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.

The pinewood nematode (PWN, Bursaphelenchus xylophilus) is known as a virulent factor of the pine wilt disease, transmitted to pinewoods by the pine sawyer beetle, Monochamus alternatus. It is very hard to discriminate B. xylophilus from B. mucronatus because these Bursaphelenchus species are genetically and biochemically very close. Therefore, it has been necessary to detect PWN-infected trees for the prevention of pine wilt disease transmission in a short time. We developed polyclonal antibodies against B. xylophilus in BalbC mice and primarily screened with ELISA. Positive clones releasing polyclonal antisera revealed B. xylophilus-specific immuno-reactivity, which were at least two times higher than that of B. mucronatus. Two clones, D9-F10 and 1F3, were finally selected and exhibited specific immuno-reactivity for B. xylophilus, not for B. mucronatus in Western blot analysis. D9-F10 clone was reactive with a 43-kDa whereas 1F3 clone with two proteins, 40- and 45-kDa. Their isotypes against mouse Ig family were identical, kappa-light chain. These results suggest that these monoclonal antibodies can be useful for the development of diagnostic kit for the pine wilt disease.

Root knot nematode species, such as Meloidogyne hapla, M. incognita, M. arenaria and M. javanica are economically most notorious nematode pests, causing serious damage to the various crops throughout world. In this study, DNA sequence analyses of the D1-D3 expansion segments of the 28S gene in the ribosomal DNA were conducted to characterize genetic variation of the four Meloidogyne species obtained from Korea and United States. PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism), SCAR (Sequence Characterized Amplified Region) marker and RAPD (Random Amplification of Polymorphic DNA) also were used to develop the methods for exact and rapid species identification. In the sequence analysis of the D1-D3 expansion segments, only a few nucleotide sequence variation were detected among M. incognita, M. arenaria, and M. javanica, except for M. hapla. The PCR-RFLP analysis that involves amplification of the mitochondrial COII and lrRNA region yielded one distinct amplicon for M. hapla at 500 bp, enabling us to distinguish M. hapla from M. incognita, M. arenaria, M. javanica reproduced by obligate mitotic parthenogenesis. SCAR markers successfully identified the four root knot nematode species tested. We are under development of RAPD primers specific to the three root knot nematodes found in Korea.

Thirteen plant essential oils were tested for their repellent activity against the bean bug Riptortus clavatus. Among the tested oils, caraway (100%) and clove bud oil (92%) significantly repelled the bean bugs at a dose of 0.142㎕/cm2 by using a Y-tube olfactometer. GC and GC-MS analyses revealed that the active components responsible for the effective repellency of caraway and clove bud oil were carvone (75%) and limonene (76.9%); eugenol (100%), isoeugenol (54.3%) and β-caryophyllene (60.0%), respectively. Of the different active fractions, eugenol was the most significant one than the other components with reference to repellent activity against the bean bugs. In the GC-EAD, limonene and carvone of caraway oil were responded to the antenna of Riptortus clavatus.

whitefly, Bemisia tabaci (Gennadius) have a wide host range including cucumber, tomato, and pepper, resulting in loss of crop yield. In this study, we tested larvicidal efficacy of several on-the-market environment–friendly agricultural materials (EFAM) to select the effective products after the target pests were stabilized in indoor rearing condition. The developmental periods of two whiteflies are as follows: in the case of T. vaporariorum, egg duration is 9.6 days, and nymph is 18.9 days, and in the case of B.tabaci, egg durationis 7.4 days, and nymph is 15.2 days under 25℃ with relative humidity (RH) of 60±5% and a photoperiod of 16L : 8D. The total period of T. vaporariorum as 5 days longer than B. tabaci. Among 22 EFAMs six products showed more than 60% of insecticide efficacy for against T. vaporariorum BTVB, BTVD, BTVG, BTVL, BTVM, and BTVS. On the other hand, seven EFAM products including showed over 60% of insecticide efficacy against B. tabaci BTVD, BTVG, BTVK, BTVL, BTVM, BTVN, and BTVU. In the case of Spodptera litura previously, xxEFAMs were tesed against 2nd instar S.litura, and EFAMs were found to have more than 90% efficacy. Test of these six EFAMs against entire larval stages were performed in this study. Although some of these products showed still more than 90% of insecticidal efficacy against up to 3rd instar larvae, the efficacy of these EFAMs sharply decreased as ages increase, result is less than 60% of efficacy of the products at most. This result indicates the difficulty to control S. litura with the on-the-market EFAMs alone under economic injury level. Collectively, it is required to find more EFAMs and find alternative method to control those insect pests tested in this study.

