이탈리안벌인 A, C, F계통과 코카시안벌인 D, V계통을 2005년부터 2007년까지 국내에서 수집하였다. 수집한 계통은 육종을 위해 격리 된 섬에서 근친교배를 통해 순계로 분리하였다. 이 연구는 꿀, 로열젤리 다수확계통 선발에 있어 개체군 선발과 육종 효율을 높이기 위해 수행되 었다. 23 개의 형태학적 특성을 평가하고 두 아종의 기존 데이터와 비교한 결과, 이탈리안벌 순계계통은 코카시안벌 순계 계통과 달리8개의 특성이 기존의 이탈리안벌과 유사해 더 많은 특성이 보존되고 있음을 알 수 있었다. 또한 국내에서 유지되고 있는 순계들은 타 지역의 동일 계통과 차 이를 보여 분리된 순계의 형태적인 특징이 확인되었다.

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.

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.

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 toxic components, including vitellogenin, which display various biological, toxicological,and pharmacological activities. However, the biological actions of vitellogenin, a venom protein in bee venom, remainlargely unknown. Here, we demonstrate that Asiatic honeybee (Apis cerana) venom vitellogenin (AcV-Vg) exhibits anti-oxidantand anti-microbial activities. AcV-Vg is expressed in the venom gland and is then secreted into venom. The recombinantAcV-Vg protein was produced in baculovirus-infected insect cells. We found that AcV-Vg reduced cytotoxicity and oxidativedamage against oxidative stress. Furthermore, AcV-Vg bound to microbial surfaces and induced structural damage in themicrobial cell walls, which, in turn, exhibited anti-microbial activity against bacteria and fungi. Together, our data demonstratedthat the bee venom protein AcV-Vg has multifunctional roles as an anti-oxidant and anti-microbial agent.

The honeybee inhibitor cysteine knot (ICK) peptide acts as an antifungal peptide and insecticidal venom toxin. However, the ICK peptide from bumblebees has not been characterized. Here, we report the molecular cloning and antifungal activity of a bumblebee (Bombus ignitus) ICK peptide (BiICK). We identified a BiICK that contains an ICK fold. The BiICK was expressed in the epidermis, fat body, and venom gland of B. ignitus worker bees. A 6.7-kDa recombinant BiICK peptide was expressed in baculovirus-infected insect cells. Recombinant BiICK peptides directly bound to Beauveria bassiana, Ascosphaera apis, and Fusarium graminearum, but they did not bind to Escherichia coli, Paenibacillus larvae, or Bacillus thuringiensis. Consistent with this finding, BiICK exhibited antifungal activity against fungi. These results demonstrate that BiICK acts as an antifungal peptide.

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.