Bumblebee, Bombus ardens ardens (Apidae: Hymenopera), is an important resource for pollination that is most widely distributed in Korea. This study utilized microsatellite markers for investigation of genetic diversity and geographic relevance of the B. a. ardens populations in Korea. Through Next Generation Sequencing analysis, we identified 10 microsatellite markers and genotyped for 107 individuals of B. ardens collected from 10 populations. At each locus the number of alleles ranged from 10 to 23; the observed and expected heterozygosities ranged from 0.8909 to 1.0000 and 0.6641 to 0.8422, respectively; and inbreeding coefficient(FIS) ranged from –0.5053 to –0.0891. Significant deviation from the Hardy–Weinberg equilibrium was not observed at any locus. Population structure analysis indicated that there are three genetic groups in Korea with each Jeongseon and Ulleung-do composed of different gene pool from the remaining other populations. Similarly, Principal coordinates analysis also showed the same pattern. FST and RST analyses showed that each Jeongseon and Ulleung-do population had a significant genetic distance from other populations. Considering these results, genetic isolation of Ulleung-do may be explained by “Oceanic island” status and Jeongseon, which showed the positive FIS (0.069) and genetic isolation may be caused by its location on the east side of Baekdudaegan and by on-going inbreeding with a small population size.

Biological activities of bombolitins from Bombus ardens, B. consobrinus, B. terrestris and B. ussurensis (bombolitinsA, C, T and U, respectively) were examined using hemolytic, anti-microbial, anti-fungal and anti-tumor activity assays.Among the four bombolitins tested, bombolitin T showed the highest hemolytic and anti-tumor activities. All bombolitinsexhibited strong anti-microbial and anti-fungal activities, and bombolitin A specifically possessed the highest anti-microbialactivity against the Gram-negative bacteria Escherichia coli. Circular dichroism spectrometry analysis revealed that allfour bombolitins had over 61.7% and 45.5% of α-helicity in 30 mM sodium dodecyl sulfate and 50% trifluoroethanolbuffers, respectively, which form lipid-membrane-mimicking environments. Bombolitin T showed the lowest IC50 valuesof 8.5 μM and 8.8 μM against SK-OV-3 and NIH-OVCAR-3 cell lines, respectively, after 72 h of treatment, but itsrelative hemolytic activity at a concentration of 200 μM was 2.3-fold higher than that of 0.1% Triton X-100. Thisstudy provides new information on the biological and molecular properties of venom peptides of bumblebees. However,further studies on reducing cytotoxicity of bombolitins are needed for designing selective anti-tumor peptides.

Bombolitin is a venom peptide originally isolated from bumblebees and possesses various biological activities, including hemolytic activity. Bombolitins exhibit amphipathic α-helical structure in lipid-membrane-mimicking environments. To investigate their pharmacological and toxicological properties, anti-tumor, anti-microbial and cytotoxic activities of bombolitins from Bombus ardens and Bombus ussurensis were evaluated. Bombolitins of the two species exhibited extremely high anti-tumor activity against ovarian tumor cells SK-OV-3 and NIH; OVCAR-3 at 25-50 μM, which is 2-fold more potent than other wasp venom peptides studied to date (Yoon et al., 2015; Yoon et al., 2016). The two bombolitins showed significantly high antimicrobial activity. However, bombolotin of B. ussurensis showed no antimicrobial activity against Escherichia coli. In addition to their high levels of anti-tumor activity, bombolitins showed considerable levels of hemolytic activity. Thus, to utilize bombolitins as a potential candidate for anti-tumor peptide drugs, further studies for reducing cytotoxic properties of bombolitns is essential.

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 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.

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.

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.