We investigated the mating age for sexual maturity and sperm quality of the bumblebee Bombus terrestris queens and males. In the mating age of sexual maturity of queen, mating rate was 6.7% at just emergence, 85.0% at 10 days of emergence, and decreased thereafter. In case of mating age of sexual maturity of male, mating rate was 38.3% at just emergence and 62.5% to 75.0% at 7 days to 20 days of emergence. The colony development at aging of B. terrestris queen and male was a similar tendency to the mating age of sexual maturity. In case of multiple mating, B. terrestris male was mated by 4 times, which was 74.3% for one time, 50.0% for two times, 22.9% for three times and 8.6% for four times. The number of spermatozoa was increased as the age of male was older until 25days after emergence. The number of spermatozoa of non-mated males of one day and 3 days after emergence was higher at 1.2 and 1.7 times than that of mated males and 18.9 and 36.6 times than that in spermatheca of mated queen. Our results indicate that period favorable for artificial insemination of B. terrestris was from 6 days after emergence for queen and 7 days after emergence for male.

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.

Among bee venom proteins, phospholipase A2 (PLA2) is critical one of bee venom components to defend against predators intruders. In this study, PLA2 gene from cDNA libarary using the venom glands of Bombus ignitus worker bees(BiVn-PLA2) was cloned and characterized. BiVn-PLA2 spans 2211 bp and consists of three introns and four exons encoding 180 amino acid residues. BiVn-PLA2 shares high levels of identity with a bumblebee, B. terristris (89% protein sequence identity), B. pennsylvanicus (88%), and a honey bee, Apis mellifera (53%). Northern blot analysis revealed that BiVn-PLA2 is expressed in venom gland, indicating that BiVn-PLA2 is one of the venom components of B. ignitus. To determine BiVn-PLA2 of venom components from venom sac, N-terminal amino acid sequencing of a putative BiVn-PLA2 (the purified 18 kDa) was performed by Edman degradation. The N-terminal amino acid sequencing of the 18 kDa protein was coincident with the N-terminal amino acid residues of the mature BiVn-PLA2 and the 18 kDa protein catalysed the hydrolysis of DBPC subs trate[1-O-(6-Dabcyl-aminohexanoyl)-2-O-(12-(5-B ODIPY-entanoyl) aminododecanoyl)-sn-glyceryl phosphatidylcholine] that is a sensitive fluorogenic probe for PLA2 activation. Western blot analysis revealed that BiVn-PLA2 is expressed in the venom gland, stored in the venom sac, and then emitted throughout sting apparatus. Finally, to test BiVn-PLA2 toxicity, BiVn-PLA2 was adjusted to a insect cell (Sf9) at different concentrations (1-30 μg/2×105 cells). The apoptotic cell death assay results showed that the cell survival decreased with increasing concentrations (1-30 μg/2×105 cells).

vitellogenin cDNA was cloned from the bumblebee Bombus ignitus. The cDNA encoding B. ignitus vitellogenin (BiVg) is 5473 bases long with an open reading frame of 1773 amino acid residues. BiVg possesses two consensus (RXXR/S) cleavage sites and has the conserved DGXR and GL/ICG motif near its C-terminus. The deduced amino acid sequence of BiVg cDNA showed significant similarity with hymenopteran Vgs (51% identity to Apis mellifera Vg, and 33-36% to other insect Vgs). The BiVg cDNA was expressed as a 200-kDa polypeptide in baculovirus-infected insect Sf9 cells. Northern and Western blot analyses showed the expression of BiVg in fat bodies of pupae and adults. In queens, the expression of BiVg was first detected in late pupal stage fat bodies, and secreted BiVg was also observed in late pupal stage hemolymph.

The bumblebees, Bombus species are valuable natural resources being utilized for greenhouse pollination. Low level of genetic variation of Bombus species has been reported previously. In this study, we sequenced complete internal transcribed spacer 2 (ITS2) of the nuclear rDNA from 100 individuals of B. ardens collected from seven localities in Korean peninsula. The ITS2 sequence of B. ardens is longest, ranging in size from 1,940 bp-1,954 bp among known in insects, which ranges approximately from 241 bp-1,728 bp. The ITS2 sequences have ~51% of C+C content and contain each two 27 bp repeats, 20 bp repeats, 33 bp repeats, and 34 bp repeats at the beginning. Such repeats were not found in other insects. Uncorrected pairwise distance among 96 sequences that were obtained from 100 individuals revealed a maximum sequence divergence of 1.03%. Genetic diversity (π) of each population ranged from 0.007801 to 0.009627, and the lowest diversity was obtained from islet population of Ulleungdo, indicating possibly small, isolation of the population. Significant level of genetic distance was only found when Ulleungdo population was compared to two other mainland populations. Except for this, overall, a very high rate of per generation migration ratio (Nm=7.1-infinite) and a very low level of genetic fixation (FST=0-0.06546) were detected between pairs of localities. Analysis of hierarchical relationships among localities consistently revealed no statistically significant structure among populations. Taken these together, the B. ardens populations on the Korean peninsula are panmictic this is consistent with our understanding of the dispersal capability.

