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.

We compared the grafting success in total of 107 rearing Apis carana queens cells, to which we grafted 540 larvae. The wax for cups we prepared from A. mellifera and A. cerana wax. The A. cerana wax cups were found that artificial queen cell cups with the internal diameter of 8.0 mm at the mouth and 8.0 mm depth were highly preferred by the bees for rearing of queens from the grafted larvae. From the 210 grafted larvae into A. mellifera wax bees accepted 30 queens cells, only (16.67 %) ; A. cerana wax bees accepted 59 queens cells (33 %) ; plastic cup bees accepted 18 queens cells, only (10 %). In the preference test the grafting success in the A. cerana wax cups were better than in the A. mellifera wax and plastic cup. The results show better acceptance of larvae grafted into the pure A. cerana wax cups for rearing A. cerana queen. A new method for rearing honey bees, A. cerana, in vivo was developed and the effects of royal jelly from A. mellifera. We used royal jelly diluted 50:50 with sterile water (The royal jelly is kept frozen until used). A small amount of royal jelly is placed at the center of each cell cup. Young A. cerana larvae were transferred into the queen cups containing ± 10 ㎍ of the Royal jelly from A. mellifera and A. cerana. The average rates of acceptance were affected significantly due to the royal jelly source in the queen cell cups. It is so workable first to produce pure A. cerana wax for making the queen cups before a beekeeper starts with grafting.

Honeybees (Apis mellifera) adapted themselves to different geographical and climatical conditions since they had been introduced in Korea. Beekeepers have tried to breed valuable lineages with artificial insemination or conventional mating techniques. However, evaluation of breeding resultants still relies on timeconsuming observation data. Genetical characterization of breeds has proven its usefulness to preserve genetic resources of livestock. In recent years, microsatellites are most commonly used to evaluate population structures and diversities of living organisms in that the characteristics of locus specificity, rich polymorphism, abundant and random distribution over the genome, and their co-dominant inheritance. Determining classic genetic distances using neutral, highly polymorphic microsatellite markers is a reliable method to investigate genetic relationships and breed differentiation. This methodology can be used to establish preservation priorities for livestock breeds. The final aim of this study was to develop potent markers for assessing genetic structure of lineages after artificial insemination. In this study, the genetic structure of ten microsatellite markers were sequenced or analysed with polymerase chain reaction for eleven European honeybee populations. The results may help to develop reliable microsatellite markers for more efficient preservation strategies of valuable honeybee breeds.

The time-zone of pollinating activity according to numbers of Apis mellifera hive released in the strawberry(Maehyang var.) houses was together from 9A.M. to 4P.M., and the peak times of pollinating activity were between 11A.M and 1P.M.. The effects on pollinating activity according to numbers of A .mellifera hive released in the strawberry houses were ordered 5bee combs(11,000heads), 4bee combs(8,800heads) and 3bee combs(6,600heads). The rate of workers lost in A. mellifera hives with 5bee combs and 4bee combs during the strawberry cultivating period were lower than that of 3bee combs. The rates of fruit set by pollinating activity according to numbers of A.mellifera hive released in the strawberry houses were same level with 99%. The fruit qualities; No. of seeds, sugar content and rate of normal fruit set were same level, but fruit weight was ordered 5bee combs in 16.9g, 4bee combs in 16.4g and 3bee combs in 15.6g. The rate of marketable fruit of 4bee combs and 5bee combs were 5% to 9% higher than that of 3bee combs, respectively.

The time-zone of pollinating activity relative to numbers released of Apis mellifera in the strawberry(Janghui var.) houses was together from 9A.M. to 4P. M., and the peak time of pollinating activity was 1P.M.. The effects on pollinating activity relative to the comb numbers in the honeybee hive released in the strawberry houses were ordered 5bee combs(11,000heads), 3bee combs(6,600heads) and 4bee combs(8,800heads). The rate of workers lost in A. mellifera hives with 5bee combs during the strawberry cultivating period were lower than those of 3bee combs and 4bee combs. The rates of fruit set by pollinating activity relative to the comb numbers in the honeybee hive released in the strawberry houses were same level with over 98%. The fruit qualities; number of seeds, sugar content and rate of normal fruit set were same level, but fruit weights was recorded 5bee combs with 40.8g, and 4bee combs and 3bee combs were showed with 37.8g. The marketing income of 5bee combs was 8% higher than that of 4bee combs and of 3bee combs, respectively.

