Search

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.

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 -1,388 to -1,208-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. This article is protected by copyright. All rights reserved.

Silkworm (Bombyx mori) pupa protein (SPP) is a high-quality source of animal protein with substantial nutritional benefits and health value. To develop an efficient extraction method for SPP that is environmentally friendly, we selected choline hydroxide ionic liquid (CH-IL) as the extraction solvent and performed orthogonal experiments to optimize the extraction conditions. We demonstrated that 3% CH-IL, a solid-to-liquid ratio (g/mL) of 1:30, an extraction temperature of 40 °C, and an extraction time of 1 h facilitated the most efficient extraction. Compared to the conventional alkali solubilization–acid precipitation method, the CH-IL extraction increased protein content by 12.14%. Protein structure analysis showed that the β-sheet content increased by 10.98% and that of disulfide bonds reduced by 16.4%. The processing properties of the CH-IL extracted protein showed that the solubility, emulsification, and foaming capacity were enhanced by 82.87%, 15.44%, and 18.97%, respectively. The physical properties of SPP remarkably improved relative to the increased stretching of the polypeptide chains. The findings of this study provide technical knowledge that will enhance the processing performance of pupal proteins.

Apidermins (APDs) are known as structural cuticular proteins in insects, but their additional roles are poorly understood. In this study, we characterized the honeybee

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.

Ascocarps of some species of the genus Cordyceps have long been used as a traditional medicine and food source for promoting human health. The compound cordycepin, isolated from C. militaris ascocarps (CE), show similar health effects to CE. In this study, we investigated and compared the anti-obesity and antidiabetic effects of dietary CE and Paecilomyces tenuipes ascocarps (PE) in mice. In addition, we investigated their effects on the expression of genes related to the regulation of obesity and diabetes. We found that dietary CE and PE suppressed body weight gain and fat accumulation in the liver and adipocyte tissues of mice fed a high-fat diet (HFD). Enzyme and lipid profiles induced by HFD returned to normal with CE or PE treatment. Dietary CE or PE reduced fasting blood glucose and serum insulin levels in HFD-fed mice. Finally, we show that CE and PE treatment restored to normal the hyperlipidemia- and hyperglycemia-related gene expressions in HFD-fed mice. These results indicate that dietary CE or PE exert their anti-obesity and antidiabetic effects by regulating adipogenesis and insulin signaling pathways. Finally, we show that dietary CE or PE have similar anti-obesity and antidiabetic effects even when included in a normal mouse diet. Although cordycepin is not found in PE, PE treatment improves lipid and glucose metabolic parameters in a manner similar to CE. We find that PE provides alternative potential therapeutic treatments for obesity and diabetes.

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 β-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.

Major royal jelly proteins (MRJPs) of honeybee royal jelly (RJ) exhibit antimicrobial and antioxidant activities. Although MRJPs of Apis mellifera RJ (AmMRJPs) responsible for antibacterial activity have been identified, AmMRJPs with antioxidant effects remain to be elucidated. Here we identified and compared the antioxidant activities of purified recombinant AmMRJPs 1–7, which are expressed in baculovirus-infected insect cells. Antioxidant assays of recombinant AmMRJPs 1–7 against H2O2 revealed that AmMRJPs reduce caspase-3 activity and oxidative stress-induced cell apoptosis and lead to increased cell viability. Consistent with these results, AmMRJPs 1–7 exhibit 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity and protect against oxidative DNA damage. These results indicate that AmMRJPs play a role as antioxidants in A. mellifera RJ.

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

Royal jelly (RJ) is a well-known functional and medicinal food for human health promotion. Major royal jelly proteins (MRJPs), which are the major protein components in RJ, exhibit antimicrobial activities. However, the identities of the MRJPs of RJ responsible for its antioxidant effects have remained unclear. Here, we report that honeybee (Apis cerana) MRJP 2 (AcMRJP2) acts as an antimicrobial and antioxidant agent in RJ. Using recombinant AcMRJP2, which was produced in baculovirus-infected insect cells, we established the antimicrobial and antioxidant roles of MRJP 2. AcMRJP2 bound to the surfaces of bacteria, fungi, and yeast, which then induced structural damage in the microbial cell walls and led to a broad spectrum of antimicrobial activities. AcMRJP2 protected mammalian and insect cells via the direct shielding of the cell against oxidative stress, which led to reduced levels of caspase-3 activity and oxidative stress-induced cell apoptosis, followed by increased cell viability. Moreover, AcMRJP2 exhibited DNA protection activity against reactive oxygen species (ROS). Our data indicate that AcMRJP2 could play a crucial role as an antimicrobial agent and antioxidant in RJ, suggesting that MRJP 2 is a component responsible for the antimicrobial and antioxidant activities of RJ.

