The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is widely utilized for greenhouse pollination in South Korea. Understanding the magnitude of genetic diversity and geographic relationships is of fundamental importance for long term preservation and utilization. As a first step, we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to the “DNA barcode” region and the complete internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA from 88 individuals collected in nine South Korean localities. The complete ITS2 sequences were longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated 112-bp long repeats. The 658-bp long mtDNA sequences provided only six haplotypes with a maximum sequence divergence of 0.61% (4 bp), whereas the ITS sequences provided 84 sequence types with a maximum sequence divergence of 1.02% (21 sites). The combination of the current COI data with those of published data suggest that the B. ignitus in South Korea and China are genetically a large group, but those in Japan can be roughly separated into another group. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population were found to exist among the South Korean populations of B. ignitus, which suggests panmixia. This finding is consistent with our understanding of the dispersal capability of the species.

Bumblebees are important pollinators of crops and wildflowers in northern temperate regions. The number of bumblebee species has declined in recent decades, particularly, in developing regions. Here, we investigated the distribution and floral preferences of founder bumblebee queens that were collected between 2000 and 2012 in Korea with the intention of gathering information useful in the conservation of native bumblebee populations. A total of 13,130 overwintered bumblebee queens were collected at 63 locations from 8 provinces; the collected bumblebees consisted of five species of native bumblebees (Bombus ardens, B. ignitus, B. hypocrita spporoensis, B. ussurensis and B. consobrinus wittenburgi) and one species of exotic bumblebee (B. terrestris). The dominant species were Bombus ardens Smith and Bombus ignitus Smith, which accounted for 7,462 (56.8%) and 3,725 (28.4%) of the collected bumblebees, respectively. As a proportional distribution of the founder bumblebee queens that were collected, B. ignitus was the most common (9.8%-78.5%), followed by B. ardens (9.8%-66.1%), B. hypocrita spporoensis (0.9%-31.4%), B. ussurensis (0.8%-22.2%), B. consobrinus wittenburgi (0.1%-3.8%) and B. terrestris (0.0%-1.1%). The bumblebee queens foraged from 7 families of plants and 18 plant species. B. ignitus bumblebees were primarily collected from Prunus serrulata Matsumura and Corydalis speciosa Max, and B. ardens bumblebees were primarily collected from Rhodldendron mucronulatum Turcz and Prunus yedoensis.

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.

Many bumblebee species have declined in number in recent decades, particularly in developing regions. Widespread declines of bumblebee species threaten the pollination levels of both wildflowers and crops. Here, we investigated the body weight and colony-developmental characteristics of Korean native bumblebee (B. ignitus) queens collected from 2000 to 2010 for conservation of native bumblebees for breeding. The average weight of 6,852 queens was 0.77 ± 0.44 g. The weight of B. ignitus queens collected in 2005 was the greatest, 0.87 ± 0.12 g, which was 1.0–1.3-fold heavier than any other year. The average oviposition rate was 81.6 ± 10.7%, and 2004 showed the highest rate, 95.0%. This value corresponded to 1.1-1.6-fold increases over the queens collected in the other years. The average rate of colony foundation was 60.9 ± 11.0%. Queens in 2008 exhibited the best performance, 75.4%, which was 1.0-1.9-fold higher than the other years. The rate of progeny-queen production averaged 27.0 ± 9.4% (Fig. 5) and peaked in 2001 at 43.2%; this value was 1.1–4.7-fold higher than other years. The average number of queens produced and number of generations begotten by queens was 27.6 ± 10.1% and 4.8 ± 2.0, respectively. Queens in 2000 averaged 9 generations of offspring, which was 1.1-3.1-fold greater than other years. These results indicate that the colony-developmental characteristics of the collected queens changed significantly between 2000 and 2010. In addition, there was no correlation between body weight and number of queens collected, although body weight was affected by collection year. Since 2008, the colony- developmental characteristics of queens have worsened.

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.

Bee venom contains serine proteases and serine protease inhibitors. In this study, we identified a bumblebee (Bombus ignitus) venom Kunitz-type serine protease inhibitor (Bi-KTI) that acts as a plasmin inhibitor. Bi-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tissue plasminogen activator. In contrast, Bi-KTI strongly inhibited plasmin, indicating that it acts as an antifibrinolytic agent. The fibrin(ogen)olytic activities of B. ignitus venom serine protease (Bi-VSP) and plasmin in the presence of Bi-KTI indicate that Bi-KTI targets plasmin more specifically than Bi-VSP. These findings demonstrate a novel mechanism by which bumblebee venom affects the hemostatic system through the antifibrinolytic activity of Bi-KTI and through Bi-VSP-mediated fibrin(ogen) olytic activities, raising interest in Bi-KTI and Bi-VSP as potential clinical agents.

