Here, we investigated antioxidant defense mechanism in the spermatheca of A. mellifera queens via RNA-seq analysis of spermathecae in both mated and virgin queens. We identified the genes encoding antioxidant proteins, which were differentially expressed in the spermatheca of mated queens. The concentrations of antioxidant proteins, such as superoxide dismutase 1 (SOD1), catalase, glutathione peroxidase (GTPX), and transferrin (Tf) together with the levels of ROS, H2O2, and iron were higher in the spermathecal fluid of mated queens as opposed to those in the spermathecal fluid of virgin queens; this indicated that increase in antioxidant protein concentration is an antioxidant defense mechanism occurring in the spermathecal fluid of mated queens against ROS; this mechanism involves conversion of ROS using antioxidant enzymes and Tf-mediated inhibition of the Fenton reaction occurring between Fe2+ and H2O2. Our data indicate that an increased expression of antioxidant proteins could facilitate prolonged storage and survival of sperms in the spermatheca of mated queens, suggesting the role of antioxidant proteins in antioxidative defense against ROS.

The embryonic genome activation (EGA) is genetically activated states that embryos make the materials such as growth factors for using themselves. EGA is various because they have many materials, different site, different stage, also different species. At this time, transcription factors are expressed. Transcription factors bind to specific DNA region, and regulate the gene expression. Thus, we check the expression of transcription factors, we can know that embryo development is very well or not. The development stages of embryos are basically the stages from fertilization to blastocyst. So, we check the embryos oocyte to blastocyst. In our experiments, we focus the early developmental transcription factors such as Cdx2, Oct4, Sox2, Nanog and E-Cadherin. Above antibody factors showed different expression sites, and there were many differentiated parts from other animal species. In addition, we compared the SCNT and parthenogenetic activation (PA) because these are same methods using electrical activation among the embryo production methods. Our results showed not only similar patterns but also different patterns between pig and mouse. Therefore, we have to investigate that different patterns of transcription factors play a role in pigs, and why occur.

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.

Bee venom, which serves as a weapon to defend the colony from predator attacks, induces an immediate local inflammatory response that causes acute redness and swelling at the site of the sting. This venom-induced inflammation is a rapid anti-predatory defense strategy of the bee against vertebrate predators. Although acute inflammation by venom from venomous arthropods, including bees, is a typical response, how venom acutely elicits inflammatory responses remains unknown. Here, we identify a novel mechanism underlying acute inflammation and provide a rationale for the presence of superoxide dismutase (SOD3) in bee venom. In mouse models, paradoxically, SOD3 in bee venom (bvSOD3) acts as a reactive oxygen species (ROS)-based harm-inducing system to promote acute inflammation. Exogenous bvSOD3 rapidly induced overproduction of H2O2 through endogenously produced superoxide by venom components, such as melittin and phospholipase A2 (PLA2), which then upregulated the expression of proinflammatory genes and promoted the acute inflammatory response. Furthermore, a more severe noxious effect by bvSOD3 elevated a type 2 immune response, and bvSOD3 immunization protected against bvSOD3-mediated toxicity. Our findings that bvSOD3 promotes an acute inflammatory response and induces a protective immune response against inflammation may offer a new approach in venom therapy/immunotherapy.

Bumblebee, Bombus ardens ardens (Apidae: Hymenopera), is an important resource for pollination that is most widely distributed in Korea. This study utilized microsatellite markers for investigation of genetic diversity and geographic relevance of the B. a. ardens populations in Korea. Through Next Generation Sequencing analysis, we identified 10 microsatellite markers and genotyped for 107 individuals of B. ardens collected from 10 populations. At each locus the number of alleles ranged from 10 to 23; the observed and expected heterozygosities ranged from 0.8909 to 1.0000 and 0.6641 to 0.8422, respectively; and inbreeding coefficient(FIS) ranged from –0.5053 to –0.0891. Significant deviation from the Hardy–Weinberg equilibrium was not observed at any locus. Population structure analysis indicated that there are three genetic groups in Korea with each Jeongseon and Ulleung-do composed of different gene pool from the remaining other populations. Similarly, Principal coordinates analysis also showed the same pattern. FST and RST analyses showed that each Jeongseon and Ulleung-do population had a significant genetic distance from other populations. Considering these results, genetic isolation of Ulleung-do may be explained by “Oceanic island” status and Jeongseon, which showed the positive FIS (0.069) and genetic isolation may be caused by its location on the east side of Baekdudaegan and by on-going inbreeding with a small population size.

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.

Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putativelow-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identifiedfrom 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 shownto act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-likedomain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putativelow-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, AcVSPIinhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial andfungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. Thesefindings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor.