Major royal jelly proteins (MRJPs) are the protein components in royal jelly (RJ). MRJPs 1–7 are detected in the honeybee Apis mellifera RJ. Although A. mellifera MRJP (AmMRJP) 2 exhibited antibacterial activity, the other MRJPs with antimicrobial activities in A. mellifera RJ remains largely unknown. Here, we compared the antibacterial activity of recombinant AmMRJPs 1–7 expressed in baculovirus-infected insect cells. Antibacterial assays of recombinant AmMRJPs 1–7 against the gram-negative bacterium Escherichia coli revealed that AmMRJPs 2–5 and 7 exhibited antibacterial activity, whereas AmMRJPs 1 and 6 displayed almost no antibacterial activity. Consistent with the antibacterial activity of AmMRJPs, AmMRJPs 2–5 and 7 are bound to bacterial cell walls. These results indicated that AmMRJPs 2–5 and 7 contribute directly to the antibacterial property of RJ, suggesting that MRJPs play a role in the antimicrobial property of RJ.

The protein component of honeybee royal jelly (RJ) is constituted by major royal jelly proteins (MRJPs). The Asiatic honeybee (Apis cerana) MRJP-4 (AcMRJP4) exhibits antimicrobial activities. In this study, we identified the antimicrobial activity of AcMRJP4-15, which is a hydrophilic peptide with 88 amino acid residues in the C-terminal of AcMRJP4 that contains a high content of Asn and positively charged amino acids. Recombinant AcMRJP4-15, which is expressed as a 15-kDa peptide in baculovirus-infected insect cells, induced structural damage to the cell walls of bacteria, fungi, and yeast. Interestingly, the antimicrobial activity of AcMRJP4-15 was greater than that of AcMRJP4, demonstrating that the antimicrobial activity of AcMRJP4 was due in large part to the C-terminal. Our data suggest that AcMRJP4-15 can function as an effective antimicrobial agent.

In this study, we isolated and characterized an immunomodulatory peptide from silkworm (Bombyx mori) pupa protein hydrolysates. Ultrasound-pretreated hydrolysates were prepared by alcalase digestion and their pro-proliferative activity was assessed with the splenic lymphocyte proliferation assay. Peptide fractions exhibiting the highest activity were purified by Sephadex-G100 and -G15 gel filtration chromatography. The structure of the purified peptides was analyzed using Liquid Chromatography Electrospray Ionisation Tandem Mass Spectrometry (LC–ESI–MS/MS); their digestive stability and their effects of the expression on immune-related cytokines were also evaluated. We purified a novel immunomodulatory peptide with a molecular weight of about 441.06 Da. The amino acid sequence was Asp-His-Ala-Val (DHAV). The splenocyte proliferation rate was 91.1% (P

Centipede venom contains a variety of proteins, peptides, and enzymes. However, the biological actions of toxin peptides in centipede venom remain largely unknown. In this study, we identified a centipede (Scolopendra subspinipes mutilans) venom toxin peptide (SsmTP) that was shown to act as a cell growth factor at low concentrations-in vitro. SsmTP was found to consist of 66 amino acids that display seven cysteine residues, which exhibited high similarity to the predicted neurotoxin. SsmTP was expressed in the venom gland of S. s. mutilans. A recombinant SsmTP peptide of approximately 5.2 kDa was produced in baculovirus-infected insect cells. Interestingly, SsmTP exhibited cytotoxicity in murine cells in a concentration-dependent manner but displayed a cell growth effect at low concentrations. SsmTP at a low concentration also protected murine cells against oxidative damage through the inhibition of caspase-1 and apoptosis. Our data indicate that SsmTP acts as a cell growth factor and a toxin peptide in a concentration-dependent manner. Consequently, our results provide evidence that the identification of SsmTP, a centipede toxin peptide, may not only elucidate the biological action of toxin peptides but also offer additional insight into the pharmacological applications of centipede venoms.

Major royal jelly proteins (MRJPs) are important protein components of bee royal jelly (RJ) and exhibit various biological and pharmacological activities. The antimicrobial activities of the royalisin and the jelleines contained within MRJP 1 and MRJP 2 in RJ have been elucidated. However, the antimicrobial effects of other MRJPs remain largely unknown. In this study, we demonstrated the antimicrobial activity of the Asiatic honeybee (Apis cerana) MRJP 4 (AcMRJP4). Recombinant AcMRJP4 was expressed as a 63-kDa protein in baculovirus-infected insect cells. We examined the antimicrobial activity of recombinant AcMRJP4 against bacteria, fungi, and yeast. The mechanisms underlying the antimicrobial activity of AcMRJP4 were assessed using western blot analysis, immunofluorescence staining, and scanning electron microscopy. Recombinant AcMRJP4 bound to the cell walls of bacteria, fungi, and yeast and induced structural damage in the microbial cell walls. AcMRJP4 has an antimicrobial role and exhibits a broad spectrum of antimicrobial activities against bacteria, fungi, and yeast. We demonstrated that AcMRJP4 functions as an antimicrobial agent with activity against bacteria, fungi, and yeast. Together, our data identified a novel function of MRJP 4 as an antimicrobial agent.