A taxonomic review of a new record, Bolitophagiella pannosa (Lewis) in Korea is presented. Description of adult is presented and also we conducted laboratory and field observations of the life history and fungal hosts of the darkling beetle, Bolitophagiella pannosa (Lewis). A fungivorous tenebrionid beetle, Bolitophagiella pannosa (Lewis), was a rare inhabitant of fungi on deciduous trees (Quercus, Robinia pseudoacacia etc.) in Korea. This species is associated with host fungi, generally order Aphyllophorales throughout its whole life. Especially both adults and larvae was inhabit on widespread fungi, Perenniporia, on deciduous trees in Korea. Apparently this species used obligately the fruiting bodies of Perenniporia medulla-panis (Fr.) Donk and Perenniporia frazinea (Fr.) Ryv. for breeding and feeding site. Development from egg to adulthood took 3-10 months in nature and about 54 days in the laboratory at 25.5-26.1℃ and 63.5-64.5% relative humidity. Both larvae and adults overwintered in their host fungi or beneath the bark of the host tree near the host fungi. Sporophores of Perenniporia medulla-panis (Fr.) Donk and Perenniporia frazinea (Fr.) Ryv. were obligate feeding and breeding sites in Korea. Description, habitus photographs of adult and instar, and illustrations of diagnostic characters are provided.

The occurrence and damage of stink bugs were monitored at South Korea from 2007 to 2008. A total of 11 stink bug species were observed in paddy field and occurrence periods of stink bugs were significantly differed by species. In occurrence period and damage of stink bug feeding in paddy field, Scotinophara lurida occurred from June, Cletus punctiger, Eysarcoris aeneus, Rhopalus maculatus, Riptortus clavatus, E. ventralis, Stenotus rubrovittatus occurred from August and Pachygrontha antennata, C. schmidti, Trigonotylus caelestialium, Paromius exiguus occurred from September. When we were classified into two region with mountain and plain area cultivated rice, generally C. puntiger population was higher in mountain area than plain area, and E. aeneus was higher in plain area than mountain area. Also, stink bug species were higher in environment condition formed with a paddy field and a waterway overgrown with weeds. Control threshold for E. aeneus in paddy field was 1-2 individuals/heading stage, and then rate of pecky rice made to 5.0% while milk stage, dough stage and yellow ripe stage made to less than 5.0%.

Two cherry tomato plant cultivars (Lycopersicon esculentum Miller, cultivars ‘Koko’ and ‘Pepe’) were supplied with high (395 ppm), medium (266 ppm) and low (199 ppm) concentrations of nitrogen to determine the influence of nitrogen fertilization on development, cultivar preference and honeydew production by greenhouse whiteflies, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae). The nitrogen, protein, andchlorophyll content of tomato leaves were higher in the high nitrogen supplied plants than in the medium or low nitrogen supplied plants, but the sugar content showed an inverse relationship. The developmental times of eggsand nymphs decreased as the nitrogen concentrations increased in both cultivars. The preference of T. vaporariorum was compared by counting the number of eggs deposited on leaves in choice and non-choice tests. In the non-choice test, no significant nitrogen treatment effects were observedbut the upper plant stratum was preferred for egg laying. In the choice test, there were significant main effects of cultivar and nitrogen concentration. T. vaporariorum laid eggs more on leaves of plants with higher nitrogen at the upper stratum. In both experiments, T, vaporariorum preferred the ‘Koko’ cultivar to the ‘Pepe’ cultivar. The honeydew production of T. vaporariorum nymphs increased with decreasing nitrogen treatment concentrations. The largest honeydew production was detected in the ‘Pepe’ cultivar grown at low nitrogen concentration. It is concluded that cultivar ‘Pepe’ had an advantage over ‘Koko’ in term of T. vaporariorum management program in tomato greenhouses.

