In Korea, the Asian honey bee (Apis cerana) and the European honey bee (Apis mellifera) (Hymenoptera: Apidae) are the two most common honey bee species. These two closely related species are known to have different sensitivity levels to various insecticides due to millennia of exposure to different pests and pesticides. It is reported that A. cerana is known to be more sensitive to several insecticides, such as amitraz, fenitrothion, and fipronil, than A. melllifera. Multiple studies investigated toxicological responses and related CYPome in A. mellifera, but little is known in A. cerana. The goal of this study is to elucidate the underlying mechanism of different toxicological responses between two bee species, with an emphasis on cytochrome P450 (P450), a significant enzyme involved in metabolic activities. The differences in basal P450 expression patterns were investigated by comparing the relative expression levels of P450 orthologs in several dissected organisms of each species. To compare the sensitivity against major insecticides, lethal doses of major insecticides relevant to both honey bee species were assessed by topical and oral ingestion bioassays. The determined sublethal doses of insecticides were applied to honey bees, and the inducibility of P450s was investigated by comparing the expression patterns of multiple P450s. From these results, this study eventually attempts to compare the toxicological differences between two Apis species with differences in induced cytochrome P450 expression levels.

새롭게 육성된 낭충봉아부패병 저항성 신품종 토종벌(Apis cerana koreana) 과 기존 농가에서 관행적으로 사육되는 토종벌 사이의 형태학적 차 이를 육안으로 확연하게 구분하는 것은 어렵지만 본 연구에서는 신품종 토종벌(A. c. koreana) 을 기존 토종벌(A. c. koreana) 품종 및 계통 간 형태학적 비교를 통해 신품종 만의 특성을 결정할 수 있는 표현형 정보를 제공하였다. 신품종 토종벌(A. c. koreana)의 외부형질을 이용한 품종 특성은 22가지의 형태학적 특성을 기하학적, 형태학적 분석 방법을 적용하고 토종벌(A. c. koreana)의 로얄젤리 생산량, 일벌, 여왕벌, 수벌의 특성을 비교 분석하였다. 본 연구 결과, 신품종 토종벌(A. c. koreana)은 기존 토종벌과 앞날개의 길이에 차이를 보였으며, 중국의 동양종꿀벌(A. cerana)과 비교한 결과, 일벌은 몸무게, 혀의 길이, 앞날개의 길이 등의 값이 높았다. 또한, 신품종 토종벌(A. c. koreana)은 A. cerana indica 보다 두 가지 부위에서 형태학적인 차이를 보였다. 그리고 신품종 토종벌(A. c. koreana)은 로얄젤리를 다른 품종과 비교하여 많이 분비하여 봉군의 발육에 긍정적인 영향을 끼쳤다. 따라서 본 연구결과는 신규 육성 토종벌(A. cerana)에 대한 형태학적 분석 방법을 이용하여 품종을 분류하는데 도움이 될 것으로 기대한다.

South Korea has over 0.38 million of managed honey bee (Apis cerana) colonies before 2009 years ago, which produce the highest quantity of honey in the Korea; however, almost colony (90%) were collapsed by Korean Sacbrood Virus (KSBV) in South Korea. Korean Sacbrood Virus (KSBV) is the pathogen of A. cerana Sacbrood disease, which poses a serious threat to honeybee A. cerana, and tends to cause bee colony and even the whole apiary collapse. Colony collapse of A. cerana was first reported on the Pyeong-Chang of the South Korea in 2009. Several scientists and governments has been tried research for cure the sacbrood disease in A. cerana colony by medicines and management techniques. Unfortunately, The sacbrood disease dosen’t improve. So, we were developed a better breed of A. cerana for resistance of sacbrood virus by selection and then artificial insemination. A. cerana breeding technique was first successful applied with A. cerana in Korean. Queens was grafted from sacbrood resistance line and then it was growing in sacbrood disease colony that was survived 100%. Altogether selected 18 queens were artificially inseminated and 2,000 drones of A. cerana in Korea was used to evaluate amount of semen collection. We are select two scabrood resistance A. cerana line (R and H). R line be used for rearing the Queen. Drone was reared in H line colony. The RH hybrid were not infected sacbrood virus even spread sacbrood virus (2×106). RH colonies have very excellent hygienic behavior, brood, and sacbrood disease resistance activity.

Sacbrood virus (SBV) caused significant colony collapse in Korean Apis cerana. Considering that hygienic behavior in honey bees confers colony-level resistance against brood diseases, we utilized this trait for selecting A. cerana colonies. In addition, the brood survival rate was evaluated after colonies were SBV-inoculated. Over four selective generations, dead brood removal and brood survivorship in selected colonies were higher than those in the unselected colonies (P < 0.01, 99.3 vs. 89.9% for removal of pin-killed pupae; P < 0.01, 99.0 vs. 63.9% for removal of SBV-killed larvae; and P < 0.01, 70.0 vs. 9.2% for brood survivorship). Following SBV-inoculation, selected colonies showed an increase in the number of surviving pupae and adults, whereas unselected colonies collapsed mostly. Our results confirm the feasibility of selecting SBV-resistant A. cerana.