Background Uridine diphosphate-glycosyltransferases (UGTs) are phase II metabolic enzymes involved in metabolism of toxins and resistance to insecticides in insect pests. Reactive oxygen species (ROS) induced by xenobiotics are important for activation of detoxification pathways. However, relationships between ROS and UGTs involved in toxin metabolism and insecticide resistance remain unclear. Results Here, involvement of dual oxidase (Duox)-dependent ROS in regulating UGT expression-mediated insecticide resistance in the brown planthopper Nilaparvata lugens was investigated. The overexpression of NlUGT386F2 contributed to N. lugens' resistance to clothianidin. Furthermore, the ROS inhibitor (N-acetylcysteine) significantly reduced the expression of NlUGT386F2 and increased the susceptibility of N. lugens to clothianidin. Silencing the ROS producer Duox significantly increased the susceptibility of N. lugens to clothianidin through the downregulation of NlUGT386F2 expression. Conclusion NlDuox-dependent ROS regulates NlUGT386F2 expression-mediated clothianidin resistance in brown planthopper. These observations further our understanding of the metabolism of toxins and of insecticide-resistance in insect pests. This article is protected by copyright. All rights reserved.

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.

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.

Insect immulectins are involved in various aspects of the innate immunity, including encapsulation, melanization, and phagocytosis. Although the silkworm Bombyx mori immulectin (BmIML) has been reported previously, the ligand and functions of BmIML have not been investigated. Here, we show the dual roles of BmIML in cuticular melanization and immunity of B. mori. BmIML recognizes carbohydrates in a Ca2 +-dependent manner and binds to Gram-negative and Gram-positive bacteria, fungi, and yeast. BmIML was expressed in the fat body during infections and localized to the hemocytes of silkworms. Additionally, BmIML was expressed in the epidermis during the prepupal stage and localized to the cuticle of silkworms. After treatment with E. coli, dopa, dopamine, or tyrosine injections, BmIML production was induced in the fat body but not in the epidermis of silkworms. Treatment with BmIML RNAi resulted in the arrest of pupal cuticular melanization. Therefore, we concluded that BmIML is involved in pupal cuticular melanization and innate immunity responses of silkworms, suggesting dual roles for BmIML.

Our previous study demonstrated that the secapin peptide from the venom of the Asiatic honeybee ( Apis cerana , AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. In the present study, we investigated the anti-microbial activity and cytotoxicity of a synthetic AcSecapin-1 peptide. Seven synthetic AcSecapin-1 peptides (AcSecapin-S1 to AcSecapin-S7) were synthesized based on the peptide sequence of AcSecapin-1. AcSecapin-S1, which consists of the 25-amino acid sequence identical to that of the mature AcSecapin-1 peptide, exhibited the highest anti-microbial activity against bacteria and fungi. This was followed by AcSecapin-S6, which was missing 10 N-terminal amino acids and 6 C-terminal amino acids from AcSecapin-S1. Furthermore, AcSecapin-S1 was not cytotoxic and did not activate macrophages. Taken together, our data demonstrated that a synthetic AcSecapin-1 peptide could exhibit anti-microbial activity without producing a cytotoxic or inflammatory response, suggesting that this synthetic AcSecapin-1 peptide can be used as an anti-microbial agent for biomedical applications.

Simple Summary To understand the mechanisms underlying long-term storage and survival of sperm in honeybee Apis mellifera queens, previous studies have elucidated the components of honeybee spermathecal fluid. However, the expression profiles of transferrin (Tf) and major royal jelly proteins 1–9 (MRJPs 1–9) in the spermatheca and spermathecal fluid of mated honeybee queens have still not been characterized. In this study, we confirmed upregulation of Tf and MRJPs in the spermatheca and spermathecal fluid of mated honeybee queens by using RNA sequencing, reverse transcription-polymerase chain reaction, and Western blot analyses. The levels of Tf and antioxidant enzymes were elevated in the spermathecal fluid of the mated queens, paralleling the levels of reactive oxygen species, H2O2, and iron. The increased levels of MRJPs, especially MRJP1, MRJP4, and MRJP6, in the spermathecal fluid of mated queens may be responsible for energy provision during sperm storage in honeybee queens. Overall, our findings indicate that Tf and MRJPs are upregulated in the spermatheca and spermathecal fluid of mated honeybee queens, providing a novel insight into antioxidant defense and energy metabolism for stored sperm in honeybee queens. Abstract 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.

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.

