Presently, We have constructed an olig-d(T) primed directional cDNA library from the silkworm Dongchunghacho, an entomopathogenic fungus, of which species is belonging to Paecilomyces tenuipes Jocheon-1. To isolate and screen genes in the fungus, 626 expressed sequence tags(ESTs) were generated by a partial sequencing from the cDNA library. Paecilomyces tenuipes Jocheon-1 cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase(Pt-GAPDH) of Paecilomyces tenuipes Jocheon-1 was cloned from the above cDNA library. The complete cDNA sequence of Pt-GAPDHis comprised of 1,014bp encoding 338 amino acid residues. The deduced protein sequence of Pt-GAPDH showed higher homology with Beauberia bassiana-GAPDH(93% amino acid identity). Hydropathy analysis revealed that Pt-GAPDH protein is hydrophilic. The major three amino acids in its composition of amino acid residues were alanine(11.54%), valine(9.47%) and glycine(8.88%). The cDNA encoding Pt-GAPDH was expressed as a 37 kDa polypeptide in baculovirus-infected insect Sf9 cells. The Pt-GAPDH gene of Paecilomyces tenuipes entomopathogenic fungus consisted of three exons and two introns coding for 338 amino acid residues, and the genomic DNA length of the gene spans 1302bp. The accession number of the gene in GenBank are GU997099 for Pt-GAPDH cDNA and GU997102 for Pt-GAPDH genomic DNA.

To evaluate the biohazard properties of an extremely low frequency electromagnetic field (ELF-EMF), we explored the physical properties of the ELF-EMF that generates the electric current induction in the secondary coil from the chamber of a primary solenoid coil. We subsequently explored the biological effects of a strong alternating electromagnetic field (EMF), ranging from 730-960 Gauss, on the mouse testis. Mice were exposed to an alternating EMF field induced by a rectangular electric current at 1, 7, 20, 40, and 80 Hertz, for 1, 3, 5, and 7 hours. The mouse testes were examined for proliferative activity and apoptosis using the in situ terminal deoxynucleotidyl transferase (TdT) method and by immunostaining of proliferating cell nuclear antigen (PCNA), respectively. We found that the electric currentm induction increased in the 6-8 Hertz range, and that exposure to an ELF-EMF induced the apoptosis of mouse spermatocytes. In situ TdT staining was found to be most prominent in 7 Hertz group, and gradually reduced in the 20, 40, and 80 Hertz groups. These data suggest that a strong EMF can induce reproductive cell death within a short time, and the harmful effects of the EMF are maximal at low frequency alternating EMFs.

Fungi belonging to the Paecilomyces spp. have recently been used as food and herbal medicines in Korea and are greatly popular as commercially available powdered supplement or dried fruiting body. Despite this acceptance and its use, little is known of the genes related to its reactive agents. Presently, We have constructed an olig-d(T) primed directional cDNA library from the silkworm Dongchunghacho, an entomopathogenic fungus, of which species is belonging to Paecilomyces spp. based on the previous identification of ITS1 and ITS2 at the molecular level and collected from Jocheon Miryang, Korea. To isolate and screen genes in the fungus, 626 expressed sequence tags(ESTs) were generated by a partial sequencing from the cDNA library. cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase(Pt-GAPDH) of Paecilomyces tenuipes- Jocheon was cloned from the above cDNA library. The complete cDNA sequence of Pt-GAPDH is comprised of 1,014bp encoding 338 amino acid residues. The deduced protein sequence of Pt-GAPDH showed higher homology with Beauberia bassiana-GAPDH(93% amino acid identity). Hydropathy analysis revealed that Pt-GAPDH protein is hydrophilic. The major three amino acids in its composition of amino acid residues were alanine(11.54%), valine(9.47%) and glycine(8.88%). The Pt-GAPDH gene of Paecilomyces tenuipes entomopathogenic fungus consisted of three exons and two introns coding for 338 amino acid residues, and the genomic DNA length of the gene spans 1302bp. The accession number of the gene in GenBank are GU997099 for Pt-GAPDH cDNA and GU997102 for Pt-GAPDH genomic DNA. More investigation works including gene expression, immunological analysis etc. will be carried continuously without hesitation after this presentation.

