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.

암반생물막의 군집구조와 생물량의 시, 공간적인 변화를 확인하기 위하여, 파도에 대한 노출이 다른 고사포와 격포에 서 11월부터 2011년 9월까지 격월로 암반조각을 채집하였다. 군집구조는 채집된 암반조각을 칫솔로 긁어 광학현미경 하에서 미세조류의 분류군별 개체수를 계수하여 분석하였고, 생물량은 NDVI, VI, 엽록소 a 농도를 측정하여 확인하였 다. 고사포와 격포의 조간대 암반생물막에서 가장 우점하는 분류군은 Aphanotece spp., Lyngbya spp.를 포함하는 남조류 였으며, 환경스트레스가 적은 조간대 하부에서는 규조류의 출현율이 높게 나타났다. 암반생물막에서 우점하는 규조류는 Navicula spp., Achnanthes spp.와 Licmophora spp.로 확인되었다. 식생지수와 엽록소 a 농도는 격포에 비해 고사포 생물막에서 높게 나타났다. 식생지수인 NDVI와 VI는 고사포에서 각각 0.49-0.40(평균 0.43), 2.64-3.22(평균 2.90)였으 며, 격포의 암반생물막은 NDVI와 VI가 각각 0.32-0.41(평균 0.38), 2.03-2.86(평균 2.48)으로 확인되었다. 엽록소 a의 농도는 고사포에서 12.79-32.87 ㎍/㎠(평균 22.84 ㎍/㎠)였고, 격포에서는 11.14-18.25 ㎍/㎠(평균 15.48 ㎍/㎠)로 식생지수와 마찬가지로 1월(겨울)에 최대, 3월(봄)에 최소인 계절 변화를 보였다. 엽록소 a 농도는 NDVI, VI와 양의 상관관계를 보여 비파괴적인 식생지수 측정방법이 파괴적인 엽록소 a 추출 방법을 대체할 수 있음을 알려준다. 결론적으 로 암반생물막은 여름보다 겨울에, 조간대 상부보다 중부와 하부에서, 파도에 보호된 해안보다 노출된 해안에서 높은 값을 보였다.

제주 연안 저서생물(해조류, 무척추동물)의 군집구조가 저서 기질 형태에 따른 차이를 파악하기 위해 신흥해역에서 2015년 2월부터 11월까지 매월 조사되었다. 암반정점에서는 해조류 57종과 무척추동물 102종이 출현하였고, 몽돌정점 에서는 해조류 42종과 무척추동물 67종이 관찰되어 암반기질에서 저서생물의 출현종수가 높은 것으로 나타났다. 해조 류의 연평균 생물량은 암반정점에서 1,601.13 g wet wt./㎡으로 몽돌정점의 448.85 g wet wt./㎡보다 약 4배 높았다. 감태는 암반정점에서 전체 생물량의 62.64% (1,002.93 g/㎡)를 차지하는 우점종이었고, 몽돌정점에서는 넓은게발, 감태, 붉은뼈까막살, 자루바다표고, 갈래곰보가 우점하였다. 저서무척추동물의 개체수 및 생물량은 암반정점에서 각각 106.9 indivi./㎡, 871.93 g/㎡이며, 몽돌정점에서는 64.6 indivi./㎡, 984.28 g/㎡로 나타났다. 무척추동물의 생물량 우점종을 파악한 결과, 암반정점에서는 소라(36.40%), 바퀴고둥(19.18%), 보라성게(13.61%)였고, 몽돌정점에서는 거 품돌산호(54.13%)와 그물코돌산호(24.28%)로 기질에 따른 우점종의 차이를 확인하였다.

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.