Chitin, a structural component of the insect peritrophic membrane (PM), is associated with chitin-binding proteins (CBPs). Insect CBPs have been studied extensively regarding their interactions with chitin in the structure and functions of the cuticle and PM. However, the functions of bee CBPs are poorly understood. In this study, we present the molecular cloning and functional characterization of a CBP from the Asiatic honeybee Apis cerana. A. cerana CBP (AcCBP) contains the peritrophin-A domain with a six-cysteine motif. AcCBP is exclusively expressed in the midgut and is localized in the PM of A. cerana worker bees. Recombinant AcCBP was expressed in baculovirus-infected insect cells, and it exhibited chitin-binding ability, thus indicating a role for AcCBP as a CBP in the PM of A. cerana worker bees. Furthermore, AcCBP bound to entomopathogenic fungal surfaces but did not show anti-fungal activity. Taken together, our data demonstrate that AcCBP functions as a CBP of the bee PM.

Silk fibroin proteins serve as biomaterials for diverse applications. Previous studies have shown that silk fibroin proteins can be used in biomedical applications for treating diabetes. In the present study, we investigated the cytotoxicity and inflammatory response induced by spider silk fibroin protein in vitro. By using recombinant AvMaSp-R spider silk fibroin protein, which consists of the 240 amino acid repetitive domain of the spider (Araneus ventricosus) silk fibroin protein and was expressed in baculovirus-infected insect cells, we tested the cytotoxicity, apoptosis, macrophage stimulation, and release of proinflammatory mediators and cytokines in vitro. We found that recombinant AvMaSp-R was not cytotoxic and did not activate macrophages. Consequently, our results provide evidence that recombinant AvMaSp-R produces a non-cytotoxic and non-inflammatory response, suggesting that recombinant AvMaSp-R silk fibroin protein could be a biomaterial for biomedical applications.

Bee venom contains a variety of peptide constituents that have various biological, toxicological, and pharmacological actions. However, the biological actions of secapin, a venom peptide in bee venom, remain largely unknown. Here, we provide the evidence that Asiatic honeybee (Apis cerana) secapin (AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. The recombinant mature AcSecapin-1 peptide was expressed in baculovirus-infected insect cells. AcSecapin-1 functions as a serine protease inhibitor-like peptide that has inhibitory effects against plasmin, elastases, microbial serine proteases, trypsin, and chymotrypsin. Consistent with these functions, AcSecapin-1 inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products, thus indicating the role of AcSecapin-1 as an anti-fibrinolytic agent. AcSecapin-1 also inhibited both human neutrophil and porcine pancreatic elastases. Furthermore, AcSecapin-1 bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi and gram-positive and gram-negative bacteria. Taken together, our data demonstrated that the bee venom peptide secapin has multifunctional roles as an anti-fibrinolytic agent during fibrinolysis and an anti-microbial agent in the innate immune response.

Bee venom contains a variety of toxic components, including enzymes, peptides, and biogenic amines. An acid phosphatase Acph-1-like protein has been identified from Asiatic honeybee (Apis cerana) venom. However, no molecular information is currently available for acid phosphatases from A. cerana venom. In this study, an A. cerana venom acid phosphatase (AcVAP) was identified. The amino acid sequence analysis of the predicted AcVAP protein revealed high identity with other bee venom acid phosphatases. An anti-AcVAP antibody was produced against a recombinant AcVAP (46-kDa) expressed in in baculovirus-infected insect cells. Northern and Western blot analyses showed that AcVAP was expressed in the venom gland and was present as a 46-kDa protein in the secreted bee venom. The enzymatic properties of recombinant AcVAP were determined using p-nitrophenyl phosphate (p-NPP) as a substrate and exhibited the highest activity at pH 4.8 and 45 °C. Taken together, our data demonstrated that AcVAP functions as a bee venom acid phosphatase.

In the present study, an epsilon glutathione S -transferase from the beet armyworm Spodoptera exigua (SeGSTe) was cloned and characterized. The SeGSTe gene consists of 746 bp full-length cDNA with a 669 bp open reading frame encoding a predicted protein of 223 amino acid residues weighing 24.517 kDa and an isoelectric point of 6.44. Furthermore, sequence analysis of SeGSTe showed 90% sequence identity to Spodoptera litura GSTe2 and 47–36% to other insect GSTs. Phylogenetic relationship analysis further revealed that SeGSTe is a novel member of the Epsilon-class of GSTs. During S. exigua development, SeGSTe is expressed in the midgut of the prepupa stage but not in the epidermis and fat body. Recombinant SeGSTe (25 kDa) purified from baculovirus-infected insect cells was able to protect super-coiled DNA from oxidative damage. It was also able to catalyze the conjugation of both 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB), but not 4-nitrobenzyl chloride (4-NBC) and 4-nitrophenethyl bromide (4-NPB), to glutathione (GSH). Additionally, recombinant SeGSTe exhibited an optimal pH of 6.0 and an optimal temperature of 40 °C. The enzyme activity of recombinant SeGSTe was sensitive to heavy metals Cu 2 + and Cd 2 + and was significantly inhibited by various kinds of pesticides including insecticide, herbicide, and fungicide. Taken together, these findings suggest that the SeGSTe may be involved in the detoxification of pesticides and protecting S. exigua against oxidative stress, which provides a molecular basis for understanding the mechanisms of insecticide resistance in S. exigua .