The time-zone of pollinating activity relative to numbers released of Apis mellifera in the strawberry(Seolhyang var.) houses was together from 9A.M. to 4P. M., and the peak time of pollinating activity was 11A.M.. The effects on pollinating activity relative to the comb number in the honeybee hive released at the strawberry houses were ordered 5bee combs(11,000heads), 4bee combs (8,800heads) and 3bee combs (6,600heads). The rate of workers lost in A. mellifera hives with 5bee combs and 4bee combs during the strawberry cultivating period were lower than that of 3bee combs. The rates of fruit set by pollinating activity relative to the comb number in the honeybee hive released at the strawberry houses were same level with over 98%. The fruit qualities; No. of seeds, sugar content and rate of normal fruit set were same level, but fruit weights were ordered 5bee combs in 37.2g, 4bee combs in 35.6g and 3bee combs in 32.6g. The marketing incomes of 4bee combs and 5bee combs were 9% to 13% higher than that of 3bee combs, respectively.

Toxicity of seven environment friendly agricultural materials (EFAM), which have been used in the domestic market were evaluated on honeybee (Apis mellifera) and asian multicolored ladybird beetle (Harmonia axyridis). Three EFAMs made from plant extract agents (Wangjoongwang Eco, Bogum Eco and Bestop Eco) and four EFAMs made from microbial utilizing agents (Worldstar Eco, Goodmorning, Bluechip and Cameleon) were investigated as EFAMs. In evaluation of toxicity on honeybee, the RT25 values of 3 EFAMs made from plant extract agents ranged from 1 to 3 days. Therefore, honeybee should be released 1-3 days after application of these EFAMs. Meanwhile, the four agricultural materials made from microbial utilizing agents did not show any mortality against honeybee. In evaluating the toxicity to adult and larva ladybird beetles, all seven EFAMs made from plant extract agents and microbial utilizing agents to show any mortality.

The infectious pathogens against honeybee (Apis mellifera) comprise a heterogeneous group of bacterial, viral, and fungal organisms including Paenibacillus larvae, Deformed Wing Virus (DWV) and Nosema apis. Many species like Paenibacillus larvae, Deformed Wing Virus (DWV) and Nosema apis have been isolated from a number of different continents, e.g. America, Asia and Europe, indicating its wide spread in whole nature. Little is known about the occurrence and distribution in the environment of these pathogens. For a more rapid, systematic and efficient monitoring of each pathogenic species against honeybee in the environment, PCR-based detection systems were developed that allows species-specific identifications of various pathogenic species with one reaction. These could be achieved by selecting specific primers from conserved regions of each species with speciesspecific DNA sequence variations. For the detection of any already known pathogen, well-developed PCR-detection system allows the specific detection of expected pathogenic species based on its specific nucleic acid sequence. Since each pathogenic species delivers a specific PCR-product of different size, bands can be distinguished very easily by simple gel electrophoresis. After the development of real-time PCR system, PCR-based specific detections of honeybee pathogens were dramatically improved their applications, from just detection to quantification of pathogens. These systems, quantitative PCR (qPCR) for the detection of honeybee pathogens, could be distinguished from previous PCR detection on the points of “real-time”, “easy” and “quantitative”. Moreover, very rapid PCR, so-called “Ultra-Rapid Real-Time PCR” were developed recently in field of pathogen-detection. Typical Honeybee pathogens such as Paenibacillus larvae, Israelli acute paralysis virus (IAPV) were successfully detected inner 7 minutes using 30 cycled Ultrarapid PCR. According to development of more rapid apparatus, even 30 cycled, 1 minute PCR seems to be possible. Ultla-Rapid PCR was currently attempted to apply for the direct detection system of all viral pathogens against honeybee from bee-samples and different environmental probes.