한국 태안군 정산포와 황도 갯벌에서 저서미세조류의 종조성과 생물량의 계절적 변화가 바지락 생장과 연관성이 있는지를 파악하기 위하여 2012년 2월부터 11월까지 계절별로 조사되었다. 연구기간 동안 저서미세조류는 총 122종이 관찰되었으며, 정산포에서 85종(계절별 30-45종), 황도에서는 92종(32-57종)이었다. 연평균 엽록소 a 의 농도는 정산포에서 79.75 ㎎/㎡였고 황도에서 151.50 ㎎/㎡였다. 갯벌 퇴적물에서 서식하는 저서미세조류의 연평균 세포수는 정산포에서 13,255 cells/㎠이었고 황도에서 15,943 cells/㎠였다. 정산포에서 Cylindrotheca closterium, Gyrosigma sp.와 Navicula sp.가 우점하였고 황도에서는 Amphora sp.1., C. closterium, Detonula pumila, Navicula sp.와 Merismopedia sp.였다. 또한, 두 연구정점에서 최대의 세포수를 나타낸 종은 Paralia sulcata였다. 정산포 바지락의 소화기관에서는 계절별로 18-31종의 미세조류가 분류되었으며, 황도 바지락 소화기관에서는 19-25종의 미세조류가 동정되었고 P. sulcata는 모든 계절에 바지락 소화기관에서 확인되었다. 바지락의 연평균 비만도는 정산포에서 연평균 0.42로 황도의 0.57에 비해 0.15가 낮았다. 결과적으로 바지락의 먹이원인 저서미세조류의 풍도와 엽록소 a 의 농도는 바지락 생장과 밀접한 관련이 있음을 확인하였다.

Marine macroalgal community structure of subtidal zones were examined at eight study sites, Jeju, Korea, from March to June 2010. A total of 182 species were identified, including 22 green, 37 brown and 123 red algae. Of them, 15 species were observed at all the study sites. Species richness of seaweeds was maximal at Seongsan with 112 species and minimal at Sinheung with 44 species. Average seaweed biomass was 735.24 g wet wt. m-2 and ranged from 165.82 g wet wt. m-2 at Sinheung to 1,160.43 g wet wt. m-2 at Bomok. Articulated coralline algae were dominant occupying 27.05% (198.92 g wet wt. m-2) of total biomass for the eight study sites in Jeju Island. Subdominant species were Ecklonia cava and Codium coactum, comprising 26.62% (195.72 g wet wt. m-2) and 8.42% (61.91 g wet wt. m-2), respectively. Vertical distribution of subtidal seaweeds in terms of biomass showed from E. cava Colpomenia sinuosa - Codium coactum Ecklonia kurome Undaria pinnatifida - Cladophora wrightiana - Peyssonnelia capensis in the subtital zone between 5~15 m depth level.

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.

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.

Apolipophorin III (apoLp-III) is a multifunctional protein that 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 consists of 193 amino acids that share high protein sequence identity to other members of the hymenoptran 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 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.

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.

2011년부터 공식적으로 수입되어 사육·유통되는 슈퍼밀웜의 국내 샘플들은 형 태 분류학적 검토를 통하여 Zophobas atratus란 종으로 밝혀졌고, Z. morio란 학명 은 이 종의 동물이명임이 확인되었다. 이 외래종은 자원 관리측면에서 국명을 ‘아 메리카왕거저리’로 신칭하였다. 이 종과 형태적으로 유사한 자생종 대왕거저리 및 사육종 갈색거저리와 DNA 바코드 분석 결과, 아메리카왕거저리와 대왕거저리는 평균 21.4%, 아메리카왕거저리와 갈색거저리는 20.9%의 염기분화율을 보여 DNA 바코드로 쉽게 종 동정 할 수 있음을 확인하였다. 아메리카왕거저리의 국내 집단은 모두 동일 일배체형을 갖고 있어 국외의 동일 지역 개체군이 국내로 유입된 것으로 추정된 반면에 갈색거저리는 동일 사육집단 내에서도 두 개의 종내 집단이 뚜렷이 구분되고 서로의 염기 분화율이 1.17-2.19%로 갭을 형성한 것으로 보아 국 내 갈색거저리 사육개체들은 서로 다른 지역 집단이 혼입되어 대량 사육에 이용되 어진 것으로 추정된다. 이번 연구를 통하여 상업적으로 도입, 이용되는 2종의 거저 리류의 분류학적 기초 정보가 국내 곤충자원 관리를 위하여 유용할 것으로 판단된다.