Osmia cornifrons is a cavity-nesting solitary species used as an apple pollinator in Korea. To elucidate the developmental characteristics of O. cornifrons , we investigated its development from the egg to adulthood, including a dormant prepupal phase and mating through indoor rearing (25 °C, 65% R.H.). The egg durations of the female and male bees were 3.6 ± 0.8 days and 3.1 ± 1.3 days, respectively. During larval development, the head widths of the 1st to 5th instars ranged from 0.7 ± 0.1 mm to 1.3 ± 0.1 mm. The peak of the growth in head width was the 2nd instar. The larval lengths ranged from 3.7 ± 0.6 mm to 13.6 ± 1.3 mm. The peak of growth was the 4th instar. The larval weights ranged from 4.5 ± 1.2 mg to 78.3 ± 16.1 mg. The peak of growth was the 3rd instars. The total larval durations of from the 1st to 5th instars for the females and males were 14.0 ± 6.0 days and 13.2 ± 5.8 days, respectively. The spinning durations of the females and males were 2.2 ± 0.7 days and 2.3 ± 0.8 days, the prepupation durations were 55.5 ± 5.9 days and 55.8 ± 2.9 days, and the pupation durations were 26.4 ± 2.1 days and 25.3 ± 2.3 days, respectively. The average longevity of the female adults and male adults was 21.8 ± 8.7 days and 24.4 ± 12.4 days, respectively. The total duration of from the egg to an adult bee of the O. cornifrons females and males was 123.5 days and 124.1 days, respectively. Mating consisted of the three following phases: the precopulatory (courtship and attempting copulation), copulation and postcopulatory phases. The mating times of the precopulatory, copulation and postcopulatory phases were 159.6 ± 288.9, 8.4 ± 7.1, 12.9 ± 4.5, and 198.8 ± 69.8 s.

Members of the glutathione S-transferase superfamily can protect organisms against oxidative stress. In this study, we characterized an omega glutathione S-transferase from Spodoptera exigua (SeGSTo). The SeGSTo gene contains an open reading frame (ORF) of 744 nucleotides encoding a 248-amino acid polypeptide. The predicted molecular mass and isoelectric point of SeGSTo are 29007 Da and 7.74, respectively. Multiple amino acid sequence alignment analysis shows that the SeGSTo sequence is closely related to the class 4 GSTo of Bombyx mori BmGSTo4 (77 % protein sequence similarity). Homologous modeling and molecular docking reveal that Cys35 may play an essential role in the catalytic process. Additionally, the phylogenetic tree indicates that SeGSTo belongs to the omega group of the GST superfamily. During S. exigua development, SeGSTo is expressed in the midgut of the fifth instar larval stage, but not in the epidermis or fat body. Identification of recombinant SeGSTo via SDS-PAGE and Western blot shows that its molecular mass is 30 kDa. The recombinant SeGSTo was able to protect super-coiled DNA from damage in a metal-catalyzed oxidation (MCO) system and catalyze the 1-chloro-2,4-dinitrobenzene (CDNB), but not 1,2-dichloro-4-nitrobenzene (DCNB), 4-nitrophenethyl bromide (4-NPB), or 4-nitrobenzyl chloride (4-NBC). The optimal reaction pH and temperature were 8 and 50 °C, respectively, in the catalysis of CDNB by recombinant SeGSTo. The mRNA expression of SeGSTo was up-regulated by various oxidative stresses, such as CdCl2, CuSO4, and isoprocarb, and the catalytic activity of recombinant SeGSTo was noticeably inhibited by heavy metals (Cu(2+) and Cd(2+)) and various pesticides. Taken together, these results indicate that SeGSTo plays an important role in the antioxidation and detoxification of pesticides.

Insect cuticular melanization is a defense mechanism involving the prophenoloxidase (proPO)-activating system. Here, we showed the differential regulation of tyrosine hydroxylase in silkworm (Bombyx mori) pupae. B. mori tyrosine hydroxylase (BmTH) was expressed in the epidermis during development or in the fat body during infection, which indicates that the differential expression of BmTH is responsible for cuticular melanization or innate immunity. The melanin of the silkworm pupal cuticle formed a melanization complex with Beauveria bassiana and this effect showed an antifungal activity, indicating that melanin in silkworm pupal cuticle plays a role in the defense against fungal pathogens. Finally, we found that the differential expression of BmTH is involved in pupal cuticular melanization and adult pigmentation as well as the innate immunity of silkworms, demonstrating a differential regulation of BmTH in a tissue-specific manner.

