Autographa californica nucleopolyhedrovirus (AcMNPV) has a large doublestrand DNA genome of approximately 134 kbp and comprises 156 open reading frames (ORFs). To elucidate DNA replication cascade of AcMNPV, we developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, 56 single ORF-truncated mutants were generated by random insertion into bAc-MK genome. These single ORF-truncated mutants were independently transfected into Sf9 cells to verify viral replication. Interestingly, both lef-1 and p48 knockout mutants showed normal viral replication in infected cells, which are reported to essential for viral replication. These results suggest that these single ORF-truncated mutants are useful for elucidation of viral replication cascade.

Through an application of plasmid capture system (PCS) to Bacillus thuringiensis plasmid DNAs, we acquired 21 polymorphic clones of putative genomic DNA of bacteriophage. The genome size of phage 1-3 (PhBT1-3) was determined to be 46,517 base pairs (bp) with 35.43% G + C content and 83% coding region. Sixty-five putative open reading frames (ORFs) with more than 50 codons were found in the new phage genome. In accordance with this genome finding, the phage particles and its DNA were confirmed from the supernatant of B. thuringiensis 1-3. Morphological characterization and infectivity assay demonstrated that PhBT1-3 belongs to the family Siphoviridae and it showed infectivity to three B. thuringiensis type strains, galleriae, entomocidus, and morrisoni. Based on these results, we screened the existence of phages in B. thuringiensis type strains by PCR with terminase small subunit-specific primers. Ten of 67 type strains showed PCR products and the similarity of those putative amino acids was more than 70%. Furthermore, we verified the existence of various shaped phages from the supernatants of 10 B. thuringiensis type cultures. In conclusion, we characterized a putative genome of phage, PhBT1-3 from B. thuringiensis 1-3, and confirmed the distribution of phages in the group of 67 B. thuringiensis type strains.

A new Bacillus thuringiensis isolate 19-22 (Bt 19-22) exhibited high anti-fungal activity against barley powdery mildew (Blumeria graminis f. sp. hordei). The cry gene content of Bt 19-22 comprised cry1Aa, cry1Ab, cry1Ac and cry1D which have high insecticidal activity against lepidopteran larvae. We tried to confer a dipteran insecticidal activity to Bt 19-22 for constructing a recombinant strain which has multiple functions, anti-fungal and dual insecticidal activity. The insecticidal cry11Aa gene of B. thuringiensis was constructed under cry1Ac promoter in an E. coli-B. thuringiensis shuttle vector (pPro11A). The plasmid, pPro11A was introduced into Bt 19-22 isolate by electroporation and four transformants which had different cry gene contents were identified by PCR with cry11Aa and cry1-type specific primers. Among them, a Bt 19-22 transformant (11A/19-22 No. 7) expressed Cry11A protein (approximately 70 kDa) successfully without change of its inherent characteristics such as Cry protein expression and antifungal activity. The insecticidal activity of 11A/19-22 No. 7 was checked against Plutella xylostella and Culex pipiens. These results suggests that the recombinant strain shows dual insecticidal activity against lepidopteran and dipteran larvae as well as antifungal activity.

The hemipteran whitefly Bemisia tabaci (Gennadius) is one of the most destructive pests damaging more than 600 agricultural crop species worldwide. The B and Q biotypes are most widely spread in Korea but they are not distinguishable based on morphological characters. Carboxylesterase 2 (Coe2) was determined to be 5.9 times more expressed in B biotype compared to Q biotype. Comparison of deduced amino acid sequences of Coe2 (595 a.a.) showed a total of 3.85% polymorphisms between B and Q types but no differences in major active sites. Quantitative real-time PCR revealed that both B and Q biotypes possess a single copy of coe2, suggesting that the overexpression of Coe2 in B biotype is likely due to overtranscription. To determine the putative role of Coe2 in insecticide tolerance, esterases were separated by native isoelectric focusing (IEF) and inhibited by various insecticides. The putative Coe2 band was apparently inhibited by pyrethroid and organophosphate insecticides, but not by imidacloprid. These findings suggest that overexpression of Coe2 confers chemical defense against pyrethroid and organophosphate insecticides, perhaps by sequestration.