The green rice leafhopper, Nephotettix cincticeps is an insect vector which transmits virus diseases of rice plant causing severe damage to the rice and its damage both quality and yield of rice. Survival rate of overwintering N. cincticeps in Alopeculus acquadis among 15 species of the winter crops and weeds was 37.8% and its most preferred. Density of N. cincticeps in environment-friendly agriculture area which cultivated Astragalus sinicus as a green manure crop in winter season increased in number wintering pass in Alopeculus acquadis and Astragalus sinicus as host plants, because of control of N. cincticeps was not enough in the previous year. Survival rate of N. cincticeps was higher mainly in habitation mixed with rice straw, Astragalus sinicus and Alopeculus acquadis as winter habitation. It warmed due to the effect of the keeping warmth inside of a rice paddy field and the bank around a rice field covered with rice straw or Astragalus sinicus. Also, habitation conditions were optimal for survival because of growing Alopeculus acquadis as the major host plant of N. cincticeps in that place.

Moths were caught by using two 22W UV black light traps per treatment during May, June, August and September in 2007 from our study sites. Our study areas of the Korea Long Term Ecological Research (KLTER) sites were Mt. Nam with two plant communities (Quercus mongolica, Pinus densiflora), Mt. Jiri with three plant communities (Q. mongolica, P. densiflora, Abies koreana), Mt. Wolak with three plant communities (Q. mongolica, P. densiflora, Q. variabilis), and Mt. Jumbong with t재 plant communities (Q. mongolica, P. densiflora). The purpose of this study was to compare the species diversity of major plant feeders, Lepidopteran species (moths) at each forest type of the regional KLTER sites. Overall, the total numbers of moth species we’ve collected from the all KLTER sites in 2007 were 670 species. For the three plant communities, Mt.Jiri (11 family, 259 species, 2372 individuals) was the highest site and Mt. Wolak (7 family, 401 species, 2243 individuals) was the next highest. For the two plant communities, Mt. Jumbong (14 family, 166 species, 1750 individuals) was highest and Mt Nam (4 family, 21 species, 142 individuals) was very few. The ordination analyses, Nonmetric multidimensional scaling (NMS) and cluster analysis, showed distinct clusters separating the assemblages of moth found at each site and each plant community of the KLTER sites (P<0.05 from MRPP). Therefore, we suggest that our sustainable monitoring will verify the distinct cluster with the forest type at each site with many replications.

We assessed the environmental risk of herbicide resistant transgenic rice (Protox) on non-target herbivore, grasshoppers (Oxya japonica japonica Thunberg). We conducted life-history experiments of grasshoppers with measuring their body weight, body length, eating amount, and feces amount between non-transgenic rice (nTR; Dongjin rice) and transgenic rice (TR; Protox rice) under laboratory conditions (Temp. 25Ð, R.H. 50-70%, Photoperiod L16:D8) in 2007. The growth of grasshoppers appeared to increase at each measuring date. We also compared the growth rate of grasshoppers between nTR and TR to examine the transgenic impact on the herbivore and we found there was no statistically signifi cant difference between the two plant types (P>0.05). We found that body weight and body length for grasshoppers were highly correlated at each of the two types of plants, nTR (0.962) and TR (0.960). The correlation of eating amount and feces amount of grasshoppers were higher nTR (0.830) than TR (0.782). The energy effi ciency of the grasshopper was not a signifi cant between nTR and TR (P> 0.05). But the molt timing of the grasshoppers for TR difference was faster than for nTR. Conclusively life-history of the grasshoppers but molt timing was not a signifi cant difference between nTR and TR. Therefore, we could conclude there was not any environment risk on herbivore from our result.