Microsatellites, a special class of repetitive DNA sequences, have become one of the most popular markers of genetic polymorphism, especially as next-generation sequencing (NGS) methods have become available to streamline the identification of such markers. Based on 45,476 contigs generated by NGS, we obtained 205 Mbp of nucleotide sequences from six inbred lines of Apis mellifera maintained at the National Academy of Agricultural Science in Korea. In total, 20,580 repeat motifs were identified. Di-nucleotide repeats, comprised mostly of [AT]n motifs, were two-fold more common than tri-nucleotide repeats. We arbitrarily selected 50 microsatellite loci with high repeat numbers and were covered by overlapping contigs. PCR amplification yielded clean fragments from 34 of these loci, and nine were selected to differentiate the inbred lines. Genotyping at these nine loci revealed 4–14 alleles per locus, with average 10.11, as well as 0.1875–0.9592 per-locus observed heterozygosity, and 0.5432–0.8669 per-locus polymorphic information content. Thus, some of these loci are profoundly variable. Phylogenetic analysis using the nine microsatellite loci separated individual honey bees from different inbred lines very well, suggesting that these markers may be used to trace the six inbred lines of A. mellifera maintained in Korea.

Nest traps are one of the most common methods to study Osmia bees. To elucidate the optimal environmental conditions of nesting sites, we investigated the effects of location, direction, altitude, and sites of nest traps on the rate of trap-nesting Osmia spp. During the collection period, the average rate of trap-nesting Osmia spp. collected within 90 days after the installation of traps was 17.0 ± 20.0%. This percentage was 2.7-fold higher than that of trap-nesting bees in 30 days after the installation of traps. The Jeongseon location exhibited the highest rates of trap-nesting Osmia spp. collected in 30 and 90 days, representing 11.1 ± 17.6% and 23.2 ± 22.5%, respectively. The direction of the nest traps did not affect the rate of trap-nesting Osmia spp. The altitude ranges of the traps were 0–199 m, 200–399 m, 400–599 m, 600–799 m and over 800 m. Interestingly, the altitude range of 600–799 m showed the highest rate of trap-nesting bees, which was 40.4 ± 3.9%. Higher altitudes seemed to correspond to higher rates of trap-nesting bees. With regards to the sites where the nests were placed, the rate of trap-nesting bees in a mud wall of an old house was 45.1 ± 25.2%, which was 3-fold higher than that of a nest in an apple orchard. The flowering plants collected at different locations during the trap-nesting activity of Osmia bees belonged to 18 families and 34 species. In conclusion, the rates of trap nests colonized by Osmia spp. were affected by altitude, site, and plant diversity.

Insect cuticular melanization is regulated by the prophenoloxidase (proPO)-activating system, which is also involved in the innate immune reaction. Here, we demonstrate how the differentiation of the proPO-activating system is regulated toward a cuticular melanization or innate immunity function in silkworm ( Bombyx mori ) pupae. Our results indicate that the differential and spatial regulation of key components, such as the proPO-activating enzyme and proPOs, primes the proPO-activating system for either cuticular melanization or innate immunity. This dual strategy for cuticular melanization in development and innate immunity upon infection demonstrates a two-pronged defense mechanism that is mediated by the priming of the proPO system.

Serine protease inhibitors play a critical role in physiological processes and immune responses by regulating serine protease activities. Here we describe the molecular cloning and antimicrobial activities of a serine protease inhibitor from the hornfaced bee, Osmia cornifrons (OcSPI). OcSPI consists of 405 amino acid residues and contains a potential reactive center loop (RCL) region in its C-terminus. Recombinant OcSPI was produced as a 64-kDa glycoprotein in baculovirus-infected insect cells and exhibited inhibitory activity against chymotrypsin. Additionally, OcSPI demonstrated inhibitory activity against microbial serine proteases, such as subtilisin A and proteinase K, but not against tissue plasminogen activator, thrombin, or plasmin. Recombinant OcSPI bound directly to Escherichia coli, Bacillus subtilis, and Beauveria bassiana and exhibited antimicrobial activity against both bacteria and fungi. Our results demonstrated the antimicrobial functions of OcSPI and suggest a role for OcSPI in the immune response of O. cornifrons bees.

