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.

We describe here the cloning and characterization of a cDNA encoding the ferritin heavy chain homologue (TeFerHCH) from the cricket Teleogryllus emma. The TeFerHCH gene spans 1,009 bp and consisted of four introns and five exons coding for 217 amino acids residues. The TeFerHCH subunit contained the conserved motifs for the ferroxidase center typical of vertebrate ferritin heavy chains and the iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5'-untranslated region (UTR) of TeFerHCH mRNA. TeFerHCH was grouped with the S type (HCH) in a phylogenetic tree. The TeFerHCH cDNA was expressed as approximately 27 kDa polypeptide in baculovirus-infected insect Sf9 cells. Northern blot analysis revealed that TeFerHCH exhibited ubiquitous expression and was upregulated by wounding and iron overload in the fatbody, suggesting a functional role for TeFerHCH in iron metabolism.

Background: Proteolytic enzymes are involved in insect molting and metamorphosis and play a vital role in the programmed cell death of obsolete organs. Here we show the expression profile of cathepsin B in the fat body of the silkworm Bombyx mori during development. We also compared the expression profile of B. mori cathepsins B (BmCatB) and D (BmCatD) in the fat body during the larval-pupal transformation of B. mori in the BmCatB or BmCatD RNA interference (RNAi) process. Results: BmCatB is ecdysone-induced and expressed in the fat body of B. mori during the molting, and the larval-pupal and pupal-adult transformations, and its expression leads to programmed cell death. In particular, BmCatB is highly expressed in the fat body of B. mori during the larval-pupal transformation and BmCatB RNAi treatment resulted in the arrest of the larval-pupal transformation. RNAi-treated BmCatB knock-down sustained the expression of BmCatD during the larval-pupal transformation. On the other hand, BmCatD RNAi up-regulated the expression of BmCatB in the fat body of final instar larvae. Conclusion: Based on these results, we conclude that BmCatB is involved in the programmed cell death of the fat body during B. mori metamorphosis and that BmCatB and BmCatD contribute collaboratively to B. mori metamorphosis

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).

Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. This strain exhibited dual insecticidal activity against Aedes aegypti as well as Plutella xylostella. Recently, we improved the donor-s of plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy), to construct E.coli-Bt shuttle vector. Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their range of sizes were approximately 10 kb. Based on the sequence analysis, they were classified in 4 groups showing similarity with 4 known plasmids, pGI1, pGI2, pGI3 and pBMB175, respectively. One of pGI3-like clones was fully sequenced and its open reading frames were analyzed. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily construct novel shuttle vectors with any Bt plasmids and this combined procedure can introduce foreign gengs into various circular DNA molecular.

The genome of a granulovirus isolated from the tobacco cutworm, Spodoptera litura, was completely sequenced. The nucleotide sequence of the Spodoptera litura granulovirus (SlGV) genome was 124,121 bp long, with a 61.2% A+T content and contained 133 putative open reading frames (ORFs) of 150 nucleotides or larger. The 133 putative ORFs covered 86.3% of the genome. Among these, 29 ORFs were conserved in most completely sequenced baculovirus genomes, 35 were granuloviruses (GVs)-specific, and 60 were present in some NPVs and/or GVs. Especially, we proved that there were 9 SlGV-specific ORFs by RT-PCR. When the phylogenic relationship was analyzed using the nucleotide sequence of granulin gene, SlGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestiac-nigrum granulovirus (XcGV) which were belonged to Type-I granulovirus. Comparative analysis of gene organization of the SlGV genome with those of other baculoviruses were carried out using blast matrix and gene order diagram.

A novel recombinant baculovirus, NeuroBactrus, was constructed to develop an improved baculovirus insecticide with additional beneficial properties such as higher insecticidal activity and recovery to wild-type baculovirus. For this, Bacillus thuringiensis crystal protein gene (cry1-5) was introduced into Autographa californica nucleopolyhedrovirus (AcMNPV) genome by fusion of polyhedrincry1- 5-polyhedrin under the control of poyhedrin gene promoter. In the opposite direction of this fusion gene, an insect-specific neurotoxin gene (AaIT) under the control of early promoter from Cotesia plutellae bracovirus was introduced by fusion of orf603 partial fragment. Western hybridization and confocal microscopy revealed that AaIT neurotoxin and Polyhedrin-Cry1-5-Polyhedrin fusion protein expressed by the NeuroBactrus and that the fusion protein occluded into the polyhedra. In addition, the fusion protein was activated as about 65 kDa of crystal protein when treated with trypsin. The NeuroBactrus showed high level of insecticidal activity against Plutella xylostella larvae and significant reduction in median lethal time (LT50) against Spodoptera exigua larvae compared to those of wild-type AcMNPV. Re-recombinants derived from the NeuroBactrus, NBt-Del5 (deleted cry1-5), NBt-DelA (deleted AaIT) and NBt-Del5A (deleted cry1-5 and AaIT; wild-type baculovirus) were generated in serial passages in vitro and in vivo. These results suggested that the NeuroBactrus could be transferred to wild-type baculovirus along with serial passages by the homologous recombination between two polyhedrin genes and two partial orf603 fragments.

