiatic honeybee (A. cerana) workers, recombinant A. cerana Vg (AcVg) protein of approximately 180 kDa was produced in baculovirus-infected insect cells. In A. cerana worker bees, AcVg was expressed in the fat body and venom gland and was present in the secreted venom. AcVg induced structural damage in microbial cell walls via binding to microbial surfaces and exhibited antimicrobial activity against bacteria and fungi. AcVg protected mammalian and insect cells against oxidative damage through direct shielding of cell membranes. Interestingly, AcVg exhibited DNA protection activity against reactive oxygen species (ROS). Furthermore, the transcript level of AcVg was upregulated in the fat body, but not in the venom gland, of worker bees with antimicrobial peptides and antioxidant enzymes in response to microbial infection and oxidative stress. Our data indicate that AcVg is involved in innate immunity upon infection and in a defense system against ROS, supporting a crucial role of honeybee Vg as an antimicrobial and antioxidant agent in the body and venom.

Dual role of the serine protease homolog BmSPH-1 in the development and immunity of the silkworm Bombyx mori.

Serine proteases and serine protease homologs are involved in the prophenoloxidase (proPO)-activating system leading to melanization. The Bombyx mori serine protease homolog BmSPH-1 regulates nodule melanization. Here, we show the dual role of BmSPH-1 in the development and immunity of B. mori. BmSPH-1 was expressed in hemocytes after molting and during the larval-pupal transformation in normal development. In contrast, following infection, BmSPH-1 was expressed in hemocytes and cleaved in the hemolymph, which resulted in the induction of PO activity. Moreover, BmSPH-1 was cleaved in the cuticle during the larval-pupal transformation and early pupal stages. In BmSPH-1 RNAi-treated silkworms, the reduced BmSPH-1 mRNA levels during the spinning stage or the prepupal stage resulted in the arrest of pupation or pupal cuticular melanization, respectively. The binding assays revealed that BmSPH-1 interacts with B. mori immulectin, proPO, and proPO-activating enzyme. Our findings demonstrate that BmSPH-1 paticipates larval-pupal transformation, pupal cuticular melanization and innate immunity of silkworms, illustrating the dual role of BmSPH-1 in development and immunity.