Major royal jelly proteins (MRJPs) are important protein components of bee royal jelly (RJ) and exhibit various biologicaland pharmacological activities. The antimicrobial activities of royalisins and the jelleines contained within MRJP 1 andMRJP 2 in RJ have been elucidated. However, the antimicrobial effects of other bee RJ MRJPs remain largely unknown.In this study, we demonstrated that the Asiatic honeybee (Apis cerana) MRJP 4 (AcMRJP4) exhibits antimicrobial activitiesagainst bacteria, fungi, and yeast. Recombinant AcMRJP4 was expressed as a 63-kDa protein in baculovirus-infected insectcells. However, some of the recombinant AcMRJP4 proteins were cleaved into two fragments (i.e., 48-kDa (AcMRJP4-48)and 15-kDa (AcMRJP4-15) proteins) by the proteolytic cleavage of the C-terminus of the recombinant AcMRJP4. Interestingly,AcMRJP4, AcMRJP4-48, and AcMRJP4-15 exhibited antimicrobial activities, with AcMRJP4-15 exhibiting the highestantimicrobial activity, followed by AcMRJP4. AcMRJP4-15, which is a hydrophilic peptide with 88 amino acid residuesthat contains a high content of Asn and positively charged amino acids, induced structural damage in the cell walls ofthe assayed bacteria, fungi, and yeast. Altogether, our data demonstrated that AcMRJP4 functions as an antimicrobial agent.

Bee venom is a complex mixture of toxic components that induces immediate local inflammatory and allergic responses. However, the presence and role of superoxide dismutase (SOD) in bee venom have not been previously investigated. Here, we provide the first demonstration that bee venom contains Cu,Zn SOD (SOD3), a novel extracellular component that promotes local inflammation. Bee venom SOD3 was shown to be an oxidant, rather than an antioxidant, that induces the inflammation-signaling molecule H2O2 in vivo. H2O2 plays a pathological role by triggering an immediate local inflammatory response. Furthermore, bee venom SOD3 significantly induced the activation of proinflammatory mediators (TNF-α and COX-2) and cytokines (IL-1β and IL-6) via the overproduction of H2O2 in mice. Our data demonstrate that bee venom SOD3 induced H2O2, which drives an immediate local inflammatory response, indicating a novel mechanism underlying bee venom-induced local inflammation.

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 first evidence that the Asiatic honeybee (Apis cerana) secapin (AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. 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 a clotting factor. AcSecapin-1 also inhibited both human neutrophil and porcine pancreatic elastases. Furthermore, AcSecapin-1 exhibited anti-microbial activity against fungi and Gram-positive and Gram-negative bacteria. Taken together, our data demonstrated that AcSecapin-1 has a multifunctional role as an anti-fibrinolytic agent, an anti-elastolytic agent, and an anti-microbial peptide, and our data suggested novel functions for the biological actions of the bee venom peptide, secapin.

Inhibitor cysteine knot (ICK) peptides exhibit ion channel blocking, insecticidal, and antimicrobial activities, but currently, no functional roles for bee-derived ICK peptides have been identified. In this study, a bee (Apis cerana) ICK peptide (AcICK) that acts as an antifungal peptide and as an insecticidal venom toxin was identified. AcICK contains an ICK fold that is expressed in the epidermis, fat body, or venom gland and is present as a 6.6-kDa peptide in bee venom. Recombinant AcICK peptide (expressed in baculovirus-infected insect cells) bound directly to Beauveria bassiana and Fusarium graminearum, but not to Escherichia coli or Bacillus thuringiensis. Consistent with these findings, AcICK showed antifungal activity, indicating that AcICK acts as an antifungal peptide. Furthermore, AcICK expression is induced in the fat body and epidermis after injection with B. bassiana. These results provide insight into the role of AcICK during the innate immune response following fungal infection. Additionally, we show that AcICK has insecticidal activity. Our results demonstrate a functional role for AcICK in bees: AcICK acts as an antifungal peptide in innate immune reactions in the body and as an insecticidal toxin in venom. The finding that the AcICK peptide functions with different mechanisms of action in the body and in venom highlights the two-pronged strategy that is possible with the bee ICK peptide.

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 factor, tyrosine hydroxylase, and porPOs, 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.

Insect-derived Kazal-type serine protease inhibitors exhibit thrombin, elastase, plasmin, proteinase K, or subtilisin A inhibition activity, but so far, no functional roles for bee-derived Kazal-type serine protease inhibitors have been identified. In this study, a bee (Apis cerana) venom Kazal-type serine protease inhibitor (AcKTSPI) that acts as a microbial serine protease inhibitor was identified. AcKTSPI contained a single Kazal domain that displayed six conserved cysteine residues and a P1 threonine residue. AcKTSPI was expressed in the venom gland and was present as a 10-kDa peptide in bee venom. Recombinant AcKTSPI Kazal domain (AcKTSPI-Kd) expressed in baculovirus-infected insect cells demonstrated inhibitory activity against subtilisin A (Ki 67.03 nM) and proteinase K (Ki 91.53 nM), but not against α-chymotrypsin or typsin, which implies a role for AcKTSPI as a microbial serine protease inhibitor. However, AcKTSPI-Kd exhibited no detectable inhibitory effects on factor Xa, thrombin, tissue plasminogen activator, or elastase. Additionally, AcKTSPI-Kd bound directly to Bacillus subtilis, B. thuringiensis, Beauveria bassiana, and Fusarium graminearum but not to Escherichia coli. Consistent with these findings, AcKTSPI-Kd showed antibacterial activity against Gram-positive bacteria and antifungal activity against both plant-pathogenic and entomopathogenic fungi. These findings constitute molecular evidence that AcKTSPI acts as an inhibitor of microbial serine proteases. This paper provides a novel view of the antimicrobial functions of a bee venom Kazal-type serine protease inhibitor.