The Osmia cornifrons bee plays an important role in pollinating fruit trees, such as apple trees. To better understand diapause and oviposition in O. cornifrons , we investigated the correlation between ovarian development and the secretion level of OcVg protein in hemolymph. During ovarian development in wintering, the number of oocytes progressively increased compared with the length of the ovaries and the oocytes. After wintering, the oocyte and ovary sizes developed up to 6 days after emergence and declined after 6 days, but the number of oocytes decreased gradually. The secretion level of OcVg protein in the hemolymph revealed that during wintering, the secretion level increased from month 1 to month 2 and then stagnated after 2 months. After diapause, the secretion level increased gradually until day 6 of the newly emerged adult from the cocoon stage and thereafter gradually declined, remaining detectable until day 30 of the adult stage. The correction analysis between ovarian development and OcVg secretion level in the hemolymph showed that during wintering, the number of oocytes positively correlated with the OcVg secretion level. After diapause, the lengths of the ovary and first oocyte and the number of oocytes showed significant changes in the OcVg secretion level and strongly correlated with the OcVg secretion level, respectively. These results suggest that there is a significant interaction between ovarian development and the secretion level of OcVg protein and that the pattern of ovarian development and the secretion of OcVg protein are stage-specific in the O. cornifrons female.

Thioredoxins (Trxs) are a ubiquitous family of antioxidant enzymes that are involved in protecting organisms against various oxidative stresses. Here, we cloned and characterized two thioredoxins, named SlTrx1 and SlTrx2, from the common cutworm Spodoptera litura. SlTrx1 and SlTrx2, respectively, consist of 988 and 606 bp full-length cDNA with 318 and 447 bp open reading frames encoding 106 and 149 amino acid residues. Furthermore, the N-terminal region of SlTrx2 contains a predicted mitochondrial localization signal (33 amino acids). A phylogenetic relationship analysis revealed that SlTrx1 is in the cytosolic thioredoxin Trx1 cluster, whereas SlTrx2 is in the mitochondrial thioredoxin Trx2 cluster. Recombinant SlTrx1 (14 kDa) and SlTrx2 (16 kDa), expressed in baculovirus-infected insect Sf9 cells, demonstrated insulin disulfide reductase activity at the same optimum temperature and pH value of 35 °C and 7.0, respectively, in vitro. During S. litura development, we found that SlTrx1 and SlTrx2 had similar transcript expression patterns and were constitutively expressed in the epidermis, fat body, and midgut, with the highest expression occurring in the sixth-instar larval stage in the epidermis and midgut. In addition, both SlTrx1 and SlTrx2 mRNA were up-regulated in S. litura after injection with H2O2, cumene hydroperoxide, indoxacarb, and metaflumizone. These results suggest that SlTrx1 and SlTrx2 function as potent antioxidant enzymes, and provide a molecular basis for the roles SlTrx1 and SlTrx2 during development and the oxidative stress response of S. litura.

Apolipophorin III (apoLp-III) is involved in lipid transport and innate immunity in insects. In this study, an apoLp-III protein that exhibits antibacterial activity was identified in honeybees (Apis cerana). A. cerana apoLp-III cDNA encodes a 193 amino acid sequence that shares high identity with other members of the hymenopteran insect apoLp-III family. A. cerana apoLp-III is expressed constitutively in the fat body, epidermis, and venom gland and is detected as a 23-kDa protein. A. cerana apoLp-III expression is induced in the fat body after injection with Escherichia coli, Bacillus thuringiensis, or Beauveria bassiana. However, recombinant A. cerana apoLp-III (expressed in baculovirus-infected insect cells) binds directly to E. coli and B. thuringiensis but not to B. bassiana. Consistent with these findings, A. cerana apoLp-III exhibited antibacterial activity against both Gram-negative and Gram-positive bacteria. These results provide insight into the role of A. cerana apoLp-III during the innate immune response following bacterial infection.

Osmia cornifrons plays a major role in the pollination of orchards, but basic information on vitellogenin and oocyte development is limited. To better understand vitellogenin in hymenopteran insects, we cloned a cDNA encoding vitellogenin from the hornfaced bee O. cornifrons. Osmia cornifrons vitellogenin cDNA contains 5477 bp with an open reading frame of 1783 amino acid residues, and has a predicted molecular mass of approximately 200.21 kDa and a pI of 6.55. Osmia cornifrons vitellogenin possesses four consensus (RXXR/S) cleavage sites and has conserved DGXR and GL/ICG motifs in the C-terminus. The deduced amino acid sequence of the O. cornifrons vitellogenin cDNA showed a 66% identity with Megachile rotundata, 53% to Apis mellifera, 51% to Bombus ignitus and 42%–30% with other hymenopteran insect vitellogenins. Phylogenetic analysis showed that O. cornifrons vitellogenin clustered with vitellogenins from Megachilidae, Apidae, Vespidae and Formicidae species but not with those from Pteromalidae, Aphelinidae or Ichneumonidae species. The expression profile of O. cornifrons vitellogenin mRNA during development revealed that O. cornifrons vitellogenin was first detected in the pupal stage and was continuously detected during the adult stage. Interestingly, O. cornifrons vitellogenin mRNA expression was low in mid-diapause, then gradually increased beginning on day 3 of the newly emerged adult stage, and subsequently declined. These results suggest that the expression level of O. cornifrons vitellogenin mRNA is stage-specific.