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.

To increase the fruit quality and productivity of the apricot, 2 species of insects, Apis mellifer, and Bombus terrestris were used for pollination in apricot field in Yeongcheon province. This study was conducted using 3 kinds of methods for pollination effect, natural condition, net-house treatment and control method. Also this experiment carried out in 2 places, vinyl-house and field condition. In case of field treatment of Apis mellifera, heating lamp experiment was added in net-house to find out the reason for harmful efficacy of low temperature early in the spring. Species of apricot, Mangum, was used in this experiment. A number of foraging activity of Apis mellifera and Bombus terrestris in vinyl-house showed higher than in field condition. But after stoping heating, showed low efficacy after showing peak in 1 PM. In field, Apis mellifera showed peak of foraging activity from 1 to 3PM, heating lamp experiment of field condition showed peak in 3 PM and then lowest tendency to evening suddenly. The fruit set rate of Apis mellifera and Bombus terrestris in vinyl-house showed higher with 92% and 89% respectively than control with 83%. In field, heating lamp treatment of Apis melliferawas was highest with 28% compared to Bombus terrestris with 25% and control with 15%. This results suppose that apricot in field was to be harmed by low temperature and frost in blooming time early in spring

We cloned venom serine proteases from two bumblebee species, Bombus hypocrita sapporoensis and B. ardens ardens. We compared the predicted mature protein sequences of these serine proteasegenes to those previously reported from other bees. Using B. h. sapporoensis venom serine protease(Bs-VSP), we identify that Bs-VSP acts as a fibrin(ogen)olytic enzyme. Bs-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, as demonstrated for B. ignitus and B. terrestrisvenom serine proteases. Our results further define roles for bumblebee venom serine proteases as fibrin(ogen)olytic enzyme, providing strong evidence that bumblebee venom serine proteases are hemostatically active proteins that are potentially promising therapeutic agents.

We present evidence that the serine protease found in bumblebee (Bombus terrestris) venom exhibits fibrin(ogen)olytic activity. Compared to honeybee (Apis mellifera) venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that B. terrestris venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease, providing significant support for thepotential use of bumblebee venom serine protease as a clinical agent.

Bee venom is a rich source of pharmacologically active substances. In this study, we identified a bumblebee (Bombus ignitus) venom Kunitz-type serine protease inhibitor (Bi-KTI) that acts as a plasmin inhibitor. Bi-KTI showed no detectable inhibitory effect on factor Xa, thrombin, or tPA. However, it strongly inhibited plasmin, although this inhibitory ability was two-fold weaker than that of aprotinin. The activities of B. ignitusvenom serine protease (Bi-VSP) and plasmin in the presence of Bi-KTI indicate that Bi-KTI targets plasmin more specifically than Bi-VSP. These findings demonstrate a novel mechanism for bee venom by which Bi-KTI acts as an antifibrinolytic agent, raising interest in Bi-KTI as a potential clinical agent.

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.

Bee venom contains a variety of protein allergens, including serine proteases. Additionally, bee venom has been used in therapeutic application through immunotherapy for bee venom hypersensitivity and venom therapy as an alternative medicine. Here we present a novel view of the application of bee venom through which bee venom serine protease exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a larger amount of a serine protease as one of its major components. Immunologically, venom serine proteases from bumblebees did not show cross-reactivity with the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. 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. However, Bt-VSP did not activate plasminogen and the fibrinolytic activity of Bt-VSP is less than plasmin. These findings offer insight into the allergic reaction sequence of bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis.

Commercially managed bees are available for pollination services and are used in large commercial fields, small gardens, or enclosures such as greenhouses and screen houses. We investigated the use rate and number of commercial bees such as bumblebees, honeybees and mason bees for the pollination of 10 major horticultural crops and fruit trees in Korea. The use rates of bumblebees for 10 major horticultural crops and fruit trees were approximately 7.9% and 2.8% in 2009, respectively. The use numbers of bumblebees as pollinators was more than 64,345 colonies which included 51,400 for 10 major horticultural crops and 12,945 colonies for 10 major fruit trees, in 2009. The use rate of honeybees as a pollinator for 10 major horticultural crops in greenhouses and fruit trees were approximately 48.0% and 7.7%, respectively. The number of hives used for 10 horticultural crops and fruit trees was estimated to be 305,216 and 32,386, respectively. The number of honeybees hives used for pollination of 10 major horticultural crops and fruit trees was estimated to be 337,602. The use numbers of honeybees as pollinators was more than 337,602 hives in 10 major horticultural crops and fruit trees, in 2009. The number of honeybee hives used as pollinators of outside crops, including many fruits and vegetables, was estimated to be more than 500,000. The value of honeybees as pollinators was estimated to be more than 8.5% to 15.0% of total Korean beekeeping products. The rate of use of mason bees for 10 major fruit trees was approximately 3.3%. The number of mason bees individuals used for 10 major fruit trees was estimated to be 1,350,000. The value of commercial insect pollinators in 2009 in Korea was estimated at more than $45 million.