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Nosema apis and Nosema ceranae in honeybee. Three sets of primers were selected from different genomic sequences to specifically amplify a 831 bp amplicon within the SSU rRNA gene, specific for both N. apis and N. ceranae (MSSR primer); a 375 bp amplicon within the SSU rRNA gene, specific for N. apis (NA primer); and a 1,131 bp amplicon within SSU rRNA gene, specific for N. ceranae (NC primer). Using the primers in conjunction (multiplex PCR) we were able to N. apis and N. ceranae and to differentiate between them. The sensitivity of this PCR assay was approximately 102 spores per milliliter. We proposed that the multiplex PCR was sensitive, specific and rapid tool that can serve as a useful differential diagnostic tool for detecting N. apis and N. ceranae in honeybee.

Since the ancient times the therapeutic application of honeybee venom (BV) is practised and persisted until the present days. Resistant bacteria are in emergence and some drugs no longer have an antimicrobial action. To purify the melittin known as antibacterial peptide, five major peptidergic subfractions were separated, purified and identified from the whole BV. We investigated the antibacterial activity of whole BV and purified melittin against Staphylococcus aureus by the minimum inhibitory concentrations (MIC) and the postantibiotic effect (PAE). The MIC of whole BV for S. aureus was 0.06 ㎍/㎖, respectively. The MIC of melittin was 0.06 ㎍/㎖ on S. aureus. The in vitro PAE of whole BV and isolated melittin were determined using E. coli and S. aureus. The PAE of whole BV against S. aureus were 3.45 h (1×MIC). The PAE of melittin against S. aureus was 4.35 h (1 × MIC). Also both whole BV and melittin killed S. aureus at 5 × MIC. The regrowth wasn't observed after 18 h. These results suggest that whole BV and melittin will be developed a novel antibacterial drug.

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Nosema apis and Nosema ceranae in honeybee. Three sets of primers were selected from different genomic sequences to specifically amplify a 831 bp amplicon within the SSU rRNA gene, specific for both N. apis and N. ceranae (MSSR primer); a 375 bp amplicon within the SSU rRNA gene, specific for N. apis (NA primer); and a 1,131 bp amplicon with in SSU rRNA gene, specific for N. ceranae (NC primer). Using the primers in conjunction (multiplex PCR) we were able to N. apis and N. ceranae and to differentiate between them. The sensitivity of this PCR assay was approximately 102spores per milliliter. We proposed that the multiplex PCR was sensitive, specific and rapid tool that can serve as a useful differential diagnostic tool for detecting N. apis and N. ceranae in honeybee.

Sacbrood virus(SBV) causes a fatal disease(sacbrood) of honeybee larvae, which fail to pupate, change color and shape, and finally die. The complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a Reverse Transcription-PCR(RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. To detect the SBV infection in Korea, we collect beekeepers from various apiaries, which the RT-PCR technique was used. And we designed SBV specific primers in conserved region of the viral genome in the GenBank database. We confirmed the SBV amplicon using cloning and sequence. Homology between determined sequences of SBV korean strain and published virus sequences were 97% in DNA sequence, and 100% in amino acid sequence. We describe the first time that presence of sacbrood virus(SBV) in Korea honey bee colonies using RT-PCR. We also developed and validated a RT-PCR assay for the detection of SBV in Korea.

Mortality of honeybees is a serious problem that beekeepers have to face periodically in Korea and worldwide. The presence of RNA viruses, in addition to other pathogens may be one of its possible causes. In this work, we detected Deformed wing virus(DWV), Israle Acute Paralysis Virus (IAPV), Black queen cell virus (BQCV), Cloudy wing virus(CWV), Kashmir bee virus(KBV), Sacbrood virus(SBV), Chronic bee paralysis virus(CBPV) in samples of korea honeybees with or without Varroa destructor and Nosema apis. The detection of viruses in all provinces, simultaneous co-infection of colonies by several viruses and the fact that 96.3% of the samples were infected with one or more virus, indicates they are widely spread in the region. Using uniplex and multiplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including DWV, IAPV, BQCV, KBV, CWV, and described the detection of mixed virus infections in bees from these colonies. Conclusively, investigated disease of the bee, and confirmed new virus that lead to bee disease, this is thought by valuable thing as data for development of beekeeping industry such as CCD(Colony Collapse Disorder)'s cause searching examination.