The toxicity of methanol extract from 28 plants toward four agricultural insect pests were examined using contact toxicity bioassay with spray methods. At a concentration of 1,000 ppm Achyranthes japonica whole body was observed strong activity against Myzus persicae. Paeonia lactiflora root and Corydalis turtschaninovii rhizome methanol extract were observed strong activity toward Tetranychus urticae. Catalpa ovata leaf and Ginkgo biloba leaf and stem methanol extracts were revealed high activity toward Nilaparvata lugens. Actium lap root methanol extract was observed moderate activity against Plutella xylostella. High or moderate insecticidal activity of plant extracts were listed in Fig.1. These plant resource, particularly plant methanol extract or insecticidal constituents, merit further as potential insect pest control agents or leads because of their great activity as a insecticide.

The rice leaf roller, Cnaphalocroci smedinalis Guenée (Lepidoptera: Pyralidae) is a leaf-feeding pest of rice world-widely distributed. For better understanding of the pest insect, geographic sequence variation of the species were performed using the mitochondrial A+T-rich region, with the samples collected from seven Korean and six Chinese localities. A total of 94 haplotypes obtained from 187 individuals showed the length variation, ranging from 339 bp to 348 bp. The maximum divergence of 4.57% appears to evidence a substantial sequence variation, indicating the applicability of this molecular marker to the study of geographic variation. Overall, a high per generation migration ratio (Nm = 3.67742 ~ infinite), a low level of genetic fixation (FST = 0 ~ 0.11969), and no discernable isolated population were noted in the most C. medinalis populations. AMOVA analysis to find out allocation of genetic variability of C. medinalis populations has shown allocation of majority of variation to the within-population, rather than among-populations and between-region, suggesting that the C. medinalis populations in both China and Korea are largely well connected. This result is consistent with current knowledge of the dispersal ability of the species. The structure analysis of the A+T-rich region has shown that the typical structural elements found in other lepidopteran insects also is well preserved in the C. medinalis A+T-rich region (i.e., a poly-T stretch and a microsatellite-like A/T repeat).

The perilla leaf pyralid moth, Pyrausta panopealis Walker (Lepidoptera: Pyralidae), is a serious pest damaging to leaf perilla. In order to establish the life parameters of P. panopealis for eventual purpose of control, the developmental span of each stage were investigated under five temperature regimes (20℃ ~ 3 0℃). In addition, the larvicial efficacy of several on-the-market environment-friendly agricultural materials (EFAMs) was tested. The width of head capsule at each larval stage measured to be the mean of 0.21, 0.32, 0.47, 0.64, and 0.98 mm, respectively. The larval period of P. panopealis was longest at 20ºC as 27.0 days and shortened as temperature goes up to 30ºC as 11.3 days. Survivorship of the larval P. panopealis was the highest at 30ºC as 80%, whereas that of other temperatures ranged from 40% (20ºC) to 62.5% (27.5ºC), indicating that the P. panopealis appears to favor higher temperature. In addition to larval period, the duration of egg, prepupa, and pupa stages also shortened as temperature goes up, whereas the duration of adult stage increased as temperature goes up: from 4.1 days at 20 ºC to 6.1 days at 30ºC. After the perilla leaf pyralid moths were successfully stabilized in indoor environment the larvicidal efficacy of the ten EFAMs that were previously selected from the result of other moth species was tested aimed at 4th instar larvae for 48 hrs. Seven of the ten tested showed more than 90% of mortality within 12 hrs and reached nearly up to 100% within 24 hrs, but the remaining three showed less than ~70%.

Cuticular hydrocarbons (CHCs) were analyzed using GC and GC-MS, and compared with developmental stages of the bean bug, Riptortus pedestris. Carbon numbers on each developmental stages differed from 14-19 in eggs to 4th nymph, 25 in 5th nymph, and over 30 in last nymph that until adult emergence. Carbon numbers are increased to 16-17 carbons over time in newly emerged adults, 18-22 in 1 and 3 days after emergence, respectively; 23-27 in 6 days after emergence; approximately 30 in over 10 days after emergence. Carbon numbers increased as passed days after emergence. Riptortus pedestris consisted of n-nonacosane on almost all developmental stages, and followed by n-hentriacontane and 13,17-;15,19-demethyltritriacontane. Eggs, however, consisted of unknown compounds with high proportion and followed by n-heptacosane, and newly emerged adults also has a high proportion of n-heptacosane. From the above results, CHCs on developmental stages of the bean bug consist of n-alkane with saturated hydrocarbons (36-65 %), followed mono- or di- methylalkanes. However, newly emerged female and male adults mostly consisted of methyl-alkane. The major constituents of CHCs on the developmental stages of Riptortus pedestris is differently proportioned, but hardly showed the difference in their composition.