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.

Glutathione S-transferases (GSTs) are multifunctional enzymes that are mainlyinvolved in the xenobiotic metabolism and protection against oxidative damage. Most studies of GSTs in insects have been focused on their role in detoxifying exogenous compounds in particular insecticides. Here, we show the expression profiles of GSTs of the bumblebee Bombus ignitus in response to oxidative stress. We identified a sigma-class GST from B. ignitus (BiGSTS). The BiGSTSgene consists of 4 exons that encode 201 amino acids. Comparative analysis indicates that the predicted amino acid sequence of BiGSTS shares a high identity with the sigma-class GSTs of hymenopteran insects such as Apis mellifera (70% protein sequence identity) and Solenopsis invicta (59% protein sequence identity). Tissue distribution analyses showed the presence of BiGSTS in all tissues examined, including the fat body, midgut, muscle and epidermis. The oxidative stress responses analyzed by quantitative real-time PCR showed that under H2O2 overload, BiGSTS and BiGSTD (identified in our previous study) were upregulated in all tissues examined, including the fat body and midgut of B. ignitus worker bees. Under uniform conditions of H2O2 overload, the expression profile of GSTs and other antioxidant enzyme genes, such as phospholipid-hydroperoxide glutathione peroxidase (Bi-PHGPx) and peroxiredoxins (BiPrx1 and BiTPx1), showed that other antioxidant enzyme genes are acutely induced at 3 h after H2O2 exposure, whereas BiGSTS and BiGSTD are highly induced at 9 h after H2O2 exposure in the fat body of B. ignitus worker bees. These findings indicate that GSTs and other antioxidant enzyme genes in B. ignitusare differentially expressed in response to oxidative stress. Taken together, our findings indicate that BiGSTS and BiGSTD are oxidative stress-inducible antioxidant enzymes that may play a role in oxidative stress response.

We investigated nesting and foraging behavior of Korean mason bees, Osmia cornifrons Radoszkowski, during apple orchard pollination. When O. cornifrons was nesting, the oviposit and store pollens were taken an average 118.54±15.88 second and constructing cell partition was an average 109.33±62.09 second. At foraging, collecting pollens was timed at an average 699.89±81.87 second, and carrying mud was measured an average 464.88±151.17 second. Nesting and foraging behavior of O. cornifrons were influenced by weather conditions, especially, temperature, luminance and wind. When the luminance was more than 20,000lux and temperature was more than 20℃, O. cornifrons was more actively nesting and foraging. But, nesting and foraging of O. cornifrons were rapidly dropped when the wind blew at the speed more than 5m/s. The correlation analysis showed that foraging behavior is weak positive correlation with luminance, temperature and nesting. On the other hand, there is negative correlation between wind and the behavior of O. cornifrons. The most effective factor was luminance among the factors affecting the weather condition by regression analysis. Therefore, if weather conditions is low luminance, low temperature and strong wind, it will be better not to use O. cornifrons during apple pollination.

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.

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of the innate immune system that recognize peptidoglycan, a unique cell wall component of bacteria. Here we cloned and characterized PGRP-S from the bumblebee Bombus ignitus (BiPGRP-S). The BiPGRP-S gene consists of four exons encoding 194 amino acid residues. Comparative analysis indicates that the predicted amino acid sequence of BiPGRP-S shares high identity with enzymatically active PGRP-S proteins and contains the amino acids required for amidase activity. BiPGRP-S in B. ignitus worker bees is constitutively expressed in boththe fat body and epidermis, and it is secreted into the hemolymph. Quantitative real-time PCR assays revealed that in both the fat body and epidermis, the BiPGRP-S gene is highly induced by an injection of Bacillus thuringiensis. In addition, recombinant BiPGRP-S expressed as a 19-kDa protein in baculovirus-infected insect cells can bind to B. megaterium and B. thuringiensis but not to Staphylococcus aureus, Escherichia coli or Beauveria bassiana. Consistent with these data, BiPGRP-S shows antibacterial activity against B. megaterium and B. thuringiensis. These results indicate that BiPGRP-S is an inducible protein that may be involved in the immune response against bacterial infection of the genus Bacillus as an amidase-type PGRP-S.

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.