Honeybee (Apis mellifera) egg-yolk protein vitellogenin (Vg) plays roles in immunity, antioxidation, and life span beyond reproduction, but it also acts as an allergen Api m 12 in venom. Here we established antimicrobial and antioxidant roles of honeybee Vg in the body and venom. Using the cDNA encoding Vg identified from Asiatic honeybee (A. cerana) workers, recombinant A. cerana Vg (AcVg) protein of approximately 180 kDa was produced in baculovirus-infected insect cells. In A. cerana worker bees, AcVg was expressed in the fat body and venom gland and was present in the secreted venom. AcVg induced structural damage in microbial cell walls via binding to microbial surfaces and exhibited antimicrobial activity against bacteria and fungi. AcVg protected mammalian and insect cells against oxidative damage through direct shielding of cell membranes. Interestingly, AcVg exhibited DNA protection activity against reactive oxygen species (ROS). Furthermore, the transcript level of AcVg was upregulated in the fat body, but not in the venom gland, of worker bees with antimicrobial peptides and antioxidant enzymes in response to microbial infection and oxidative stress. Our data indicate that AcVg is involved in innate immunity upon infection and in a defense system against ROS, supporting a crucial role of honeybee Vg as an antimicrobial and antioxidant agent in the body and venom.

Several Mites are currently the most serious threat to the world bee industry. The ectoparasitic honey bee mites was originally confined to the Asian honey bee(Apis cerana etc.). Varroa destructor and Tropilaelaps clareae has plagued European honey bees, Apis mellifera. Differences in mite tolerance are reported between two honey bee species A. mellifera and A. cerana. We were amplified antimicrobial peptide cDNA genes (Defencin, Abaecin, Royalisin, Apidaecin and Hymenoptaecin) by RT-PCR. We explored the transcriptional response to mite parasitism in A. mellifera 4th instars larvae which differ in susceptibility to V. destructor and T. clareae, comparing parasitized and non-parasitized 4th instars larvae (worker and Drone) from same hive. Differential gene expression of worker bees and Drone bees induced by mites (V. destructor and T. clareae) infection was investigated by northern blot. Mites (V. destructor and T. clareae) parasitism caused changes in the expression of genes related to sex distinction. Bees tolerant to mites (V. destructor and T. clareae) were mainly characterized by differences in the expression of genes regulating antimicrobial gene expression. It provides a first step toward better understanding molecular expression involved in this differential sex distinction host-parasite relationship. We were detected bee virus in A. mellifera, comparing parasitized and non-parasitized 4th instars larvae (worker and Drone). Therefore, this result was demonstrated that mites were another possible route of horizontal transmission, as several viruses were detected in mites and their hosts.

South Korea has over 38 millions of managed honey bee (Apis cerana) colonies before 2009 years ago, which produce the highest quantity of honey in the Korea; however, almost colony (99%) were collapsed by Korean Sacbrood Virus (KSBV) in South Korea. Korean Sacbrood Virus (KSBV) is the pathogen of A. cerana Sacbrood disease, which poses a serious threat to honeybee A. cerana, and tends to cause bee colony and even the whole apiary collapse. Colony collapse of A. cerana was first reported on the Pyeong-Chang of the South Korea in 2009. Symptoms of KSBV include the rapid transmission of larval stage honeybees (A. cerana), many dead larvae found in the bottom of hive and comb. Honeybees (A. cerana) are a very important species because they provide a number of pollination services for various ecosystems in some provinces (ex. jeon-nam, jeon-buk province). They are also extremely important organisms within human society, both agriculturally and economically. The fact that a direct cause has been determined suggests that colony collapse is a complex problem with a combination of natural and anthropogenic factors. Possible instigators of colony collapse include: wax moth, viral and fungal diseases, increased population, decreased genetic diversity, climate changing and a variety of other factors. The interaction among these potential causes may be resulting in immunity loss for honeybees and the increased likelihood of collapse.

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 traditional use of insects as food continues to be widespread in tropical and subtropical countries and to provide significant nutritional, economic and ecological benefits for rural communities. Specially, Bee brood serves as a food source to humans in many countries although limited data exists concerning its nutrient composition. Bee brood (pupa and larvae) were analyzed for Carbohydrate, Saturated fatty acid, Cholesterol, protein, fat, fiber, minerals, and vitamins. Bee brood was high in protein(46.4%～46.73%), fat(18.84%～ 20.75%),carbohydrate(24.66 %～35.79 %), Folic acid(222.30 ㎍/100g), and vitamins. Differentially, folic acid had been contained by high density in pupa of drone. While low in iron, bee brood was a good source of folic acid, and carbohydrate. The fat was composed mostly of saturated and mono-unsaturated fatty acids. The present data suggest bee brood to be an excellent source of many valuable nutrients including energy, amino acids, many essential minerals, and B-vitamins. These data suggest bee brood could be a valuable source of nutrients to various populations.

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.

Sacbrood virus(SBV) causes a fatal disease(sacbrood) of honeybee larvae, which fail to pupate, change color and shape, and finally die. The complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a Reverse Transcription-PCR(RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. To detect the SBV infection in Korea, we collect beekeepers from various apiaries, which the RT-PCR technique was used. And we designed SBV specific primers in conserved region of the viral genome in the GenBank database. We confirmed the SBV amplicon using cloning and sequence. Homology between determined sequences of SBV korean strain and published virus sequences were 97% in DNA sequence, and 100% in amino acid sequence. We describe the first time that presence of sacbrood virus(SBV) in Korea honey bee colonies using RT-PCR. We also developed and validated a RT-PCR assay for the detection of SBV in Korea.

Viruses of the honeybee, Apis mellifera L. are known to reside at low levels in colonies, typically showing no apparent signs of infection. Chronic paralysis virus(CBPV) is known to induce significant losses in honey bee colonies. The pathology is characterized by clusters of trembling, flightless, crawling bees and by individual bees, sometimes hairless, standing at the hive entrance. A minusstrand-specific RT-PCR was used to assess viral replication. This is the first report on the infection of CBPV in Korea. Using (-)RT-PCR, 27 apiaries in korea were screened for the honeybee viruses, with positive colonies being analysed for viral genetic diversity. We got 550-nt PCR product from CBPV genomic RNA. Nucleotide sequences were aligned to the complete CBPV genomic RNA sequence deposited in the GenBank database and was revealed 96%(AM-CBPV) identity, respectively. Sequence comparison with other CBPV and honeybee virus.

Mortality of honeybees is a serious problem that beekeepers have to face periodically in Korea and worldwide. The presence of RNA viruses, in addition to other pathogens may be one of its possible causes. In this work, we detected Deformed wing virus(DWV), Israle Acute Paralysis Virus (IAPV), Black queen cell virus (BQCV), Cloudy wing virus(CWV), Kashmir bee virus(KBV), Sacbrood virus(SBV), Chronic bee paralysis virus(CBPV) in samples of korea honeybees with or without Varroa destructor and Nosema apis. The detection of viruses in all provinces, simultaneous co-infection of colonies by several viruses and the fact that 96.3% of the samples were infected with one or more virus, indicates they are widely spread in the region. Using uniplex and multiplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including DWV, IAPV, BQCV, KBV, CWV, and described the detection of mixed virus infections in bees from these colonies. Conclusively, investigated disease of the bee, and confirmed new virus that lead to bee disease, this is thought by valuable thing as data for development of beekeeping industry such as CCD(Colony Collapse Disorder)'s cause searching examination.