농촌진흥청 개발 품종인 토종벌 신품종(질병저항성 우수계통)에 대해 전남지역에서의 형질특성(봉세발달, 마리당 수밀력, 청소력 등)을 조사하였다. 질병저항성 계통(RS)으로 육성된 토종벌의 유밀기 마리당 수밀력은 들어오는 일벌의 무게가 80.89±8.95mg, 나가는 일벌의 무게가 63.56±8.90mg으로 조사되었고, 비저항성계통(NRS)은 각각 83.22±1.39, 66.67±1.20mg으로 조사되어 RS에서 그 무게차가 0.7mg 더 높았으나 통계적 유의차는 없었다. Pin killed test에 의한 사충제거능력(청소력)은 제거하지 못한 유충잔존율이 RS에서 12시간과 24시간 경과시 14.00±10.39와 7.00±3.46으로, NRS에서는 20.33±14.29, 13.33±10.41%로 조사되었으며, 특히 RS와 NRS의 사충 비제거율이 24시간 경과하였을 때 4.67±2.08, 8.33±7.77%로 RS가 우수한 특성을 보였다. 신규여왕벌 입식 후 봉군세력발달은 NRS에 비하여 RS의 일벌수, 번데기 및 유충수의 발달율이 안정적이고 다소 빠르게 증가하였다.

South Korea has over 0.38 million of managed honey bee (Apis cerana) colonies before 2009 years ago, which produce the highest quantity of honey in the Korea; however, almost colony (90%) were collapsed by Korean Sacbrood Virus (KSBV) in South Korea. Korean Sacbrood Virus (KSBV) is the pathogen of A. cerana Sacbrood disease, which poses a serious threat to honeybee A. cerana, and tends to cause bee colony and even the whole apiary collapse. Colony collapse of A. cerana was first reported on the Pyeong-Chang of the South Korea in 2009. Several scientists and governments has been tried research for cure the sacbrood disease in A. cerana colony by medicines and management techniques. Unfortunately, The sacbrood disease dosen`t improve. So, we were developed a better breed of A. cerana for resistance of sacbrood virus by selection and then artificial insemination. A. cerana breeding technique was first successful applied with A. cerana in Korean. Queens was grafted from sacbrood resistance line and then it was growing in sacbrood disease colony that was survived 100%. Altogether selected 18 queens were artificially inseminated and 2,000 drones of A. cerana in Korea was used to evaluate amount of semen collection. We are select two scabrood resistance A. cerana line (R and H). R line be used for rearing the Queen. Drone was reared in H line colony. The RH hybrid were not infected sacbrood virus even spread sacbrood virus (2×106). RH colonies have very excellent hygienic behavior, brood, and sacbrood disease resistance activity.

RNA interference (RNAi) has been considered as an alternative strategy to control agricultural pest whereby double-stranded RNA triggers a potent and specific inhibition of its homologous mRNA. Since small dsRNAs are required for various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-quality dsRNA. In this study, Bacillus thuringiensis (Bt) based dsRNA production platform was established under control of sporulation-dependent promoter and vp1 gene of Sacbrood virus (SBV) was introduced. The dsRNA against vp1 gene produced from the Bt suppressed the replication of SBV. In addition, the dsRNA was assembled into inulin coated-nanoparticle to increase stability of dsRNA in environment.

Control mating is important aspect in bee breeding programs. The technique of artificial insemination is the possible one that can surely control mating of the selected drones with the virgin queen. This is the first time applied artificial insemination technique to control mating of A. cerana in Korea. Altogether 18 queens were artificially inseminated, and 2,000 drones of Korean A. cerana were used to evaluate amount of semen collection. Semen of A. cerana is much difficult to separate from mucus in comparing with A. mellifera. The average amount of semen can be collected from one A. cerana drone was 0.09 μl, whereas the A. mellifera was more than 6 times (0.58 μl semen per A mellifera drone). Obtaining 1 μl of semen have to collect from 11.94 drones that successful semen ejection and have to kill 17 A. cerana drones. Queens artificially inseminated with 4 μl of semen (once insemination) or 8 μl of semen (twice insemination, each with 4 μl of semen) started laying egg later than naturally mated queens 5.3 and 2.5 days, respectively. The onsets of oviposition of artificially inseminated queens were 12.5 to 15.3 days. Queens received twice inseminations started laying eggs 2.8 days earlier than those received only once insemination. Artificially inseminated queens produced exclusively brood and were similar as the naturally mated ones. The brood production of the queens received once insemination with 4 μl of semen was insignificantly different than those received twice inseminations or naturally mated ones, suggesting that one artificial insemination with 4 μl of semen is favorable.

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.

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.

RNA interference (RNAi) has been considered as an alternative strategy to control agricultural pests whereby double-strandedRNA triggers a potent and specific inhibition of its homologous mRNA. Since small double-stranded RNAs are requiredfor various RNAi applications, there is a need for cost-effective methods for producing large quantities of high-qualitydsRNA. Bacillus thuringiensis produces much insecticidal proteins with expression of their encoding genes being drivenby sporulation-dependent promoters. To develop dsRNA mass-production platform utilizing Bt, the pHT1K-EGFP plasmidvector which has cyt1Aa sporulation-dependent promoter was constructed. The transcriptional level of target gene (EGFP)is higher 113 times than Bt reference gene (ssPE). It was applied to protect honeybee from Sacbrood virus, so targetgene was replaced to SBV-vp1. By ingestion of Bt-derived dsRNA to honeybee shows positive effect on SBV suppression.