본 연구는 RGB, 초분광 센서를 이용하여 시기별 사과 잎의 엽록소와 질소 함량을 예측하여 사과 나무 잎의 질소 영양을 진단하기 위해 수행되었다. 분광 데이터는 사과나무 ‘홍로 /M.9’ 2년생을 대상으로 고해상도 RGB와 초분광 센서로 촬 영 후 영상처리를 통해 취득하였다. 식물체 데이터는 촬영이 끝난 직후 엽록소와 잎 질소 함량을 측정하였다. 엽록소 측정 기의 SPAD meter, RGB 센서의 개별 파장, 컬러 식생지수 및 초분광 센서의 214개의 파장과 식물체 데이터를 이용하여 회 귀분석을 실시하였다. 엽록소와 잎 질소 함량 데이터는 시기 와 상관없이 질소 시비량에 따라 통계적으로 유의한 차이가 나타났다. 잎은 시기가 지나면서 잎에 있던 영양분이 과실로 전이되어 색이 옅어졌으며 RGB센서의 경우 Red파장에서 시 기와 상관없이 통계적으로 유의한 차이가 나타났다. 초분광 센서의 경우 두 시기 모두 질소 시비 수준에 따라 가시광 영역 보다 비가시광 영역에서 차이가 크게 나타났다. 반사값를 이 용하여 식물체 특성의 예측 모델 결과 엽록소, 잎 질소함량 모 두 초분광 데이터를 이용한 부분최소제곱 회귀분석을 이용하 였을 때 성능이 가장 높게 나타났다(chlorophyll: 81% / 63%, leaf nitrogen content: 81% / 67%). 이러한 원인은 RGB 센서 에 비해 초분광 센서는 좁은 FWHM과 400－1,000nm의 넓 은 파장 범위를 가지고 있어 질소 결핍에 의한 스트레스로 인 해 작물의 분광학적 해석이 가능했을 것으로 판단된다. 추후 분광학적 특성을 이용하여 전 생육 시기의 수체 생리, 생태 모 델 개발 및 검증 그리고 병해충 진단 등 연구를 통해 고품질, 안 정적인 과실 생산 기술 개발에 기여될 것으로 사료된다.

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.

세라마이드는 일반적으로 화장품 산업에서 피부의 보습 및 피부장벽강화에 도움을 주는 성분으로 많은 연구와 개발이 활발하게 이루어지고 있다. 이번 연구에서는 일반적인 합성 세라마이드가 아닌 달맞이꽃오일에서 얻은 천연 세라마이드를 함유한 보습크림이 피부의 보습 및 경피수분손실량의 변화에 어떤 영향을 미치는지를 확인하였다. 세라마이드를 함유한 보습크림이 세라마이드들 함유하지 않은 보습크림과 비교하여 피부보습력과 경피수분손실량에서 우수한 결과 값을 나타내었음을 확인하였다.

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.

국내 판매되고 있는 사슴벌레종[넓적사슴벌레(Dorcus titanus), 참넓적사슴벌레(Serrognathus consentaneus), 남방넓적사슴벌레(D. titanus jejuensis, 가칭), 애사슴벌레(D. rectus) 및 왕사슴벌레(D. hopei)]과 부산 지역에서 채집한 넓적사슴벌레 12개체와 경남 의령군에서 채집한 1개체의 미토콘드리아 DNA 중 cytochrome oxidase I(COI) 를 이용하여 계통을 비교분석하였다. 아울러 NCBI에 기등록된 북한, 일본, 대만, 필리핀, 인도네시아 및 태국의 넓적사슴벌레의 COI 서열을 이용하여 추가 분석하였다. 먼저 판매되고 있는 사슴벌레종은 넓적사슴벌레, 남방넓적사슴벌레 및 참넓적사슴벌레의 group I과 애사슴벌레 및 왕사슴벌레의 group II로 구분되었다. 부산지역 및 의령에서 채집한 넓적사슴벌레의 경우 COI 및 COII로 분석 결과 북한에서 기보고 된 3개체를 포함해 유전적 변이에 있어 큰 차이가 나타내지 않았다. 또한 한반도와 일본 일부지역에서 높은 상동성을 확인하였는데 그 외 아시아 지역에 서식하는 넓적사슴벌레와는 다른 그룹을 형성함을 확인하였다. 이러한 결과들은 국내 서식 사슴벌레 종의 유전적 분석과 함께 외래 유입종을 판별하는 근거자료로 활용될 수 있을 것이다.

Major royal jelly proteins (MRJPs), important protein components of bee royal jelly (RJ) and exclusive nourishments for queen, exhibit various biological and pharmacological activities. RJ is one of the most studied bee products, but the crucial roles for MRJP2 as an antimicrobial and antioxidant agents remain largely unknown. Here we demonstrated the antimicrobial and antioxidant functions of the Asiatic honeybee (Apis cerna) MRJP2 (AcMRJP2). Recombinant AcMRJP2 of approximately 53 kDa was expressed in baculovirus-infected insect cells, and it exhibited antimicrobial activity against bacteria, fungi, and yeast via binding to microbial surfaces and inducing structural damage in microbial cell walls. AcMRJP2 protected mammalian and insect cells against oxidative damage through shielding of cell membranes. Interestingly, AcMRJP2 exhibited DNA protection activity and DPPH radical-scavenging activity. Altogether, our data demonstrated that AcMRJP2 functions as antimicrobial and antioxidant agents.

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.

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

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.

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.

In insects, serine proteases are involved in a variety of physiological processes including digestion, development, and immunity. Bombyx mori serine protease homolog BmSPH-1 regulates nodule melanization and is recruited into nodules from the hemolymph by B. mori lipopolysaccharide-binding protein. Here, we show the dual role of BmSPH-1 in development and immunity of B. mori. BmSPH-1 was expressed in the hemocytes during larval-pupal transformation and localized to the cuticle of silkworms, which indicates that BmSPH-1 is secreted from hemocytes and then transported to the cuticle via the hemolymph. BmSPH-1 was proteolytically activated in the cuticle during larval-pupal transformation and the early pupal stage. BmSPH-1 RNAi resulted in the arrest of larval-pupal transformation and pupal cuticular melanization. Furthermore, the expression of BmSPH-1 was up-regulated in the hemocytes during infection. Taken together, we found that BmSPH-1 is involved in larval-pupal transformation and pupal cuticular melanization as well as the innate immunity of silkworms, which indicates that BmSPH-1 is responsible for either development or immunity.

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.

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 mason 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.

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 seconds.