Bumblebee, Bombus terrestris is widely used to pollinate various crops, especially tomato, in greenhouses. Mason bee, Osmia cornifrons, which is using as apple pollinator. Here we investigated characteristics on pollinating activity of B. terrestris and O. conifrons at different weather conditions in apple orchard. The foraging activity of O. conifrons was more sensitive than that of B. terrestris at weather conditions including temperature, illumination intensity and wind velocity. The pollinating activity of O. conifrons was very low than that of B. terrestris at a low temperature, cloudy, rainy and windy weather. Therefore, Our results indicated that B. terrestris was suitable for apple pollinator than O. conifrons, especially in low temperature and bad weather.

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators since the outbreak of honeybee collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites, which affect the life span and fecundity of their host, have been discovered in B. terristris. In order to detect the microsporidian pathogen, Nosema Spp. in the field populations of B. terristris, we collected adults and isolated their genomic DNA for diagnostic PCR. The PCR primers specific for Nosema Spp. were newly designed and applied to gene amplification for cloning. Only small subunit ribosomal RNA(SSU rRNA) gene of N. ceranae was successfully amplified and sequenced among examined genes, which indicates that N. ceranae mainly infects the examined field population of B. terristris. To detect of SSU rRNA gene, two regions of SSU rRNA gene were selected by primary PCR analysis and further analyzed in quantitative real-time PCR(qRT-PCR). The qRT-PCR analysis demonstrated that SSU rRNA of N. ceranae was detected at concentrations as low as 0.85 ng/μl genomic DNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of N. ceranae infection in the field population as well as risk assessment of B. terristris.

To improvement the fruit quality of the korean raspberry fruit, 3 species of bee, Bombus terrestris, Apis mellifera and Osmia cornifrons were used for pollination in korean raspberry fruit field from Korea. This study was also conducted using 3 kinds of methods for pollination effect, natural condition, net-house and control method. Blooming time of korean raspberry fruit shows always in the middle of the May, foraging behavior of bees used in this examination showed different tendency each other. Especially pollinating time of B. terrestris was 2.73sec and transfer time between flower was 2.64sec. Rate of korean raspberry fruit setting pollinated with B. terrestris, A. mellifera, O. cornifrons were showed 83.1%, 80.5%, 77.5% respectively. And, rate of fruit setting of the untreated control and the control (natural condition pollination) were 36.2% and 76.0%. Therefore, korean raspberry fruit set in the case of B. terrestris compared control was superior to 230%, 109% in contrast to natural pollination showed, A. mellifera and O. cornifrons were no significant difference over natural pollination. The quality of the products of korean raspberry fruit pollinated with B. terrestris showed the highest quality as a result fruit diameter 19.5±0.85, fruit weight 2.7±0.32, sugar content 11.2±1.48 Brix%, seed no. of 86.8±10.15. The rate of productivity increase using the Bombus terrestris was 21% in contrast to natural pollination.

To improvement the fruit quality of the korean raspberry and the blueberry fruit, 3 bee species, Bombus terrestris, Apis mellifera, and Osmia cornifrons were used for pollination. The rates of korean raspberry fruit set in the case of pollination with B. terrestris, A. mellifera, and O. cornifrons were 83.1%, 80.5% and 77.5%, respectively. In addition, the rates of fruit set in the case of the untreated control and the control(pollination under natural conditions) were 36.2% and 76.0%, respectively. The products of korean raspberry fruit pollinated with B. terrestris had the highest quality, with a fruit diameter of 19.5±0.85mm; fruit weight, 2.7±0.32g; sugar content, 11.2±1.48 Brix%; seed no., 86.8±10.15. The rate of productivity increase obtained using the B. terrestris was 21% greater than that obtained using natural pollination. And, the rate of blueberry fruit setting pollinated with B. terrestris, A. mellifera, O. cornifrons were showed 87.7%, 89.6%, 66.5% respectively. And, rate of fruit setting of the untreated control and the control(natural condition pollination) were 15.4% and 80.3%. The rate of productivity increase obtained using the B. terrestris was 9%∼17% greater than that obtained using natural pollination.

