Artificial insemination is a technique to transfer instrumentally sperm from the male into the female’s reproductive system. A key factor among artificial insemination techniques is gathering sperms. Here, a method for collecting sperm of bumblebee male by pressing was firstly developed. This method has stage of separating reprodutive tract of male, separating accessory testis, separating vas deferens, pressing vas deferens with cover-glass and collecting sperm with syringe. The developed method was 2.8 fold higher in rate of collecting sperm than that of existing method. Among 1M NaCl-, Insect ringer solution-, Phosphate buffered saline(PBS)-collecting sperm solutions, PBS was exhibited the best result as 17.2 sperms/cell, which corresponded to 1.6-1.8 fold increased over the result of other collecting sperm solution. Consequently, we think that this method is very important technique to save time for collecting sperm, to keep activity of sperm and to reduce contamination of sperm.

Bumblebees are important pollinators of crops and wildflowers. Bumblebees generally produce one generation per year. One of the key stages for year-round rearing of bumblebees is breaking diapause. To evaluate the effects of a combination method of CO2-narcosis and cold treatment to break the diapause of B. ignitus and B. terrestris queens, we determined whether this method affected their ability to establish a colony after the diapause break. The diapause treatment regimes that were utilized were CO2(CO2– narcosis), CT-1M(cold treatment at 5°C for 1 month), CT-1M-CO2(CO2–narcosis after cold treatment for 1 month), CT-2M-CO2(CO2–narcosis after cold treatment for 2 months), CT-2M (cold treatment for 2 months), CT-2.5M-CO2(CO2–narcosis after cold treatment for 2.5 months) and CT-2.5M (cold treatment at 5°C for 2.5 months). In view of the effects on the colony developmental characteristics of B. ignitus queens, the most favorable diapause treatment was CT-1M-CO2. A combination method of CO2– narcosis and cold temperature treatment yielded better results than that of single CO2–narcosis or cold temperature treatment on the colony development of diapause-broken B. ignitus queens. In the case of B. terrestris queens, we concluded that a combination method of CO2 and cold temperature treatment yielded better results than that of a single cold-temperature(up to2 months) treatment. In conclusion, the findings of the present study indicated that the combined application of CO2 and cold temperature was a favorable method for the colony development of diapause-broken B. ignitus and B. terrestris queens compared with only CO2–narcosisorcoldtemperaturetreatments. A combination method of CO2 and cold treatment reduced the side effect of CO2–narcosis and shortened the duration of cold treatment by at least 1month.

Bumblebees are important pollinators for wild flora and agricultural production. The introduction of bumblebees into greenhouses for pollination has become widespread in recent years and demand increases annually. Bumblebees give farmers the opportunity to decrease their pollination labor costs and offer a good crop yield, both in quantity and in quality. Colonies of the large bumblebee Bomus. terrestris have been imported into many countries. There has been some anxiety associated with the introduction of B. terrestris. Here, we investigated the distribution of founder bumblebee queens that were collected between 2000 and 2012 in Korea, and we show the interspecific hybridization between bumblebee species, B. terrestris and B. ignitus, under laboratory conditions. As results, 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%-1.1%). In indoor conditions, interspecific, cross-mated queens of B. ignitus or B. terrestris produced offspring but did not create a colony, and when a hybrid male B. terrestris copulated with a virgin queen of B. terrestris, the queen oviposited but did not initiate a nest. Further study of interspecies hybridization and its development will ultimately provide important information on the ecological impact to native bumblebees.

Recently, the flies are used to pollinate onion for seed production in Korea. However, the use of flies for pollination has caused complaints by local residents in onion seed production owing to sanitation issues and smell. Therefore, we studied the effects of pollination with bumblebee (Bombus terrestris) and flies(e.g., houseflies, flesh flies, blue bottle flies and blowflies) at small net(1.8m×2.3m×1.8m) in the greenhouse(66m2) for onion seed production. Although the number of flies was about 70 times than number of bumblebee workers, flies and bumblebees showed equivalent pollination efficiency. Therefore, pollination by B. terrestris can be used as an alternative approach in seed production of onion. An analysis of the most appropriate colony of B. terrestris for seed production of onion in small net showed that colony composed of fifty to eighty workers per small net was the most efficient level. When early maturing onion cultivars ‘Salad-5’ and ‘Yeonsinwhang’, and mid-late maturing onion cultivar ‘Colossus’ were pollinated by B. terrestris and files in small net, B. terrestris showed rather efficient pollination efficiency than flies or was equivalent to flies. In addition, difference among onion cultivars was affecting pollination effect by flies but was not affecting pollination effect by B.terrestris.

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.