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles forBi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.

Glutathione S-transferases (GSTs) are multifunctional enzymes that are mainlyinvolved in the xenobiotic metabolism and protection against oxidative damage. Most studies of GSTs in insects have been focused on their role in detoxifying exogenous compounds in particular insecticides. Here, we show the expression profiles of GSTs of the bumblebee Bombus ignitus in response to oxidative stress. We identified a sigma-class GST from B. ignitus (BiGSTS). The BiGSTSgene consists of 4 exons that encode 201 amino acids. Comparative analysis indicates that the predicted amino acid sequence of BiGSTS shares a high identity with the sigma-class GSTs of hymenopteran insects such as Apis mellifera (70% protein sequence identity) and Solenopsis invicta (59% protein sequence identity). Tissue distribution analyses showed the presence of BiGSTS in all tissues examined, including the fat body, midgut, muscle and epidermis. The oxidative stress responses analyzed by quantitative real-time PCR showed that under H2O2 overload, BiGSTS and BiGSTD (identified in our previous study) were upregulated in all tissues examined, including the fat body and midgut of B. ignitus worker bees. Under uniform conditions of H2O2 overload, the expression profile of GSTs and other antioxidant enzyme genes, such as phospholipid-hydroperoxide glutathione peroxidase (Bi-PHGPx) and peroxiredoxins (BiPrx1 and BiTPx1), showed that other antioxidant enzyme genes are acutely induced at 3 h after H2O2 exposure, whereas BiGSTS and BiGSTD are highly induced at 9 h after H2O2 exposure in the fat body of B. ignitus worker bees. These findings indicate that GSTs and other antioxidant enzyme genes in B. ignitusare differentially expressed in response to oxidative stress. Taken together, our findings indicate that BiGSTS and BiGSTD are oxidative stress-inducible antioxidant enzymes that may play a role in oxidative stress response.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of the innate immune system that recognize peptidoglycan, a unique cell wall component of bacteria. Here we cloned and characterized PGRP-S from the bumblebee Bombus ignitus (BiPGRP-S). The BiPGRP-S gene consists of four exons encoding 194 amino acid residues. Comparative analysis indicates that the predicted amino acid sequence of BiPGRP-S shares high identity with enzymatically active PGRP-S proteins and contains the amino acids required for amidase activity. BiPGRP-S in B. ignitus worker bees is constitutively expressed in boththe fat body and epidermis, and it is secreted into the hemolymph. Quantitative real-time PCR assays revealed that in both the fat body and epidermis, the BiPGRP-S gene is highly induced by an injection of Bacillus thuringiensis. In addition, recombinant BiPGRP-S expressed as a 19-kDa protein in baculovirus-infected insect cells can bind to B. megaterium and B. thuringiensis but not to Staphylococcus aureus, Escherichia coli or Beauveria bassiana. Consistent with these data, BiPGRP-S shows antibacterial activity against B. megaterium and B. thuringiensis. These results indicate that BiPGRP-S is an inducible protein that may be involved in the immune response against bacterial infection of the genus Bacillus as an amidase-type PGRP-S.

Background: Proteolytic enzymes are involved in insect molting and metamorphosis and play a vital role in the programmed cell death of obsolete organs. Here we show the expression profile of cathepsin B in the fat body of the silkworm Bombyx mori during development. We also compared the expression profile of B. mori cathepsins B (BmCatB) and D (BmCatD) in the fat body during the larval-pupal transformation of B. mori in the BmCatB or BmCatD RNA interference (RNAi) process. Results: BmCatB is ecdysone-induced and expressed in the fat body of B. mori during the molting, and the larval-pupal and pupal-adult transformations, and its expression leads to programmed cell death. In particular, BmCatB is highly expressed in the fat body of B. mori during the larval-pupal transformation and BmCatB RNAi treatment resulted in the arrest of the larval-pupal transformation. RNAi-treated BmCatB knock-down sustained the expression of BmCatD during the larval-pupal transformation. On the other hand, BmCatD RNAi up-regulated the expression of BmCatB in the fat body of final instar larvae. Conclusion: Based on these results, we conclude that BmCatB is involved in the programmed cell death of the fat body during B. mori metamorphosis and that BmCatB and BmCatD contribute collaboratively to B. mori metamorphosis