The bumblebee, Bombus terrestris, has played an important role as one of the alternative pollinators. Recently, pathogens and parasites affect the life span and fecundity of their host and been isolated from B. terristris. In order to detect viral infection in the field populations of B. terristris, we collected adults and isolated total RNA for reverse transcriptase-polymerase chain reaction (PCR). The PCR primers specific for several viruses such as deformed wing virus, Israel acute paralysis virus, Kashmir bee virus and black queen cell virus (BQCV) were newly designed and applied to gene amplification for cloning. Only BQCV was successfully amplified and sequenced, which suggests that BQCV may mainly infects the examined field population of B. terristris. To detect of capsid protein gene of BQCV, 4 selected regions were analyzed by primary PCR and 1 region was successfully amplified, which was further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis demonstrated that BQCV was detected at concentrations as low as 0.1ng/μl total RNA. This result suggests that the detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of BQCV infection in the field population as well as risk assessment of B. terristris.

To substitute for bluebottle fly, Chrysomyia megacephala which is being used for pollinator in mango fruit, and improve the pollinating effect of mango fruit which is also being increased as high value added crop recently in Jeju island of korea, 2 kinds of pollinator were used in analyzing and surveying of foraging activities on mango fruit in Seogwipo province. This study was conducted using 3 species of pollinator, Apis mellifera, Bombus terrestris and Chrysomyia megacephala with 3 treatment in vinyl-house condition respectively. Species of mango fruit, Irwin, was used in this experiment. A number of foraging activity of Apis mellifera and Bombus terrestris in hive showed highest 11 AM, and showed normal foraging activity in high temperature condition(28℃). Pollinating ratio of Bombus terrestris was shown 100% and over 95% in case of Apis mellifera. This ratio suggest that the 2 species of insects is effective as pollinator on mango fruit compared with bluebottle fly. Daily pollinating activity of Apis mellifera and Bombus terrestris was shown peak in 11 AM, but showed even activity from 9 AM to 3 PM in case of Chrysomyia megacephala. The pollinating characteristics of 3 species depends on illuminance but temperature, especially in case of Bombus terrestris was more affected by change of illuminance. Visiting time of Bombus terrestris and Apis mellifera on this flower was shown 2.8 and 3.4 seconds respectively. But Chrysomyia megacephala showed longer 10 times with 32.5 seconds than other insects. This results suppose that Chrysomyia megacephala showed as resting behavior for almost time on the flower not foraging activity to pollinate.

Bombus terrestris has played an important role in the pollination in agricultural fields for the alternatives in colony collapsing in the honeybee. Recently, some pathogens or parasites such as viruses, bacteria, mites have been discovered in B. terristris, which affects its life span and fecundity. In order to detect a microsporidian, Nosema apis. in the field population, we collected honeybees and isolated genomic DNA. PCR primers specific for 16S ribosomal RNA (16S rRNA) were synthesized and applied to gene amplification for cloning and quantitative real-time PCR (qRT-PCR). The amplified gene was cloned and sequenced to confirm the 16S rRNA gene. qRT-PCR analysis showed the detection limit of 16S rRNA of Nosema apis was approximately 0.5 ng/μl genomic DNA. This result suggests that detection via qRT-PCR can be applied for the diagnosis of pathogen infection.

Currently, honeybee colonies are not stable and suffer from the infection of pathogens, affecting the pollination. For the alternatives to this difficulty, Bombus terrestris has been imported and used for pollination in agricultural fields. Although imported insects for pollination are very useful, the potential risk exposing to novel pathogens has been raised. To assess the risk primarily, we designed and synthesized PCR primers for detection of pathogens and parasites in B. terrestris. The samples were obtained from companies importing B. terrestris or field collections and genomic DNAs not showing physical shearing were purified. PCR for detection of pathogen- or parasite-specific gene revealed several DNA fragments were amplified in expected molecular size including Kashmir Bee Virus, Varroa jacobsoni, V. rindereri, Acarapis woodi and Aspergillus flavus. These amplified DNA fragments are in the process of cloning for DNA sequencing to confirm the target gene amplification. We also have plans to optimize the PCR conditions for each amplified target gene and try to develop biomarkers for diagnosis.

체리의 경우 유과기나 과실수확기에 비가 잦을 경우 각종 곰팡이에 의한 과실썩음병과 열과가 발생하여 고품질의 과실을 생산하기 어렵다. 따라서 우리나라와 같이 장마기가 있는 기후조건에서는 농약살포 횟수를 줄이고 고품질의 과실을 안정적으로 생산하기 위해서는 반드시 비가림 시설재배가 필요하다. 국내에서 체리의 개화시기는 4월 상 중순으로 개화기 저온에 의해 자연상태의 꿀벌을 통한 화분매개는 결실율이 불안정하며, 봄철에는 화분매개용 꿀벌 봉군을 구입하기가 어렵다. 따라서 본 연구에서는 최근 시설재배 방울토마토, 애호박 등에서 화분매개용으로 많이 이용되고 있는 서양뒤영벌을 비가림 재배 조건에서 꿀벌을 대신하여 체리에 대한 화분매개 곤충으로 이용코자 하였다. 방화활동력은 서양뒤영벌의 경우 한 꽃에 약 6초 정도 머물러 분당 11개 정도의 꽃을 방문하였고, 꿀벌의 경우 한 꽃에 약 15초씩 머물러 분당 4~5개 정도의 꽃을 방문하였다. 또한 결실율과 과실특성에 있어 두 벌 처리구 간에 통계적인 유의성은 없었다. 따라서 비가림 체리 재배에서 개화기에 화분매개용으로 꿀벌을 대신하여 서양뒤영벌을 이용하더라도 어떠한 부정적인 영향은 없었다.