Bumblebees are widely used to pollinate various greenhouse crops. Among the different bumblebee species, Bombus ignitus is indigenous to Korea, China, Japan and Russia. B. ignitus undergoes one generation per year, and artificial hibernation is essential for year-round rearing of the bumblebee. Keeping the queens under low-temperature conditions for several months is an effective method for terminating their diapause and promoting colony development. In the present study, we investigated how cold temperature affects the artificial hibernation of B. ignitus queens. Under chilling temperatures of -2.5°C, 0°C, 2.5°C and 5°C with constant humidity >80%, the queens stored at 2.5°C exhibited the highest survival rates, which were 74.0% at one month, 67.0% at two months, 60.0% at three months, 46.0% at 4 months, 33.0% at 5 months and 24.0% at 6 months. Lower survival rates were observed at 0°C, 5°C, 7.5°C and 12.5°C. At 2.5°C the colony developmental characteristics after diapause were 1.2- to 1.5-fold greater than those when queens were stored at 5°C. Thus, 2.5°C and 70% R.H. were the most favorable chilling temperature and humidity conditions for terminating the diapause of B. ignitus queens.

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.

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.

Insect-killing fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana ERL1170 induced melanization of yellow spotted longicorn beetles, Psacothea hilaris as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm, Spodopetra exigua larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment

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.

Insect-killing (entomopathogenic) fungi have high potential for controlling agriculturally harmful pests. However, their pathogenicity is slow and this is one reason for their poor acceptance as a fungal insecticide. The expression of bumblebee, Bombus ignitus, venom serine protease (VSP) by Beauveria bassiana (ERL1170) induced melanization of yellow spotted longicorn beetles (Psacothea hilaris) as an over-reactive immune response, and caused substantially earlier mortality in beet armyworm (Spodopetra exigua) larvae when compared to the wild type. No fungal outgrowth or sporulation was observed on the melanized insects, thus suggesting a self-restriction of the dispersal of the genetically modified fungus in the environment. The fungal transformant also shows mammal fibrinolytic activity, by which the transformant can be used pharmaceutically. The research is the first use of a multi-functional bumblebee VSP to significantly increase the speed of fungal pathogenicity, while minimizing the dispersal of the fungal transformant in the environment.

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.

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.

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.