Varroa destructor Anderson & Trueman is the most injurious parasitic pest of honeybee in the world. Varroa mites had been originally external parasites of Asian honeybee (Apis cerana Fab.) in south eastern Asia. They jumped to European honeybees (Apis mellifera L.) by 1963. Since then they have killed millions of European honeybee colony, which might be susceptible to them, in Asia, Europe, America, and Africa. Also in Korea since Varroa mites were first found in 1968, they have been destructive pests in most of A. mellifera apiaries. Varroa destructor commonly infesting the European honeybees was classified in 2000 as a different species from the Varroa jacobsoni originally identified on Asian honeybees. Varroa mites not only feed the haemolymph of bees, but also introduce virulent viral diseases, and interrupt the development of bee colony. The other external parasitic mite, Tropilaelaps clarea Delfinado & Baker, which was introduced in 1994 from China, has widely spread and also brought damages on honeybees.

This study was surveyed the effects by pollinating activity of Apis mellifera and Bombus terrestris released in the paprika vinyl-houses. The foraging activity and behaviour of A. mellifera and B. terrestris visited on the paprika flowers were nearly alike. The pick times of pollinating activity by A. mellifera and B. terrestris were showed the hightest at 11:00 and 15:00, and 09:00 to 11:00, respectively. The rate of fruit set by A. mellifera and B. terrestris released for pollinating paprika were same level with 94%, and these rate were higher than the fruit setting rate which was 92% by fan operated. The qualities of paprika produced by pollinators released were higher than those by fan operated. And weight per fruit, number of seeds per fruit and economical profit per 2,310 ㎡ were over 10% higher than those by fan operated. Therefore the economical effects by the pollinating activities of A. mellifera and B. terrestris released in the paprika vinyl-houses were obviously demonstrated.

본 연구는 꿀벌의 월통에 얄맞은 실내 온도인 를 유지하면서 환기를 계속할 수 있는 저온양봉사에서 꿀벌의 실내월동 방법을 확립코자 수행되었다. 환기 팬의 작동 시간으로 실내 온도가 로 조절되고 환기가 행하여지는 저온양봉사에서 서양종 봉군을 공시하여 1997년부터 1999년까지 실내월동 시험을 수행한 성적은 다음과 같다. 실내월동 봉군의 폐사율은 1997~1998년 월동기간 중에 6.3~7.1% 그리고 1998~1999년 월동기간 중에 5~10%였다. 실내월동 시 월동 기간 중 봉군의 무게 감량 비율은 강군이 10.6~10.7% 그리고 약군이 10.2~11.7%였다. 월동 후 봉세 증가율은 강군의 경우는 136.1~142.3%였고, 약군의 경우는 128.0~136.5%였다.

본 연구는 여왕봉의 능력을 검정하여 우량여왕봉을 선발하고 또한 금후 우리나라의 여왕봉 능력검정체계 확립에 필요한 자료를 제시하고자 1988년 9월부터 1989년 8월까지 1년간에 걸쳐 대구 근교 양봉장에서 사양관리중인 서양종 봉군(Apis millifera) 30군을 대상으로 실시되었다. 각종 능력에 대한 검정결과를 요약하면 다음과 같다. 월동전 소상의 평균 무게는 이었고 월동기간 중 봉군당 평균 의 중량이 감소되었다. 봉군증식에 의한 계상설치는 4월 17일부터 5월 5일까지 행하여 졌으며 30군의 단상 봉군 가운데서 13군만이 계상이 설치되어 계상이용성이 낮았다. 화분수집 능력을 나타내는 24시간 동안의 화분수집량은 군당 평균 이었으며, 분봉성의 강약을 나타내는 왕대형성수는 군당 평균이 개이었다. 봉교생산성은 보통이었고, 외역 활동후 소상으로 돌아오는 외역봉 수는 1분간 군당 마리이었다. 산란능력을 나타내는 봉개소비면적은 군당 평균이 이었고 석고병 감염율은 30.8%이었다. 아카시아 유밀기에 있어서 2차에 걸친 총 채밀량은 군당 평균이 이었다.