Bee venom is a rich source of pharmacologically active substances. In this study, we characterized a B. terrestris venom Kunitz-type serine protease inhibitor (Bt-KTI). Bt-KTI consists of two exons encoding 82-amino acids (aa), including a predicted 24-aa signal peptide and a 58-aa mature peptide. Recombinant Bt-KTI was expressed as a 6.5-kDa peptide in baculovirus-infected insect cells. Bt-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tissue plasminogen activator. In contrast, Bt-KTI strongly inhibited plasmin, indicating that it acts as a plasmin inhibitor. The electrophoretic mobility shift assay showed that Bt-KTI binds to plasmin, indicating the formation of a plasmin-Bt-KTI complex. These results demonstrate that Bt-KTI acts as an antifibrinolytic agent, suggesting a role for Bt-KTI as an anti-bleeding agent.

Bumblebees are widely used to pollinate various crops, especially tomato, in greenhouses and fields. Here, we investigated whether different sugar solutions have any effects on the oviposition and colony development of Bombus terrestris queens. The types of sugar solution used were a white sugar solution, brown sugar solution, dark brown sugar solution, and prepared sugar solution; all solution contained with a 40% sugar concentration and 0.3% sorbic acid. Among these sugar solutions, B. terrestris queens reared on the white sugar solution exhibited the best results; the rates of oviposition, colony foundation and progeny-queen production were75.0± 11.0, 33.0 ± 9.6 and 21.7 ± 7.3, respectively, which corresponded to 1.1-33.0-fold increases over the results of other sugar solutions. Queens reared on the prepared sugar solution did not found any colonies. The death rate within one month was lowest for the queens reared on the white sugar solution at 26.1 ± 10.9%, which was 1.2–2.0-fold lower than the rate for the other sugar solutions. Furthermore, the numbers of progeny produced by queens reared on the white sugar solution, 131.4 ± 38.8 workers and 51.0 ± 40.6 queens, were also higher, corresponding to 1.1-1.2-fol d increases compared to queens raised on the other sugar solutions. Therefore, the white sugar solution was the favorable sugar solution for the egg-laying and colony developmental characteristics of B. terrestris queens.

The large bumblebee, Bombusterrestris, indigenous to Europe and adjacent area, and used extensively for high-value crop pollination, has been artificially introduced to several parts of the world. Here we show the interspecific hybridization between the bumblebee species, B.hypocrita sapporoensis and B.terrestris, under laboratory conditions. The mating and oviposition percentages resulting from the interspecific hybridization of a B.terrestris queen with a B. h. sapporoensis male were higher than those resulting from the intraspecific mating of B. h. sapporoensis. Furthermore, a competitive copulation experiment indicated that the mating of B. h. sapporoensis males with B.terrestris queens was 1.2-fold more frequent than the mating of these males with B. h. sapporoensis queens. The interspecific hybridization of a B.terrestris queen with a B. h. sapporoensis male produced either B.terrestris workers or the B.terrestris male phenotype, and the hybridization of a B. h. sapporoensis queen with a B.terrestris male produced B. h. sapporoensis males. Our results indicated that interspecific hybridization occurred between B. h. sapporoensis and B.terrestris. The results suggests that such hybridization will have a negative competitive impact and will cause genetic contamination of native bumblebees.

Bombus terrestris was released for pollination of apple flowers at apple orchards in Uileumgol, Milyang-sity, mid spring, 2008. Numbers of out-going bees and in-coming bees were 38heads at 10 a.m. and 23heads at 4 p.m., respectively. 75% bees per total in-coming bees collected the pollens about 10 a.m. and 63% bees per total in-coming bees collected the pollens around 4 p.m.. The SEM photography(A) showed that 82.7% of a pollen load were collected on apple flowers and 17.3% of a pollen load were collected from another plants and trees flowers. The another SEM photography(B) showed that 89.1% of a pollen load were collected on apple flowers and 10.9% of a pollen load were collected from another plants and trees flowers. Therefore these results show that B. terrestris mainly pollinates on the apple flowers but the bee seems to visit flowers of other plant species from time to time.