The black soldier fly (BSF), Hermetia illucens, is known as a beneficial insect and feeds on organic materials derived from animals and human, resulting in reduction of food waste and conversion of organic materials. Despite of a lot of study about the BSF, there is a less information about composition of digestive enzyme of the BSF larva. Experimentally, there is no evidence about characterization of digestive enzyme of the BSF. We investigated biochemical property of digestive enzyme released from the salivary and gut of the BSF. Through digestive enzyme assay, we found that the BSF has amylase, lipase and protease activity in gut extracts, resulting in that the BSF belong to polyphagous insect group. In the BSF gut, trypsin-like protease activity showed one peak at various temperature and pH condition. This result means the BSF has probably a similar form of trypsin-like enzymes. On study of comparison of enzyme activity between the BSF and the housefly using the apiZYM kit, the BSF had more strongly digestive enzyme activity than one of the housefly about leucine arylamidase, alpha-galactosidase, beta-galactosidase, alpha-mannosidase and alpha-fucosidase. This finding supports that the BSF can ingest raw waste far more efficiently than any other known species of fly as reported previously.

Apolipophorin-III (apoLp-III) is a hemolymph protein whose function is to facilitate lipid transport in an aqueous medium in insects. 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 the apoLp-III in insects. To investigate the genes which have a relationship with apoLp-III in fall webworm larvae, we reduced 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 Hyphantria cunea larvae. The observed effect of Hc apoLp-III RNAi suggests that Hc apoLp-III is related to the action/expression of antioxidants, especially MnSOD.

A new insect member of the STAT family of transcription factors (HcSTAT) 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 transcribed in hemocyte, fat body, midgut, epidermis, and Malpighian tubule. Especially, hemocyte and Malpighian tubule showed transcriptional activation of HcSTAT upon Gram-negative and -positive bacteria challenge. Gram-negative and -positive bacteria challenge specifically results in nuclear translocation of HcSTAT protein and induction of DNA-binding activity that recognizes a STAT target site in H. cunea hemocyte. In vivo treatment with sodium orthovanadatetranslocates HcSTAT to the nucleus in hemocyte cells.

The phylogenetic relationships among true butterfly families (superfamily Papilionoidea) have been a matter of substantial controversy, and that debate has led to several competing hypotheses. Two of the most compelling of those hypotheses involve the relationships of (Nymphalidae + Lycaenidae) + (Pieridae + Papilionidae) and (((Nymphalidae + Lycaenidae) + Pieridae) + Papilionidae). In this study, approximately 3,500 nucleotide sequences from cytochrome oxidase subunit I (COI), 16S ribosomal RNA (16S rRNA), and elongation factor-1 alpha (EF-1α) were sequenced from 83 species belonging to four true butterfly families, along with those of eight outgroup species belonging to the skipper family (superfamily Hesperioidea). These sequences were subjected to phylogenetic reconstruction via Bayesian Inference (BI), Maximum Likelihood (ML), and Maximum Parsimony (MP) algorithms. All phylogenetic analyses among the four true butterfly families strongly indicated a sister relationship between the Nymphalidae and Lycaenidae on one hand, and relatively strongly indicated a sister relationship between the Pieridae and Papilionidae on another hand, thus supporting the hypothesis: (Nymphalidae + Lycaenidae) + (Pieridae + Papilionidae).

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 beetle family Ciidae, represented by Octotemnus laminifrons Motschulsky, 1860 and Octotemnus japonicus Miyatake, 1954 is presented for the first time in Korea. The beetle family Ciidae Leach is a relatively moderate family, which comprises about 640 described species in 42 genera worldwide. This family is distributed throughout the world. Ciids are minute fungus-feeding beetles, which are associated with the mycelia and fruiting bodies of wood-rotting fungi. These beetles are considered mycetobiont because they depend upon the basidiocarps for food and breeding throughout their life span. And also ciids are important components of the saproxylic fauna of many forests ecosystems and a number of species are considered rare or threatened and are associated with old growth forests or undisturbed forest conditions. A habitus photographs of adults, description, illustrations of diagnostic characters, and host fungi are provided.