The bumblebee is an important pollinator of various greenhouse crops, especially for tomatoes and there has been increasing interest in commercial use of the insects for pollination. Recent advances in commercial rearing of the European bumblebee (Bombus terrestris) made it possible to package bumblebee for crop pollination. Bumblebees are distributed world widely including alpine, cool temperate and even arctic environments of the northern continents. We chose B. ignitus out of seven Korean native bumblebees, because the species showed the best results both in artificial multiplication and in pollinating ability. Now, we are studying an artificial year-round mass rearing of B. ignitus selected as the most reliable native species in crop pollination. Therefore, we investigated the optimum temperature and humidity, effect of photoperiod and CO2-treatment, facilitating effects of helper, and artificial hibernation of B. ignitus to establish year-round mass rearing of B. ignius. The experimental regimes of temperature and humidity were defined as 23℃, 27℃ and 30℃ under a constant humidity of 65% R.H., and 50%, 65% and 80% R.H. under a constant temperature of 27℃, respectively. Among the temperature regimes, 27℃-rearing showed the best results, i.e., the rates of colony initiation, colony foundation and progeny-queen production at 27℃ were 83%, 63% and 46%, respectively, which corresponded to 2.2-5.5 times the respective values at other temperature regimes. The numbers of progeny produced at 27℃-rearing, 164±33 workers, 553±174 males and 33±48 queens were also higher, corresponding to 21.8 and 1.5 times those at 23℃ and 30℃, respectively. In terms of humidity, 65% R.H. was favorable for big colony formation. Under the same humidity, the rates of colony initiation, colony foundation and progeny-queen production were 85%, 70% and 50%, respectively, and the number of progenies reached 180±30 workers, 578±179 males and 35±38 queens. Therefore, 27℃ and 65% R.H. were determined to be the favorable environmental conditions for colony development of B. ignitus in indoor rearing. It was investigated whether developmental characteristics of foundation queens of B. ignitus collected in the 4 localities in Korea would be affected by the first oviposition days of them. The first ovipostion day was classified as 1 - 4 days (immediate early), 5 - 6 days (early), 7 - 10 days (delayed early), 11 - 20 days (medium), 21 - 40 days (late), and above 41days (very late). The queen that had the early first oviposition day, i.e., laid eggs so early after starting to be raised indoors, showed much higher rate of colony foundation and progeny-queen production and much shorter period of colony foundation and worker emergence. Besides, the numbers of worker and progeny-queen emerged from the queen that had the early first oviposition day were higher than those of the queen that had the late first oviposition. In results, the queen that had the early first oviposition day could make colony stronger and could make colony formation period shorter, therefore, the first oviposition day of foundation queen was proved to be a criterion for the selection of super colonies when B. ignitus is raised indoors. It was investigated whether or not such helpers as worker bee, bee-cocoon and egg-cup etc, have any effects on oviposition and colony foundation of the bumblebee queen, B. ignitus. Among the helpers tested, the callow workers of B. ignitus and B. terrestris showed the most remarkable effects on the oviposition rates to 92% and 88%, respectively. The live cocoon as a helper improved oviposition rate over 60%. A narcotized old worker 10 days-aged after emergence, showed similar effects to a callow worker on the colony development such as oviposition rate, colony foundation and progeny-queen production. On the other hand, dried cocoon, callow honeybee worker or egg-cup did not show a positive effect as a helper. In the number of workers recruited to a foundation queen, two workers showed better effect than one worker on the colony development, with no difference between two and more. The effect of photoperiodic regimes on the oviposition and colony development of B. ignitus queens was examined with 0L, 8L, and 16L under 2 7℃ and 65% R. H. Among these photoperiod regimes, the oviposition rate at 8L and 16L was 80.2% and 83.1%, respectively, which was 12-15% higher than that at the dark condition (0L). Duration up to first oviposition at 8L and 16L was 17.5 days and 16.5 days, respectively, which was 2-3 days shorter than that at 0L. The colony foundation rate at 8L and 16L was 9.2% and 10.4%, respectively, which corresponded to 1.7-2.0 fold the value at 0L. In addition, the rate of progeny-queen production at 8L and 16L was also two fold higher than that at 0L. Taken there together, the light conditions (8L and 16L) rather than dark condition (0L) were more suitable for oviposition and colony development for B. ignitus in the indoor rearing condition. We investigated mating conditions of photoperiod, illumination and temperature during mating periods, care temperature of queen before mating, mating period and number of queen per mating cage to improve mating rate of B. ignitus. Among photoperiodic regimes of 12L, 14L and 16L during mating periods, queen mated at 14L showed better results than at 12L and 16L in egg-laying characteristics and colony development. In case of illumination during mating periods, intensity of 1000 lux was more effective than at intensity of 100 lux and 2000 lux in mating B. ignitus queen. Mating temperature and care temperature of queen before mating favorable for B. ignitus queen were 22-25? and 19?, respectively. The period need to mating B. ignitus queen was 3 days, and the number of queen suitable per mating cage of 55× 45× 65 ㎝ was 30. The effect of CO2-treatment on interrupting diapause of B. ignitus was examined to provide a means for year-round rearing of the bumblebee. When mated young queens were exposed to 65% or 99% CO2 for 30 min daily during two consecutive days, oviposition rate increased to 75% and 77%, respectively, comparing 50% in CO2-untreated queens. At the same time, the days needed to first oviposition shortened to 17-18 days in CO2-treated queens, comparing to 30 days in CO2-untreated queens. CO2-treatment at the second day after mating was appropriate to the oviposition and colony development. CO2-treatment showed a positive effect on the oviposition and colony development, but less than them of over-wintered queen in numbers of produced progeny. It can be concluded that CO2-treatment to B. ignitus is insufficient to produce commercial grade bumblebee colony in spite of its capability for promoting oviposition, because the treatment failed to form a big colony. Artificial hibernation is essential for year-round rearing of the bumblebee, B. ignitus that undergoes one generation per year. It is known that keeping the queens in low temperature for two or three months is effective to terminate their diapause and develop the colony. Temperature, time and surroundings to keep the queens during artificial hibernation were investigated. Among the tested temperatures, -2.5°C, 0°C, 2.5°C, and 5°C, the optimum temperature was 2.5°C. At the temperature (2.5°C), survival rate after chilling of the queens was high and colony development thereafter was enhanced. The proper time to initiate chilling queen was 10 to 14 days after adult eclosion, and the survivability of the queens after chilling was good during the upper period. For the surrounding to keep the queen during artificial hibernation, we proposed the method to preserve them in a bottle filled with perlite and keep it around 80% R. H.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is one of the most notably utilized for greenhouse pollination in Korea. In order to understand the nature of genetic relationships, gene flow, and population structure of the species we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to “animal barcode” region and the complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (nrDNA) collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.61% (4 bp), the ITS sequences provided 84 sequence types with the maximum sequence divergence of 1.02% (21 sites), confirming better applicability of the ITS sequences to the study of intraspecific variation. The complete ITS2 sequences of B. ignitus were shown to be longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated repeats. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population/ population group were noted to exist among populations of B. ignitus on the basis of both molecules, thus suggesting that the B. ignitus populations on the Korean peninsula are panmictic, which is consistent with our understanding of the dispersal capability of the species