Bumblebees are widely used to pollinate various crops, especially tomato, in greenhouses and fields. An artificial hibernation is essential for year-round rearing of the bumblebee that undergoes one generation per year. Here, we investigated whether a cold temperature and humidity affect artificial hibernation of the bumblebee queen Bombus terrestris. In cold temperature regimes of 0℃, 2.5℃, 5℃, 7.5℃ or 12.5℃ under a constant humidity 70% over, the queens stored at 2.5℃ was the best performance in survival rate, which was 74.0% in one month, 67.0% in two months, 60.0% in three months, 46.0% in 4 months, 33.0% in 5 months, and 24.0% in 6 months and lowed in the order of 0℃, 5℃, 7.5℃ and 12.5℃. And also, the colony developmental characteristics after diapause was 1.2-1.5 fold higher than those at queens stored at 5℃. In terms of cold humidity, the queens hibernated at 70% under 2.5℃ was the best performance in survival rate, which was 93.3±3.4% in one month, 83.3±0.0% in two months, 76.7±0.0% in 3 months and 36.7±12.1% in 5 months and lowed in the order of 50% and 90%. The rates of oviposition, colony foundation and progeny-queen production of queens hibernated at 70% were 80.8%, 30.8% and 30.8%, respectively. These values correspond to 1.7-3.3 fold that at queens stored at 50%. Therefore, 2.5℃ and 70% R.H. were the favorable cold temperature and humidity conditions for diapauses-break of B. terrestirs queen.

Bee venom contains a variety of peptides and enzymes, including serine proteases. Here we describe the molecular cloning and characterization of a serine protease (Bt-VSP) isolated from the venom of the bumblebee Bombus terrestris. The Bt-VSP gene consists of six exons encoding a 358-amino acid protein. The form of Bt-VSP detected in bee venom was the 34-kDa mature protein, which is created by cleavage of the catalytic domain of Bt-proVSP between Arg111 and Val112. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. The finding that Bt-VSP acts as a fibrin(ogen)olytic enzyme is similar to a previous finding that Bi-VSP, a venom serine protease of B. ignitus, exhibits fibrin(ogen)olytic activity. We also compared major venom components in honeybee and bumblebee, and found that bumblebee venom contains a larger amount of serine protease. Furthermore, unlike bumblebee venom, which exhibits fibrin(ogen)olytic activity owing to the presence of a serine protease, it is likely that honeybee venom lacks fibrin(ogen)olytic activity.

Bumblebees are widely used to pollinate crops in greenhouses and fields. Here, we investigated whether different wake-up treatments during a short period of 1-3 days just before indoor rearing has any effects on oviposition and colony development of CO2-treated Bombus ignitus queens and artificially hibernated B. terrestris queens The wake-up regimes were defined as 16L for 1 day (16L-1), 16L per day for 3 days (16L-3), 24L for 1 day (24L-1), or 24D for 1 day (24D-1). Among these wake-up treatments, the oviposition rate and preoviposition period of B. ignitus queens reared at 24L-1 were 16.7-25.1% higher and 1.0-3.5 days shorter than other wake-up treatments. B. terrestris queens reared at 24L-1 also showed the best results for egg-laying characteristics, which were 8.9-18.8% higher for oviposition and 0.6-3.5 days shorter for preovipostion period than other wake-up treatments. Furthermore, B. terrestris queens reared at 24L-1 were 17.5% and 13.8% higher in rate of colony foundation and queen production, respectively, than other wake-up treatments. These results show that the most favorable wake-up treatment just before rearing for egg-laying and colony developmental characteristics of B. ignitus and B. terrestris queens was 24L-1. Overall, our findings indicate that a wake-up treatment just before rearing was effective for colony initiation and colony development of bumblebee queens.

About 50,000 hives of Bombus terrestris are used for pollination of tomato and other economical crops (fruit vegetables and fruit trees) in Korea. Therefore this study was conducted to find out the effective use of B. terrestris and its economic effect in the major beefsteak-tomato cultivation areas from February to August in 2008. The pollinating activities, rate of bite-mark and lifespan of B. terrestris"s hive according to all beefsteak-tomato varieties (Super Momotaro, Zeus42, Mascara and Super Sunroad, 500 ㎡, 9,600～12,000plants) were apparent by regular supplement of pollen (5～6g/3 days) to the hive of B. terrestris. Especially, Super Momotaro and Super Sunroad variety were most distinctive in the effects by regular pollen supplement.