The bumblebee, Bombus ardens, is a valuable natural resource, and is most notably utilized for greenhouse pollination. In order to gain a greater understanding of the population genetic structure and the genetic diversity of this species, we sequenced a portion of the mitochondrial COI gene corresponding to the “DNA Barcode” region (658 bp) from 160 individuals collected over 15 Korean localities. Uncorrected pairwise distances among the eight haplotypes suggested low intraspecific genetic diversity, with a maximum sequence divergence of 0.3%. Such a low level of intraspecific genetic diversity was further reflected in local populations, particularly to islet populations, such as those of Youngheungdo, Jakyakdo, and Ulleugdo, which had zero genetic diversity. Geographically, one haplotype (BARBA01) was widespread and dominant, with a frequency of 90.6% (145 among 160 individuals). Other haplotypes were restricted to one to three localities and had low frequency. Overall, a very high rate of per generation female migration ratio (Nm= 4.6 to infinite) and a very low level of genetic fixation (FST= 0 to 0.099) were detected between pairs of localities, suggesting that the B. ardens population sonthe Korean peninsula are panmictic, which is consistent with our understanding of their dispersal capability.

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.

Transferrin and ferritin are iron-binding proteins involved in transport and storage of iron as part of iron metabolism. Here, we describe the cDNA cloning and characterization of transferrin (Bi-Tf) and the ferritin heavy chain subunit (Bi-FerHCH), from the bumblebee Bombus ignitus. Bi-Tf cDNA spans 2,340 bp and encodes a protein of 706 amino acids and Bi-FerHCH cDNA spans 1,393 bp and encodes a protein of 217 amino acids. Comparative analysis revealed that Bi-Tf appears to have residues comprising iron-binding sites in the N-terminal lobe, and Bi-FerHCH contains a 5’UTR iron-responsive element and seven conserved amino acid residues associated with a ferroxidase center. The Bi-Tf and Bi-FerHCH cDNAs were expressed as 79 kDa and 27 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot analysis revealed that Bi-Tf exhibits fat body-specific expression and Bi-FerHCH shows ubiquitous expression. The expression profiles of the Bi-Tf and Bi-FerHCH in the fat body of B. ignitus worker bees revealed that Bi-Tf and Bi-FerHCH are differentially induced in a time-dependent manner in a single insect by wounding, bacterial challenge, and iron overload.

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.

To understand geographic genetic variation of the species and relationships among populations of the bumble bee, Bombus ardens, that is utilized as green house pollinator we expanded our investigation by sequencing somewhat longer mitochondrial DNA (mtDNA) fragment, covering some uninvestigated regions within the species distribution, and analyzing the sequence data in terms of population genetic structure. For the purpose of study, a portion of mitochondrial COI gene, corresponding to "DNA Barcode" region (658 bp) was sequenced from 160 individuals of B. ardens collected over 15 localities in Korea. The sequence data revealed overall relatively low genetic diversity within species, with a maximum sequence divergence of 0.3%. Geographically, one haplotype (BARBA01) was found in all localities surveyed, with the frequency of 91% (145 among 160 individuals), whereas other haplotypes were found in a locality mostly as a single individual, suggesting that haplotype distribution can be summarized as coexistence of widespread, one dominant haplotype and regionally restricted, other haplotypes. Overall, very high rate of per generation female migration (Nm = 4.6 ~ infinite) and very low level of geographic substitution (FST = 0 ~ 0.099) among localities were characteristic. Although some populations were genetically subdivided from the remaining localities in the hierarchical analysis, there was regional polarity on this subdivision. Taken together with gene flow estimates, the nature of genetic divergence of the bumble bee populations is characterized as one that possessing low genetic diversity, high gene flow, and wide spread of one dominant haplotype, consistent with the previous finding. To have further detailed information of this valuable genetic resource, further longer and variable molecular portion is under investigating.

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.

A thioredoxin peroxidase (TPx) gene was cloned from the bumblebee, Bumbus ignitus. The B. ignitus TPx (BiTPx) contains an open reading frame of 585 bp encoding 195 amino acid residues and possesses two cysteine residues that are characteristic of 2-Cys subgroup of peroxiredoxin family. The deduced amino acid sequence of the BiTPx cDNA showed 90% identity to Apis melifera (AmTPx-1), 80% to Aedes aegypti (AaTPx), and 78% - 47% to other insect 2-Cys TPx. Northern blot analysis revealed the presence of BiTPx transcripts in all tissues examined. Western blot analysis showed the presence of the BiTPx in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting the BiTPx is not secretable. The cDNA encoding BiTPx was expressed as a 27-kDa polypeptide in baculovirus-infected insect Sf9 cells. The purified recombinant BiTPx was shown to reduce H2O2 in the presence of electrons donated by dithiothreitol and shown to be active in the presence of thioredoxin as electron donor.

