Hyphantria cunea is a fall webworm is considered an agricultural pest. It is a major pest of many board-leaved trees. H. cunea nucleopolyhedrovirus (HcNPV) and H. cunea granulovirus (HcGV) were isolated from the fall webworm cadavers in Korea. To better understand HcNPV and HcGV, their genomic sequences were determined, analyzed and compared to two viruses together. The entire nucleotide sequence of the HcNPV genome was fully sequenced using 454 pyrosequencing. The genome of the HcNPV was 131,302 bp with a 45 % G+C content. Computer assisted analysis predicted 146 open reading frames (ORFs) of 50 or more amino acids that showed minimal overlap. Further more, when the phylogenetic relationship was analyzed, HcNPV was closely related to Orgyia pseudotsugata MNPV (OpMNPV) which belong to Group I NPV. The HcGV genome was 114,557 bp with a 39% G+C content and contained 130 putative ORFs of 50 or more amino acids. When phylogenetic relationships were analyzed, HcGV was closely related to Xestia c-nigrum granulovirus, which belong to the Type-II GV. HcNPV shares 48 ORFs with HcGV. The most significant difference between HcNPV and HcGV is fgf gene. HcNPV contains one fgf gene, whereas HcGV contains three fgf genes. The presence of fgf reduces the time and efficient systemic infection it takes the virus to kill its host. The difference of fgf number from HcNPV and HcGV suggested that different affect for the speed of systemic infection.

Actias artemis is a members of the family Saturniidae, also known as wild silkmoths, have impressive color and size. In 2012, estimation of Actias artemis (Lepidoptera: Saturniidae) abundance in HECRI was conducted using the mark-release-recapture (MRR) method (Jolly, 1965) from mid to late May. Seven sampling events were accomplished from 19 May, 21 May, 22 May, 24 May, 26 May, 28 May and on 30 May, during the main flight of the species. Marking was made by writing numbers in the hind wing of each individual moths. Most collections were undertaken by a team of experienced four or six researches of HECRI using light trap (mercury lamp: 250W). Seven female and 58 male moths were captured in study site. The effective population size of Actias artemis was 24.9 and heterozygosity was more than 97%. Seven marked moths were recaptured, resulting in 9.7% of recapture rate. The estimated population size of A. artemis showed a peak by 133 individuals on 22 May and then declined. The estimated adult numbers of A. artemis using MRR method from minimum 168 to maximum 5,332 (p<0.05).

The polyhedrin is responsible to form polyhedra of nucleopolyhedrovirus(NPV) and highly conserved in most completely sequenced in lepidopteran NPVs. Previously, we have reported that the substitution of polyhedrin of Autographa californica NPV(AcNPV) with that of Spodoptera exigua NPV(SeNPV) or Bombyx mori NPV(BmNPV) result the change of polyhedra morphology. In this study, we investigated the influence of changed polyhedra morphology to the virulence of AcNPV. The recombinant AcNPVs were propagated in Spodoptera frugiperda clone 9, 21 cells and S. exigua larvae. Each collected recombinant polyhedra were used in bioassays using S. exigua larvae. The recombinant AcNPVs show that difference virulence according to the polyhedra morphologies. Internal and external morphological features of each recombinant AcNPV were also compared on the electron microscope. Our results suggest that the morphology of polyhedra influence the virulence of NPV and is well worth considering for the development viral insecticide.

Three species of the genus Neolygus Knight - N. hakusanensis (Yasunaga, 1991), N. roseus (Yasunaga, 1991) and N. zhugei (Yasunaga, 1991) - are recognized for the first time in Korea. An identification key to the eleven Korean Neolygus species is presented. Some illustrations of male genitalic structures are also provided.

Rice black-streaked dwarf virus (RBSDV), a member of the genus Fijivirus within the family Reoviridae, is the causative agent of maize rough dwarf and rice black-streaked dwarf diseases, both of which can lead to severe yield losses in east Asia. Although molecular approaches such as RT-PCR have potential for detection and diagnosis of this virus infections, their impact on high throughput certification is still limited. Therefore, the development of an antibody-based assay for rapid and effective diagnosis of RBSDV is preferable. In this study, we collected RBSDV from rice with rough dwarf disease and its complete nucleotide sequences of 10 genomic segments encoding 12 non-overlapping ORFs were determined. Among 12 ORFs, ORF1, 2 and 12 showed high level of similarities with the RdRp, major core protein and major outer shell protein, respectively. These ORFs were expressed as polyhedrin fusion protein or full-length soluble protein using baculovirus expression system for the preparation of specific antibody against RBSDV, which could be useful for the detection and diagnosis of this virus.