Bumblebee venom serine protease inhibitors have been shown to inhibit plasmin activity. In this study, a bumblebee (Bombus ignitus) venom serine protease inhibitor (BiVSPI) that acts as an antimicrobial factor was identified. BiVSPI is a 55-amino acid mature peptide with ten conserved cysteine residues and a P1 methionine residue. BiVSPI was expressed in the venom gland and was present as an 8-kDa peptide in venom. Recombinant BiVSPI expressed in baculovirusinfected insect cells exhibited inhibitory activity against chymotrypsin, but not trypsin. BiVSPI also exhibited inhibitory activity against microbial serine proteases, such as subtilisin A (Ki 6.57 nM) and proteinase K (Ki 7.11 nM), indicating that BiVSPI acts as a microbial serine protease inhibitor. In addition, BiVSPI was also shown to bind directly to Bacillus subtilis, B. thuringiensis, and Beauveria bassiana, but not to Escherichia coli. Consistent with these results, BiVSPI exhibited antimicrobial activity against Gram-positive bacteria and fungi. These findings provide novel evidence for the antimicrobial function of this bumblebee venom serine protease inhibitor.

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.

In arthropods, an immune challenge triggers a serine protease cascade that leads to the activation of prophenoloxidase (proPO)-activating factors (PPAFs), which are also called proPO-activating enzymes (PPAEs) or proteinases (PAPs). PPAFs are activated by cleavage between their clip and serine protease domains. Once activated, PPAFs convert proPO to phenoloxidase (PO), which then catalyzes the production of quinones to form melanin. In this study, we identified a Bombyx mori PPAF(BmPPAF) that involves in the pupal melanization. In the fat body, expression of BmPPAF was detected on day 1 to 3 of the pupal stage. RNA interference (RNAi)-mediated BmPPAF knock-down inhibited pupal melanization, resulting in the delay of pupal melanization. Based on these results, we concluded that BmPPAF is involved in the melanization of pupal stage in silkworm metamorphosis.

Spider silks hold great potential as biomaterials with extraordinary properties. Here we report cloning and characterization of the major ampullate silk protein gene from the spider Araneus ventricosus. A cDNA coding for the partial major ampullate silk protein (AvMaSp) was cloned from A. ventricosus. Analysis of the cDNA sequence shows that AvMaSp consists of 240 amino acids of a repetitive region and 99 amino acids of a C-terminal non-repetitive domain. The peptide motifs found in spider major ampullate silk proteins, (A)n, (GA)n, and (GGX)n, were conserved in the repetitive region of AvMaSp. Phylogenetic analysis further confirmed that AvMaSp belongs to the spider major ampullate spidroin proteins. The AvMaSp-R cDNA, which contains sequences encoding for 240 amino acids of a repetitive domain, was expressed as a 22 kDa polypeptide of soluble form in baculovirus-infected insect cells. Recombinant AvMaSp-R was degraded abruptly by trypsin. However, AvMaSp-R was stable at 100 °C for at least 30 min. Additionally, the AvMaSp-R was stable at various pH values from 2 to 12 for at least 1 h. Taken together, our findings provide the molecular structure and biochemical property for A. ventricosus major ampullate silk protein as a biomaterial.

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.

Classical swine fever virus (CSFV) envelope glycoprotein E2 is the main target for inducing neutralizing antibodies and protective immunity in swine. Here, we report a novel strategy forthe large-scale production of a CSFV E2 subunit vaccine that demonstrates a high immunogenic capability in the larvae of a baculovirus-infected silkworm, Bombyx mori. We constructed a recombinant B. mori nucleopolyhedrovirus (BmNPV) that expressed recombinant polyhedra together with the N-terminal 179 amino acids of CSFV E2 (CSFV E2ΔC). BmNPV-E2ΔC-infected silkworm larvae expressed an approximately 44-kDa fusion protein that was detected using both anti-polyhedrin and anti-CSFV E2 antibodies. Electron and confocal microscopy both demonstrated that the recombinant polyhedra were morphologically normal and contained CSFV E2ΔC. The CSFV E2ΔC antigen produced in BmNPV-E2ΔC-infected silkworm larvae reached 0.68 mg per ml of hemolymph and 0.53 mg per larva at 6 days post-infection. Mice that were immunized with the granule form of recombinant polyhedra or the soluble form of the fusion protein elicited CSFV E2 antibodies, which indicated that the recombinant polyhedra carrying CSFV E2ΔC were immunogenic. The virus neutralization test showed that the serum from mice that were treated with recombinant polyhedra or the soluble form of the fusion protein contained significant levels of virus neutralization activity. These results demonstrate that the present strategy can be used for the large-scale production of CSFV E2 antigen and that the recombinant polyhedra containing CSFV E2ΔC as a granule antigen can be used as a potential subunit vaccine against CSFV.