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.

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.

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.

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.

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.

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.

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.

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

Bumblebees are widely used to pollinate crops in greenhouses and fields. Here we firstly developed an apparatus for the oviposition induction of the bumblebee Bombus ignitus using electricity. The apparatus consists of boxes for colony initiation, part of temperature control, part of heat transfer, and moving shelf. The result shows that the rates of oviposition and colony foundation in the newly developed apparatus are respectively 3.9% and 5.2% higher than in the existing apparatus using hot water. More importantly, the newly developed apparatus is 75% cheaper in costs and can more save energy than existing apparatus. These results indicate that the newly developed apparatus could serve as an effective apparatus for the oviposition induction of B. ignitus.

Deformed wing virus (DWV) of honeybees (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. Here, we report for the first time the occurrence of DWV-infected bumble bees (Bombus terrestris). For the present study, the detection of DWV virus from the adult bumble bee, death adult bumble bee, mail bumble bee, pupa and larva to the infection cycle was investigated in the same colony. Using specific RT-PCR protocols for the detection of DWV followed by sequencing of the PCR products we could demonstrate that the bumble bees were indeed infected with DWV. The virus was detected from diseased insects, and its partial DWV gene was cloned and sequenced. The partial DWV gene encoding the polyprotein is 711-nt of 235 amino acid residues. The deduced nucleotide sequence of the polyprotein partial gene of DWV showed 96.9%, 96.2%, 96.8%, and 96.5% homology to other structure polyprotein partial gene of DWV, respectively. Phylogenetic analysis further conformed that the deduced nucleotide sequence of the polyprotein partial gene of DWV divided to the outside tree. We describe the first time that presence of Deformed wing virus(DWV) from bumble bee(Bombus terrestris) in korea using RT-PCR.

We report for the first time the occurrence of DWV-infected bumble bees (Bombus ignitus). For the present study, the detection of DWV virus from the female and male bumble bee was investigated in the same colony. The Deformed wing virus (DWV) of honeybee (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. Using specific RT-PCR protocols for the detection of DWV followed by sequencing of the PCR products we could demonstrate that the bumble bees were indeed infected with DWV. The virus was detected from Bombus ignitus, and its partial DWV gene was cloned and sequenced. The partial DWV gene encoding the polyprotein is 711-nt of 235 amino acid residues. The deduced nucleotide sequence of the polyprotein partial gene of DWV showed 96.9%, 96.2%, 96.8%, and 96.5% homology to other structure polyprotein partial gene of DWV from insects, respectively. Phylogenetic analysis further conformed that the deduced nucleotide sequence of the polyprotein partial gene of DWV divided to the outside tree. We describe the first time that presence of Deformed wing virus(DWV) from bumble bee(Bombus terrestris) in korea using RT-PCR.

Phospholipid-hydroperoxide glutathione peroxide (PHGPx) is an antioxidant enzyme that reduces lipid hydroperoxides in biomembranes. Here, we cloned and characterized cys-PHGPx from the bumblebee Bombus ignitus (Bi-PHGPx). The Bi-PHGPx gene consists of 4 exons, encoding 168 amino acid residues with a canonical cys-codon at residue 45 and active site residues Gln82 and Trp134. Recombinant Bi-PHGPx, expressed as a 19 kDa protein in baculovirus-infected insect cells, exhibited enzymatic activity against PLPC-OOH and H2O2 using glutathione as an electron donor. Tissue distribution analyses showed the presence of Bi-PHGPx in all tissues examined. Bi-PHGPx transcripts were upregulated by stresses, such as wounding, H2O2 exposure, external temperature shock, and starvation. Under H2O2 overload, the RNA interference (RNAi)-induced thioredoxin peroxidase (BiTPx1)-knock-down B. ignitus worker bees showed upregulated expression of Bi-PHGPx in the fat body. These results indicate that Bi-PHGPx is a stress-inducible antioxidant enzyme that acts on phospholipid hydroperoxide and H2O2.

Bee venom contains a variety of toxic enzymes and peptides. One of the major components of bumblebee venom is bombolitin, which is the most abundant venom constituent and biologically similar to melittin. Here, we first show the molecular cloning and antimicrobial activity of the venom bombolitin from the bumblebee Bombus ignitus. The B. ignitus venom bombolitin gene consists of 2 exons, encoding 56 amino acid residues. The bombolitin purified from B. ignitus venom was the 2104 Da mature peptide with 18 amino acid residues, which are created by cleavage of the probombolitin domain between Ala38 and Leu39. We examined the pattern of bombolitin expression to confirm that it is a component of bumblebee venom. B. igniutus venom bombolitin exhibits venom gland-specific expression. We also investigated the venom bombolitin for antimicrobial properties against bacteria and fungi. The venom bombolitin showed high antibacterial activity against both Gram-negative and Gram-positive bacteria. Most interestingly, the venom bombolitin showed high antifungal activity against Fulvia falva, a leaf mold, and Alternaria radicia, a black rot. These antimicrobial profiles of B. ignitus venom bombolitin reported herein will be useful in the application for potential antimicrobial agents.

We cloned and characterized two peroxiredoxins (Prxs), BiPrx1 (a 1-Cys Prx) and BiTPx1 (a 2-Cys Prx) from the bumblebee Bombus ignitus. The BiPrx1 gene consists of 5 exons, encoding 220 amino acid residues with one conserved cysteine residue. The BiTPx1 gene consists of three exons, encoding 195 amino acid residues with 2 conserved cysteine residues. Recombinant BiPrx1 (27 kDa) and BiTPx1 (25 kDa), expressed in baculovirus-infected insect Sf9 cells, reduced H2O2 in the presence of electrons donated by dithiothreitol. Unlike BiTPx1, however, BiPrx1 did not show reduction activity when thioredoxin was used as the electron donor. Both BiPrx1 and BiTPx1 protected super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Tissue distribution analyses showed the presence of BiPrx1 and BiTPx1 in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting that BiPrx1 and BiTPx1 are not secretable. When H2O2 was injected into B. ignitus bees, BiPrx1 and BiTPx1 transcripts were acutely up-regulated in the fat body tissues. We also demonstrated regulation of BiPrx1 and BiTPx1 expression via reduction of transcript levels in the fat body with RNA interference (RNAi). Under H2O2 overload, the RNAi-induced BiPrx1 knock-down B. ignitus worker bees showed up-regulated expression of BiTPx1. Reciprocally, BiTPx1 RNAi knockdowns showed up-regulated BiPrx1 expression in the fat body. These results indicate that loss of expression of BiPrx1 or BiTPx1 is compensated by up-regulation of expression of the other peroxidase in response to H2O2 overload.