Cystatins are involved in various physiological and cellular processes, including immune responses, protein homeostasis, signaling pathways, and apoptosis. Thus far, no Cystatins have been identified from spider venom. In this study, we report the cloning and characterization of a spider venom-derived Cystatin from Araneus ventricosus (AvCystatin). The AvCystatin gene contains an open reading frame of 405 bp encoding a predicted protein of 134-amino acids with a 16-amino acid signal peptide. Sequence alignment and structural modeling indicated that AvCystatin is closely related to family 2 Cystatins. Endogenous AvCystatin was present as an 18-kDa peptide in spider venom. Recombinant AvCystatin, expressed in baculovirus-infected insect cells, showed inhibitory activity against papain (Ki 86.73 nM), but not trypsin and chymotrypsin, defining a role for AvCystatin as a spider venom-derived cysteine protease inhibitor. Furthermore, recombinant AvCystatin exhibited stability against high temperatures and pH extremes, but had no effects on the growth of the entomopathogenic fungi Beauveria bassiana. These data demonstrate that AvCystatin is a novel member of the family 2 Cystatins and provide new insight for the future investigations of spider venom-derived Cystatins.

Ascoviruses are a recently described family and the traditional plaque assay and end-point PCR assay have been used for their titration. However, these two methods are time-consuming and inaccurate to titrate ascoviruses. In the present study, a quick method for the determination of the titer of ascovirus stocks was developed based on ascovirus-induced apoptosis in infected insect cells. Briefly, cells infected with serial dilutions of virus (10-2-10-10) for 24 h were stained with trypan blue. The stained cells were counted, and the percentage of nonviable cells was calculated. The stained cell rate was compared between virus-infected and control cells. The minimum-dilution group that had a significant difference compared with control and the maximum-dilution group that had no significant difference were selected and then compared each well of the two groups with the average stained cell rate of control. The well was marked as positive well if the stained cell rate was higher than the average stained cell rate of control wells; otherwise, the well was marked as negative wells. The percentage of positive wells were calculated according to the number of positive. Subsequently, the virus titer was calculated through the method of Reed and Muench. This novel method is rapid, simple, reproducible, accurate, and less material-consuming and eliminates the subjectivity of the other procedures for titrating ascoviruses.

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor.

Bee venom contains a variety of peptides and enzymes, including acid phosphatases. An acid phosphatase has been identified from European honeybee (Apis mellifera) venom. However, although the amino acid sequence is known, no functional information is currently available for bee venom acid phosphatase Acph-1-like proteins. In this study, an Asiatic honeybee (Apis cerana) venom acid phosphatase Acph-1-like protein (AcAcph-1) was identified. The analysis of the predicted AcAcph-1 amino acid sequence revealed high levels of identity with other bee venom acid phosphatase Acph-1-like proteins. Recombinant AcAcph-1 was expressed as a 64-kDa protein in baculovirus-infected insect cells. The enzymatic properties of recombinant AcAcph-1, determined using p-nitrophenyl phosphate (p-NPP) as a substrate, showed the highest activity at 45 °C and pH 4.8. Northern and western blot analyses showed that AcAcph-1 was expressed in the venom gland and was present as a 64-kDa protein in bee venom. In addition, N-glycosylation of AcAcph-1 was revealed by tunicamycin treatment of recombinant virus-infected insect Sf9 cells and by glycoprotein staining of purified recombinant AcAcph-1. Our findings show that AcAcph-1 functions as a venom acid phosphatase. This paper provides the first evidence of the role of a bee venom acid phosphatase Acph-1-like protein.

Drosophila Niemann–Pick disease type C2 (NPC2) proteins play roles in sterol homeostasis, steroid biosynthesis, and innate immune signaling pathways. In this study, a bee (Apis cerana) NPC2a protein (AcNPC2a) that might function in innate immune reactions was identified. AcNPC2a consisted of 148 amino acids, which included six conserved cysteine residues. Recombinant AcNPC2a protein (expressed in baculovirus-infected insect cells) bound directly to live Escherichia coli, Bacillus thuringiensis, and Beauveria bassiana; however, AcNPC2a did not show antimicrobial activity against these microorganisms. Nevertheless, the expression of AcNPC2a was significantly induced in the fat body of A. cerana worker bees after injection with E. coli, B. thuringiensis, or B. bassiana. Our data suggest a role for AcNPC2a in innate immunity that is induced in response to microbial challenge and binds directly to the cell walls of bacteria and fungi. These findings provide insight into the role of AcNPC2a during the innate immune response following bacterial and fungal infection.