Mamestra brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) was isolated from naturally infected Mamestra brassicae (Lepidoptera: Noctuidae) larvae in Korea. Restriction endonuclease fragment analysis using EcoRI, PstI, and BamHI estimated that the total genome size of MabrNPV-K1 is about 150 Kb. The full genome sequences of MabrNPV-K1 were determined, analyzed and compared to those of other baculoviruses. The MabrNPV-K1 genome consisted of 152,471 bp and had an overall G + C contents of 39.90 %. Computer-assisted analysis predicted 159 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. The gene content and arrangement in MabrNPV-K1 were most similar to those of Mamestra configurata nucleopolyhedrovirus-B (MacoNPV-B), including three polh, p10 and lef-8 gene homologues. The MabrNPV-K1 genome contains four homologous repeat regions (hr1,hr2,hr3,hr4) that account for 3.1% of the genome. The genomic positions of MabrNPV-K1 regions hr1– hr4 are conserved with the genomic positions of MacoNPV-B hr1–hr4. This indicates that the position of MabrNPV–K1 hrs is conserved with regard to both the upstream and downstream genes. Given that hrs share higher similarity within a virus strain than any hrs between species, this evidence further indicates that hrs play a fundamental role in viral life cycle and replication process appears to be tightly linked to functional conservation. The dot plot analysis, percent identity of the gene homologues and a phylogenetic analysis suggested that MabrNPV-K1 is a Group II NPV that is closely related to MacoNPV but with a distinct genomic organization.

Mamestra brassicae (cabbage moth) is a common European moth of the order Lepidoptera and the family Noctuidae. The larval stage is highly polyphagous and is known to feed on more than 70 species of host plants from 22 families, including Brassica species, lettuce, onion, potato, pea, tomato and apple. M. brassicae has become a significant pest also in Asia due to the damage caused to agriculturally and economically important Brassica crops. It is difficult to control M. brassicae using chemical insecticide because of its rapid development of resistance. The objective of our study, therefore, was the mass production and formulation of a local strain of M. brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) for the development of viral insecticide to control it. In production efficiency of MabrNPV-K1 using M. brassicae larvae, the mortality of the 3rd instar larvae was 100% when inoculated with 1.0 × 105 PIBs/larva and the yield of MabrNPV-K1 was maximal. Regarding the mortality, yield of polyhedra, inoculation doses and required time, the 1.0 × 104 PIBs/larva at 30°C was determined as optimal conditions producing polyhedra efficiently. To formulate MabrNPV-K1, feeding toxicities of various supplements including spreader and ultraviolet (UV) -protectant were determined. Tinopal UNPA-GX which is UV-protectants was effective for protection of polyhedra from UV and showed the increased mortality when added with 1% concentration. Other supplements did not influence significantly the mortality of MabrNPV-K1. Formulated MabrNPV-K1 with several supplements showed higher pathogencity than un-formulated MabrNPV-K1.

Entomopathogenic fungi are widely available as biological control agents for controlling insect pests in agriculture and forestry. The fungal culture broth contains various pathogenesis-related components such as blastospores, mycelium and insecticidal enzymes such as chitinase, Pr1- and Pr2-proteases, which have been reported to play an important role in penetrating insect cuticles. In this study, we tried to evaluate the utility of culture broth from Beauveria bassiana SFB-205 to control lepidopteran pests. High level of insecticidal activity correspond to over 90% of mortality were observed when the culture broth of B. bassiana SFB-205 was inoculated to the Spodoptera litura larvae together with the B. thuringiensis K1. The freeze-dried culture broth showed synergistic effects in insecticidal activity against larvae of S. exigua and S. litura when treated with corresponding baculoviruses, SeNPV and SlNPV. Active ingredient of the B. bassiana SFB-205 culture broth was identified to chitinase, which have truncated form by insertional mutation compared to previously reported chitinases.

The silkworm (Bombyx mori), as an industrial insect, possesses a high economic value. Casual discrimination and accumulated genetic information of silkworm varieties are essential ground for the practical utilization and long-term conservation. In this study, nine available microsatellite loci were successfully genotyped from ~50 silkworm strains preserved in Korea. According to genotyping analysis, we obtained 3 ~ 16 alleles per locus, with an average of 7.4, the observed heterozygosity ranging from 0.04 to 0.98, and the polymorphic information content (PIC) ranging from 0.06 to 0.88, revealing that some loci are highly variable. Among 54 strains 13 strains were casually identified by the presence of 17 strain-specific apomorphic alleles. Furthermore, 30 among remaining strains contained strain-specific allele combinations that are also apomorphic to each strain, allowing us to discriminate each of these from other strains by genotyping of multiple loci. These results collectively suggest that the silkworm microsatellite DNA is actually and potentially important molecular marker for the discrimination of the silkworm strains that are preserved as hundreds in Korea, as more loci are genotyped.

The diamondback moth, Plutella xylostella is a one of the most important pests of various cruciferous crops and has a geographically wide ranging habitat. The heavy dependence on chemical pesticides has created severe pesticide resistance problems. In recent years, Bacillus thuringiensis product have been widely used for P. xylostella control bus genetic resistance in populations to some B. thuringiensis strains, compounded by cross-resistance to several different B. thuringiensis toxins, has also been identified. Such recent resistance problems serve to emphasize the urgent need for alternative control agents and their use within an integrated pest management approach. Baculoviruses have been used as agents for the biological control of certain insect pest species. the granuloviruses (GVs), based on the structure of the occluded virus and the occlusion body (OB). Several reports have showed P. xylostella granulovirus (PxGV) as a promise control agent for P. xylostella. However, it is very difficult to study GV because its OB, granule, has very small size and could be observed exactly under the electron microscopy (EM). This study was performed to develop rapid quantification method for granule of PxGV. After the exact quantification of granule with latex beads using EM, the universal extraction method of viral DNA was established for consistent experiment. The number of granules was calculated by the quantification of PCR products for granuline gene using spectrophotometer and densitometer. This novel calculation method for granule would be useful to study GV.