Plasmid capture system (PCS) was developed for easy cloning and manipulation of circular double-stranded DNA from various sources. Recently, we improved PCS system (named PCS-LZ) to clone relatively large-sized DNA molecules (30-150 kb). PCS-LZ donor consists of a Mini-F replicon and a kanamycin resistance marker between Tn7L and Tn7R regions. Both replicon and marker gene of PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition and the transposed DNAs can replicate in E. coli cells by transformation. White/blue screening by LacZ expression is also available to avoid backgrounds. Up to now, we acquired various circular DNA clones from four sources such as plasmids of B. thuringiensis, bacteriophage genome isolated from B. thuringiensis, genome segments of Cotesia glomerata bracovirus, and polymorphic genomes of Autographa californica nucleopolyhedrovirus. Among them, interestingly, the genome clones of bacteriphage (Ph1-3) were screened from the PCS transposition with plasmids of B. thuringiensis 1-3 strain. The genome of Ph1-3 was fully sequenced (46517 bp) and open reading frames were analyzed. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Ph1-3 showed infectivity to B. thuringiensis type strains such as subsp. galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phage in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and their sequence similarity was more than 70%. Conclusively, we expect this PCS-LZ system would be a powerful tool for genomic analysis and mutagenesis study at the area of invertebrate pathology and further its application will be enlarged to the vertebrate pathology area.

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.

Reactive oxygen species (ROS) is toxic to living organisms, because its high reactivity causes oxidative damage to proteins, nucleic acids, and lipids. Superoxide dismutase (SOD) is an enzyme facilitating the removal of superoxide anions from living organisms. This study focused on the cloning of MnSOD cDNA from Hyphantria cunea and its induction upon bacterial infection and various stresses. The open reading frame of MnSOD is composed of 645 bp, encoding 215 amino acid residues. The theoretical molecular mass and pI of putative MnSOD was evaluated to be 24276 Da and 9.14, respectively. The MnSOD from H. cunea is highly similar to human MnSOD (59.5%) as well as Bombyx mori MnSOD (76.2%). MnSOD showed no big induction upon bacterial infection and stresses, compared to that of Cu/ZnSOD.

Apolipophorin-Ⅲ (apoLp-Ⅲ) is a hemolymph protein whose function is to facilitate lipid transport in an aqueous medium in insect. Recently, apolipophorin-Ⅲ in Galleria mellonella and Hyphantria cunea was shown to play an unexpected role in insect immune activation. We show here a novel possible function/role of apoLp-Ⅲ in insects. To investigate the genes which have a relationship with apoLp-Ⅲ in fall webworm larvae, we reduction of endogenous Hc apoLp-Ⅲ mRNA levels in larvae via RNA interference (RNAi). The RNAi-mediated Hc apoLp-Ⅲ reduction resulted in the reduction of antioxidants, like MnSOD, catalase, and glutathione S transferase as well as immune proteins. In particular, expression of MnSOD commonly decreased in fat body, midgut, and hemocytes following the knockdown of Hc apoLp-Ⅲ, which induced an elevated level of superoxide anion in H. cunea larvae. The observed effect of Hc apoLp-Ⅲ RNAi suggests that Hc apoLp-Ⅲ is related to the action/expression of antioxidants.

Innate immunity responses are triggered by the immune challenge and therefore involve signaling processes. The cellular response is initiated by hemocytes and mainly involves phagocytosis and encapsulation of intruders by these cells. To address whether Hc-STAT is activated upon bacterial challenge, we examined the subcellular location of STAT protein in hemocyte by immunostaining. A new insect member of the STAT family of transcription factors (Hc-STAT) has been cloned from the lepidopteran, Hyphantria cunea. The domain involved in DNA interaction and the SH2 domain are well conserved. The gene is transcribed at a low level during all stages of development, and the protein is present in hemocytes, fat body, midgut, epidermis, and Malphigian tuble (Mt). Especially, hemocytes and Mt showed transcriptional activation of Hc-STAT upon Gram (-) bacteria and fungal challenge. Gram (-) bacteria and fungal challenge specifically results in nuclear translocation of Hc-STAT protein and induction of DNA-binding activity that recognizes a STAT target site in H. cunea hemocyte. In vitro treatment with pervanadate translocates Hc-STAT to the nucleus in hemocyte cells. Here we report the first evidence for the involvement hemocyte JAK/STAT pathway upon microbial infection in lepidopteran insect.

The complete nucleotide sequences of the mitochondrial genome (mitogenome) from the white-spotted flower chafer, Protaetia brevitarsis (Coleoptera: Cetoniidae) was determined. The 20,319-bp long circular genome is the longest among the completely sequenced arthropods. This extraordinary length of the genome stemmed from 5,654-bp long A+T-rich region composed of twenty-eight 117-bp tandem repeats, seven 82-bp tandem repeats, and each two 19-bp and 38-bp tandem repeats. The P. brevitarsis contains a typical gene complement, order, and arrangement identical to most common type found in insects. The P. brevitarsis COI gene does not have typical ATN codon. Thus, we also designated it as AAC (asparagine), which is found in the start context of all sequenced Polyphaga within Coleoptera. All tRNAs showed stable canonical clover-leaf structure of other mt tRNAs, except for tRNASer (AGN), DHU arm of which could not form stable stem-loop structure. The 5bp-long motif sequence (TAGTA) that has been suggested to be the possible binding site for the transcription termination peptide for the major-strand also was found betweent RNASer (UCN) and ND1, as have been detected in all sequenced coleopteran insects.