시설재배 작물의 주요 화분매개곤충인 땅뒤영벌의 난황단백질(Vitellogenin; Vg) 유전자를 클로닝하고 휴면 및 사회성에 따른 발현 양상을 조사하였다. 클로닝한 Vg 유전자의 일부분은 꿀벌 및 기생벌의 Vg와 높은 유사성을 나타냈다. Northern blot을 통해서 Vg 유전자의 발현 패턴을 조사해 본 결과 우화한 여왕벌은 교미를 했으나 휴면 전 단계에는 난소가 발달하지 않았고 Vg 발현이 아주 저조했다. 그러나 3개월 동안 저온에서 휴면기간을 지내고 난 뒤에 상온에서 산란유도를 했을 시에는 Vg 유전자의 발현이 증가하였다. 여왕벌의 휴면 전 단계(우화후 6일간)와 여왕벌의 지배를 받지 않은 같은 나이의 일벌을 대상으로 Vg 유전자의 발현패턴을 비교한 결과, 우화한 이 후 여왕벌은 Vg 발현이 저조한 반면 일벌은 증가하였다. 또한 여왕벌의 몸무게에 따라서 Vg의 발현이 차이가 있었다. 즉, the heavy group(>0.9g)은 Vg 발현이 저조한 반면 휴면에 들어가지 않는 the light group(<0.7g)은 증가하였다. 땅뒤영벌의 유약호르몬인 JH-III를 먹이에 혼합하여 휴면 전 단계인 여왕벌을 습식시킨 결과 0.1μM 농도에서 Vg 유전자의 발현이 촉진되었다. 하지만 다른 농도에서는 그 영향이 나타나지 않았다. 본 연구를 통하여 Vg 유전자의 발현패턴은 여왕벌의 휴면생리와 관련하여 차이가 있었을 뿐만 아니라 사회성에 의한 영향도 관찰할 수 있었다. 또한 유약호르몬의 습식에 의한 Vg의 발현이 유도되는 것으로 보아 호르몬처리에 의한 여왕벌의 휴면타파를 예상할 수 있으며 휴면기작 연구를 위한 분자 마커로서 활용할 수 있을 것으로 기대된다.

In order to select the superior lines of bumblebee queens(Bombus terrestris) in oviposition and colony development which has some characteristics of diapause with adult type, undiapaused bumblebee queens of 3rd generation were surveyed in room condition compared to queen treated with CO2 and artificial hibernation. Oviposition rate and preovipositional days of undiapaused queens was lower in efficacy with 46.7% and shows same trend in preovipositional days with 52.8 days than the other treatment with CO2 anesthesia and artificial hibernation with 60.0%, 86.7%, 26.1 days and 26.6 days respectively. In continuous experiment, we also surveyed ovipositional characteristic and colony development of three types of queens emergenced from undiapaused queens and treated queens with CO2 and artificial hibernation. Although undiapaused queens in 1st generation were more effective in the same condition than treated queens, but the undiapaused queens of 3rd generation emergenced from undiapaused queens of 2nd generation were very low trend in all checkpoints compared with treated queens in same generation. This results was shown that the queens treated with CO2 or artificial hibernation need to be in successive mass rearing continuously in the year round.

We investigated mating conditions about care temperature of queen before mating, mating period, and number of queen per mating cage to improve mating rate of Bombus terrestris. Among 19℃, 22℃ and 25℃, care temperatures of queen before mating, queen cared at 19℃ was more effective than those at 22℃ and 25℃ in death rate during care and mating periods, and colony development. In case of mating period, oviposition rate and preoviposition periods at queen mated during 3 days were 89.3% and 11.4 days, respectively, which was higher and earlier than those of queen mated during 5 days and 7 days. The rate of worker emergence, colony foundation and progeny-queen production at 3 daysmated queen were also 2.0-11.6% higher than those at queen mated during 5 days and 7 days. In number of queen per mating cage, the rate of worker emergence, colony foundation and progeny-queen production queen mated at mating cage with 10 queens and 30 males were 41.5%, 25.9% and 23.2%, respectively. These values correspond to 1.5-6.8 folds those queen mated at cage with 20 queens and 30 queens. Therefore, we supposed that care temperature favorable for B. terrestris queen was 19℃ and the period need to mating was 3 days, and the number of queen per mating cage (55× 45× 65㎝) was 10.