Bumblebees are important pollinators in greenhouse and have colonized all parts of the World. In Korea, the value of bumblebees is increasing as pollinator. However, the more recent use of reared colonies may ultimately allow pathogens to spread to peripheral areas for bumblebees. Generally, bumblebees are hosts to a large number of parasites which are mites, flies, protozoa, fungi, virus and bacteria. For most of these, very little is known about their effects against host insect, epidemiology or evolutionary ecology. So, we report pathogenic bacteria isolated from Bombus terrestris and B. ignitus at first time in Korea. Bacillus fusiformis and Klebsiella oxytoca are isolated from B. terrestris and confirmed with 16S rRNA gene nucleotide comparison in NCBI genebank. Pathogenicity of B. fusiformis was 35~40% against B. terrestris. Pantoea dispersa and K. oxytoca are isolated from B. ignitus and confirmed with 16S rRNA gene nucleotide comparison in NCBI genebank. Pathogenicity of these species were 35~40% against B. ignitus. These pathogenicity are considered as low-level.

To understand geographic genetic variation of the species and relationships among populations of the bumble bee, Bombus ardens, we sequenced a portion of mitochondrial COI gene, which corresponds to "DNA Barcode" region (658 bp) from 101 B. ardens individuals collected over 11 localities in Korea. The sequence data were used to investigate genetic diversity within populations and species, geographic variation within species, phylogeographic relationship among populations, and phylogenetic relationship among haplotypes. A total of nine haplortpes were found, but they very close to each other (a maximum sequence divergence of 0. 304%). Summarized, overall moderate to low genetic diversity within populations and species was characteristic, concordant with the high potential to disperse of B. ardens in Korea. There was no clear regional subdivision was observed and relatively high rate of gene flow among localities and low FST was characteristic.

The large bumblebee, Bombus terrestris, which is indigenous to Europe and is used extensively for high-value crop pollination, has been artificially introduced throughout the world. Here we show the interspecific cross-mating between bumblebee species, B. terrestris and B. ignitus under laboratory conditions. The mating and oviposition rates of interspecific cross-mating of B. terrestris queen with B. ignitus male are higher than those of intraspecific matings of B. ignitus. Furthermore, the competitive copulation experiment indicates that cross-mating of a B. ignitus male with B. terrestris queen is 1.8-fold more frequent than with a B. ignitus queen. The cross-mated B. ignitus queen with B. terrestris male produced B. ignitus workers and B. ignitus male offspring, and the cross-mated B. terrestris queen with B. ignitus male produced both B. terrestris males and B. ignitus male offspring. The genetic tests using a portion of the mitochondrial COI gene for the parent and hybrid offspring indicate that mitochondrial DNA was maternally inherited. Our results indicate that interspecific cross-mating occurred between B. ignitus and B. terrestris, which suggests that the cross-mating is not an exclusive force against the negative impact of competition and genetic contamination on native bumblebees.

We compared colony development and survival rate after artificial hibernation between the bumblebees, Bombus terrestris, produced from domestic and foreign companies. The number of worker and queen produced from foundation colony of three domestic and one foreign companies was 232.4-270.8 and 62.0-181.8, respectively, but there was no statistical difference. in colony development of B. terrestris queens which were terminated diapause by CO₂ narcosis at different companies, oviposition rate, colony foundation and progeny-queen production of bumblebees produced from domestic companies were superior to those from foreign company. But foreign company in the number of worker and queen was superior to domestic companies. On the other hand, survival rate and colony development of B. terrestris queens, which were terminated diapause by artificial hibernation at three domestic and two foreign companies, were no significant differences. These results indicate that oviposition, colony foundation, progeny-queen production, and survival rate after artificial hibernation showed no differences between the bumblebees produced from domestic and foreign companies.

The pollinating activity of bumblebee, Bombus ignitus was investigated at the blossom of peach [Prunus persica (L.) Batsch] planted in vinyl house. The major time of foraging activity by B. ignitus was between 08:00 and 7:00, and the peak time of pollinating activity by B. ignitus was 11:00 in the peach vinyl house from February 28 to March 5 in 2002. The rate of fruit set and the rate of marketable fruit by foraging activity of B. ignitus were much better than artificial pollination, and the effect of labor reduction by the pollinating activities of B. ignitus released against the artificial pollination was took remarkable profits over 93%. Therefore, the foraging activity of B. ignitus in peach vinyl house brought satisfactory results among the rate of fruit set, the rate of marketable fruit and the effect of labor reduction against the artificial pollination.

Pure Bombus ignitus venom samples were submitted to two-dimensional gel electrophoresis. A total of 64 excised spots were analyzed by mass spectrometry. Three main proteins resulted in the identification have not been described in other bee venoms before. Dose-dependence against human carcinoma (Hep3B, BT-20, A549 and AGS) were observed from 1 ng/㎖ to 100 ng/㎖. Expecially, the treatment of 100 ng/㎖ B. ignitus venoms showed the highest cytotoxicity with 55% against hepatocellular carcinoma (Hep3B). The B. ignitus venoms showed strong antimicrobial activities against Enterococcus faecium and Shigella sonnei, and practically antimicrobial activity against the other microorganisms tested. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of E. faecium and S. sonnei, were 0.256 ug/㎖, respectively.