This study was performed to investigate the seasonal occurrence, developmental characteristics of each nymphal stages with different temperatures (20, 25, 30℃), longevity and fecundity of ussur brown katydid, Paratlanticus ussuriensis, damaging by outbreaks in the orchard areas of Bitan-ri, Yeongdong, Chungbuk. Paratlanticus ussuriensis occurred from late-March to late-August with peak of mid-May. Newly emerged nymphs appeared from March and do damaged fruit orchards with peak of mid-May when P. ussuriensis existed as 4th and 5th nymphal stages. P. ussuriensis adult occurred from early-June to mid-Aug. with peak of mid-July. Total density of P. ussuriensis was showed highest in mid-May. P. ussuriensis goes through nymphal stages to 7th nymph, the ovipositor began exposed to outside from the 4th instar and the body weight increased heavily from this stage and the wings were observed from 6th instar. Developmental period was longer as increased the nymphal stages. Sex ratio of collected insect was showed as 0.57; females more than males. As increased the temperature, developmental period was to be short. Preoviposition was also to be short as 5.0, 4.3, and 3.4 days at 20, 25, 30℃, respectively, and fecundity increased as 69.0, 87.1, and 104.3 at 20, 25, 30℃, respectively. Longevity of male and female at 25℃ was showed the longest with 35.7, and 32.9 days and showed the shortest with 30.1 and 28.1 days at 30℃, respectively. The difference of developmental period in male and female were showed longer in female without relation of temperature. The eggs laid were frequently distributed 3 to 4 cm from soil surface, and showed the behavior laying eggs intensively when early oviposition period.

Sex pheromone production in lepidopteran is stimulated and regulated by a pheromone biosynthesis activating neuropeptide (PBAN). A cDNA of PBAN receptor (Plx-PBANR) isolated from female pheromone gland of the diamondback moth (DBM, Plutella xylostella (L.) encodes 338 amino acids. Plx-PBANR has conserved biochemical motifs and 7 transmembranes, indicating it belongs to G-protein coupled receptor family. Plx-PBANR expression was only found in female pheromone gland, demonstrating that pheromone gland is the only molecular target of Plx-PBAN. Human uterus carcinoma (HeLa) was stably transfected with Plx-PBANR gene and its expression was confirmed by RT-PCR analysis. Plx-PBANR expressing cells increased level of Ca2+ influx when challenged with Plx-PBAN and Hez-PBAN from Heliothis zea. When RNAi fragment for PBANR was injected into pupae, suppression of PBANR expression was confirmed by RT-PCR and maintained for at least 2 days at post-emergence. Injection of RNA fragment into pupae for inhibition of Plx-PBANR expression also inhibited mating behavior, revealing that reproductive organ of the female has no spermatocyte and that there are no successful reproductive behaviors. These results suggest of PBANR expression affects the molecular biological events of PBAN and eventually suppresses mating behavior.

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.

Since the ancient times the therapeutic application of honeybee venom (BV) is practised and persisted until the present days. Resistant bacteria are in emergence and some drugs no longer have an antimicrobial action. To purify the melittin known as antibacterial peptide, five major peptidergic subfractions were separated, purified and identified from the whole BV. We investigated the antibacterial activity of whole BV and purified melittin against Staphylococcus aureus by the minimum inhibitory concentrations (MIC) and the postantibiotic effect (PAE). The MIC of whole BV for S. aureus was 0.06 ㎍/㎖, respectively. The MIC of melittin was 0.06 ㎍/㎖ on S. aureus. The in vitro PAE of whole BV and isolated melittin were determined using E. coli and S. aureus. The PAE of whole BV against S. aureus were 3.45 h (1×MIC). The PAE of melittin against S. aureus was 4.35 h (1 × MIC). Also both whole BV and melittin killed S. aureus at 5 × MIC. The regrowth wasn't observed after 18 h. These results suggest that whole BV and melittin will be developed a novel antibacterial drug.

The Black Soldier Fly (BSF, Hermetia illucens) was widely distributed throughout Korea. This insect was mainly found in the vicinity of and in cattle sheds, manure sheds, living waste dump grounds, and food waste dump grounds. This fly is a kind of a beneficial fly because BSF adults do not go into houses, they do not regurgitate on human food, they do not bite, bother or pester humans in any way and they are not associated in any way with the transmission of disease. But their greatest attribute lies their ability to eat and digest raw waste. They can devour, for example, a large, raw, Irish potato and others in just a few hours. Unlike many other flies, since the BSF larvae have very powerful mouth parts and digestive enzymes, they can ingest raw waste far more efficiently than any other known species of fly. On this study, to investigate whether feeding strategy of the BSF larvae involves extra-oral digestion or not, and to better understand this process, the salivary glands and a few tissue from the BSF were dissected and subjected to morphological and preliminary enzyme characterization.