Bacillus thuringiensis 1-3 (Bt 1-3), isolated from Korean soil sample, showed high insecticidal activity against Plutella xylostella. Recently, we improved plasmid capture system donor-s (PCS-S) by inserting attB sites including lacZ between transposable elements (designated as pTroy), to reduce background and construct E. coli-Bt shuttle vector. Through in vitro transposition with total plasmid DNA of Bt 1-3, at least 6 different size plasmids of Bt 1-3 were cloned. Among them, 47 clones which have approximately 10 kb plasmid in size were sequenced and 5 contigs were assembled. These contigs showed partial similarity with two known plasmids, pGI3 or pBMB175, separately. These cloned plasmids will acquire erythromycin resistance by BP recombination reaction with pDonrattPEm vector. After transformation into Bt cells, final erythromycin resistant Bt cell might contain novel E. coli-Bt shuttle vector. This scheme proposes that pTroy and pDonr-attPEm system can easily construct new shuttle vector by in vitro transposition, BP reaction, and erythromycin selection with any Bt plasmids.

Recently, we constructed a novel recombinant baculovirus genome, bEasyBac, enabling easy and fast generation of pure recombinant baculovirus without any purification step. In the bEasyBac, bacteriophage lambda site-specific attachment (att) sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus (CpBV) ORF3005 early promoter to negatively select against non-recombinant background. The bEasyBac could replicate in host insect cells only when the barnase gene was replaced to gene of interest by in vitro transposition. When the bEasyBac was transposed with pDualBac-EGFP and the EGFP expression efficiency along passage was investigated, the resulting recombinant virus, EasyBac-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, AcEGFP, which was constructed using bAcGOZA system, whereas, the non-purified AcEGFP showed quite reduced level of EGFP along passages. Moreover, no non-recombinant backgrounds were detected from unpurified EasyBac-EGFP stocks. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established. These results suggest that the bEasyBac has an effective benefit enabling for high-throughput baculovirus expression vector without purifying recombinant virus.

To determine the characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), CA, which was isolated from the serum of an infected pig in 2006, we investigated the nucleotide sequence and expression of the structural ORFs (ORFs 2 to 7) using the bApGOZA system. We found that the structural ORFs 2 to 7 of CA consisted of 3188 nucleotides that were the same as those formed from VR-2332. Comparison of the CA with the other strains revealed nucleotide sequence identity ranging from 89.8 to 99.5%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The CA strain was closely related to the other North American genotype strains but formed a distinct branch with high bootstrap support. Additionally, expression levels of the PRRSV proteins in Sf21 cells were strong or partially weak. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.

The porcine reproductive and respiratory syndrome virus (PRRSV) has six structural proteins which encoded by ORFs 2 to 7 are designated as GP2, 3, 4, 5, M and N, repectively. In this study, we determined the expression of each protein using novel transfer vector, pBmKSK4 which has the polyhedrin promoter of BmNPV and 6xHis tag. The recombinant transfer vector was co-transfected into Bm5 cells along with bBpGOZA DNA. Recombinant virus was purified by plaque assay and amplified in Bm5 cells. Expression of each protein was identified by SDS-PAGE and Western blot analysis using anti-6xHis monoclonal antibody. The expression levels of the structural proteins in Bm5 cells were stronger than the expression system using pBacPAK9 transfer vector in Sf21 cells. As expected, GP5 was expressed at low levels from its structural properties and its toxicity for cells. In addition, each recombinant protein was purified using Ni-NTA spin columns. The ability to produce each protein in the baculovirus system indicates that these could be major candidates for the development of a vaccine against PRRSV.

The nucleotide sequence of the Spodoptera litura granulovirus (SlGV) genome was determined and analysed. It was 124,121 bp long, with a 61.2% A+T content and contained 133 putative open reading frames (ORFs) of 150 nucleotides or larger. The 133 putative ORFs covered 86.3% of the genome. Among these, 29 ORFs were conserved in most completely sequenced baculovirus genomes, 44 were granuloviruses (GVs)-specific, 4 were nucleopolyhedroviruses (NPVs)-specific, and 56 were present in some NPVs and/or GVs. Especially, we proved that there were 9 SlGV-specific ORFs in 44 GV-specific ORFs by RT-PCR. Chitinase and cathepsin genes involved in the liquefaction of the infected hostwere not found in the SlGV genome, which explains why SlGV-infected insects do not degrade in a typical manner. When the phylogenic relationship was analyzed using the nucleotide sequence of granulin gene, SlGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestia c-nigrum granulovirus (XcGV) which were belonged to TypeI granulovirus.

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.