Three acetylcholinesterases (AChEs) were identified from the pinewood nematode, Bursaphelenchus xylophilus. Sequence comparison with known AChEs in conjunction with three-dimensional structure analysis suggested that all BxAChEs share typical characteristics of AChE at the major catalytic structures. BgAChE3 was most predominantly transcribed and then followed by AChE1 and AChE2. Immunohistochemistry using anti-BxAChEs antibodies revealed that BxAChE1 is most widely distributed whereas BxAChE2 exhibits more localized distribution in neuronal tissues. BxAChE3 was detected from entire body together with some limited tissues, including mouth parts and alimentary lining, and determined to be the only soluble AChE, suggesting its localization in hemolymph or/and extracellular space. Kinetic analysis of in vitro expressed BxAChEs revealed that BxAChE1 has the highest substrate specificity whereas BxAChE2 has the highest catalytic efficiency with BxAChE3 having the lowest catalytic efficiency. Interestingly, presence of BxAChE3 in the pool of BxAChEs significantly reduced the inhibition of BxAChE1 and BxAChE2 by inhibitors. Knockout of BxAChE3 by RNAi significantly increased the toxicity of nematicides, suggesting the protective role of BxAChE3 against these toxicants. Based on several features, including tissue distribution, expression level, substrate kinetics and inhibition property, it appeared that BxAChE1 is the major AChE with the function of postsynaptic transmission whereas BxAChE3 has been evolved to acquire the function of chemical defense, perhaps intrinsically against secondary toxic compounds from host pine trees, such as α-pinene and limonene. BxAChE2 appears to play a role in post-synaptic transmission in specialized neurons but its detailed physiological function still remains to be elucidated.

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.

Among the cultured products of Beauveria bassiana SFB-205 (KCCM 10892P), the supernatant showed the highest insecticidal activity against 2nd instars of Aphis gossypii (Aphididae) nymphs under glasshouse condition. The enzymes in the supernatant were confirmed as active materials, and the chitinase was finally selected as a QC factor for commercial production. However, the chitinase activity in the supernatant decreased by 11-folds due to the thermal stress at 50℃ for 2 h. To obtain thermal stability, the chitinase in the supernatant was adsorbed to a precipitable material and the pellet was freeze-dried (PCT/KR2007/005886). The adsorbent-A showed about 92.7% of harvesting efficiency which was higher than the other candidates. The chitinase activity of the freeze-dried powder was kept up about 82.0% of initial activity for the same thermal stress. Subsequently, an optimal formulation recipe was established to maximize long-term storage stability and efficacy. SFB-205 oil-based formulation was stable up to 18 months at room temperature. It showed 96.1% efficacy against 2nd instars of A. gossypii nymphs at 1 day after the treatment in the glasshouse. This novel approach can be a promising method to develop competitive biopesticies in the entomopathogenic fungi, even though it needs to be intensively studied.

The purpose 01' pl'esent study was to examine the molecular events in apoptosis by CoCl2, mimicking hypoxic cond ition and recovering effects by LED ir l'adiation on Human SH-SY5Y neuroblastoma cells The SOUl'ce 0 1' light for ir l'adiation was a continuous-wave LED emitting at a wavelenl양h of 590 nm, and manufactured that ene rgy density was 5 mW!cm2 on sample surface, After ir l'adiation, cell viabi lity was measured with BrdU , cell morphol ogy was examined with Diff- Quik staining, cell signaling was monitored with various apoptosis-related molecules using RNase Pl'otection Assay(RPA) , W11en treated with CoC12, apoptotic induction was found in the SH-SY5Y cells in a concentration-dependent and time-dependent manner , Diff-Quik s taining was revealed that DNA fragmentation re presented apoptosis was examined in CoC12-tl'eated group, Moreover, RPA assay of SH-SY5Y cclls lIs ing val'iolls apoptosis-related molecllles showed that the apoptotic cell population was mcreased J-loweve. there was sorne signifïcant change in LED irradiatied cells aftel' treatement of CoC12 The main mechanism for Lhese a poptosis appearecl to be mito c hondriεt - m ecliated pathway, such as cytochrome- c‘ caspase-9, caspase-3, pro-apototic protein ßax, anti-apototic protein Bcl-2, and death receptor• mediated pathway, such as Fas, cas pase- 8, a ncl TNFRl These results demonstrate that CoCI2 induce apoptosis in SH-SY5Y via different dual apop tosis pathway through death receptor pathway as well as mitochondria- dependent pathway and LED irradiation can recl llces the CoCl2-induced apoptosis by blocking their internal signaling pathway