Three species, Allobethylus tomoae Terayama, Cephalonomia tarsalis (Ashmead), and Laelius yamatonis Terayama of the subfamily Epyrinae are recorded for the first time in Korea. Both sexes of A. tomoae Terayama, C. tarsalis (Ashmead) and female of L. yamatonis Terayama are briefly described with diagnostic morphological illustrations for each species based on the Korean specimens. Male of the genus Allobethylus is discovered for the first time from the word.

The bumblebee, Bombus ignitus (Hymenoptera: Apidae), is a valuable natural resource that is one of the most notably utilized for greenhouse pollination in Korea. In order to understand the nature of genetic relationships, gene flow, and population structure of the species we sequenced a partial COI gene of mitochondrial DNA (mtDNA) corresponding to “animal barcode” region and the complete internal transcribed spacer 2 (ITS2) of the nuclear ribosomal DNA (nrDNA) collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.61% (4 bp), the ITS sequences provided 84 sequence types with the maximum sequence divergence of 1.02% (21 sites), confirming better applicability of the ITS sequences to the study of intraspecific variation. The complete ITS2 sequences of B. ignitus were shown to be longest among known insects, ranging in size from 2,034 bp ~ 2,052 bp, harboring two duplicated repeats. Overall, a very high per generation migration ratio, a very low level of genetic fixation, and no discernable hierarchical population/ population group were noted to exist among populations of B. ignitus on the basis of both molecules, thus suggesting that the B. ignitus populations on the Korean peninsula are panmictic, which is consistent with our understanding of the dispersal capability of the species

tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for Bursaphelenchus xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2,304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions or bands via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two non-identical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

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.

Seeking to improve the weak resolution of deeper divergences in an initial study based on five nuclear genes (6.6kb total) in 123 exemplars, we nearly tripled the total sequence (to 26 genes, 18.4 kb total) in one third (41) of the taxa. The expanded, deliberately incomplete data matrix consistently increased bootstrap support for previously-identified groupings, while introducing no contradictory groupings of the kind that missing data have been predicted to produce. To test the relative effectiveness of “more genes” versus “more taxa” sind that we compared two largely complete matrices, the initial 5 gene × 123 taxon and the 26 gene × 41 taxon data sets, that contain roughly equal amounts of sequence. The “more genes” data set yielded consistently, sometimes dramatically higher bootstrap support that is generally not attributable to taxon number alone. We also found that a gene-rich taxon subset provides reassuring evidence of strong underlying signal that is not obvious in subsequent larger analyses, helping to encourage and guide the search for deep relationships amid the noise of expanded taxon sampling.

In this paper, Fe-TiO2 and Fe-fullerene/TiO2 composite photocatalysts were prepared with titanium (IV) n-butoxide (TNB) by a sol-gel method. TiO2, Fe-TiO2 and Fe-fullerene/TiO2 were characterized by scanning electron microscopy (SEM), Transmission electron microscope (TEM), specific surface area (BET), X-ray diffraction analysis (XRD) and energy dispersive X-ray spectroscopy (EDX). The photocatalytic activities were evaluated by the photocatalytic oxidation of methylene blue (MB) solution. XRD patterns of the composites showed that the photocatalyst composite contained a typical single and clear anatase phase. The surface properties shown by SEM presented a characterization of the texture on Fe-fullerene/TiO2 composites and showed a homogenous composition in the particles for the titanium sources used. The EDX spectra for the elemental identification showed the presence of O, C and Ti elements. Moreover, peaks of the Fe element were observed in the Fe-TiO2 and Fe-fullerene/TiO2 composites. The degradation of MB solution by UV-light irradiation in the presence of photocatalyst compounds was investigated in complete darkness. The degradation of MB concentration in aqueous solution occurred via three kinds of physical phenomena: quantum efficiency of the fullerene; organo-metallic reaction of the Fe compound; and decomposition of TiO2. The degradation rate of the methylene blue solution increased when using Fe-fullerene/TiO2 compounds.