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 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 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

The purpose of this study was to investigate the effects of the collection time, co-culture and sperm penetration of canine oocytes on in vitro maturation and fertilization. The oocytes were cultured in TCM-199 media containing hormonal supplements (10% FCS, 10 IU/ml HCG, 10 IU/ml PMSG) at 5% CO2, 95% air, 38℃. The in vitro maturation rate to MⅡ stage of in vitro oocytes recovered from ovaries that collected at follicular, luteal and inactive phases of the reproductive phase for 44～72 hrs were 19.2%, 12.2%, and 6.0%, respectively. Follicular phases oocytes had a significantly higher in vitro maturation rate than oocytes collected at luteal and anestrus stage (p<0.05). The in vitro maturation rates to the MII stage of canine oocytes after 48 hrs of culture with glutathione, pyruvate, or glutathione + pyruvate were 12.5%, 10.7%, and 17.5%, respectively. This was higher than that in both alone or the combination of the two compared to the control group (19.0%). The sperm penetration rates of in vitro matured oocytes by fresh and frozen semen were 29/80 (36.3%) and 18/80 (22.5%), respectively. Although there are limited reports about canine oocytes co-culture and in vitro fertilization, our results on in vitro maturation is comparable to the results from other researches.

Diamondback moth (Plutella xylostella L.) belonging to genus Lepidoptera is a notorious pest of cruciferous crops worldwide. We evaluated the bioinsecticidal activity of the liquid cultures (LB and NB) of a bacterial strain, Serratia sp. EML-SE1, isolated from a diseased diamondback moth. The pathogenicity of a bacterial strain to diamondback moth was confirmed by the following procedures: treatment of liquid culture on cabbage leaves, ingestion of inoculated cabbage and mortality response. For the test, twenty 3rd instar larvae of diamondback moth were placed on the Chinese cabbage leaf in a round plastic cage (Ø 10 × 6 cm) and sprayed with the liquid cultures. After 72 hours, insecticidal activity of LB and NB cultures of Serratia sp. against P. xylostella larvae showed 91.7% and 88.3%, respectively. In addition, the bioinsecticidal activity on potted cabbage with 14 leaves in a growth cage (165 × 83 × 124 cm) also was similar to that of plastic cage experiment. Summarized, the Serratia sp. EML-SE1 may be a potent candidate as a bioinsecticidal agent to control diamondbac kmoth.

In order to understand the nature of population genetic structure of the notorious pest insect, Spodoptera litura (Lepidoptera: Noctuidae), a total of 159 individuals of the tobacco cutworm collected from six Korean and five Chinese localities were cloned and sequenced their mitochondrial A+T-rich region. A total of 90 haplotypes ranged 324 bp ~ 372 bp in length and 0.30% ~ 4.85% in sequence divergence. Compared to the preliminary mitochondrial COI gene sequence data that provided nearly identical sequences in all samples, the A+T-rich region provided substantially high sequence variation, validating the applicability of this molecule to the study of intraspecific variation. Overall, a very high per generation migration ratio (Nm = 5.91 ~ infinite), a very low level of genetic fixation (FST = 0 ~ 0.077796), and no discernable isolated population were noted to exist among populations of S. litura, although some significant genetic differentiations were found between several pairs of populations. These results collectively suggest that the S. litura populations in both Korea and China are very well interrelated to each other. The structural analysis revealed that the S. litura A+T-rich region harbored two stretches of the [TA(A)]n sequence, the poly-A stretch, and a stem-and-loop structure that are well found in other lepidopteran A+T-rich region. Furthermore, the region harbors the 13 ~ 19-bp long T stretch and 5-bp long motif that have been suggested to function as a possible recognition site for the initiation of replication of the minor strand of mtDNA also were found.

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) with the individuals collected from Korean localities. Although the 658-bp long mtDNA sequence provided only six haplotypes with the maximum sequence divergence of 0.456% (3 bp, from 91 individuals), but the ITS sequences provided 33 sequence types with the maximum sequence divergence of 0.78% (16 bp, from 35 individuals), confirming better applicability of the ITS sequences to the study of intraspecific variation. The ITS2 sequences of B. ignitus were shown to be the longest among known insects, ranging in size from 2,034 bp ~ 2,045 bp and harbored two duplicated repeats, indicating unusual structure of B. ignitus ITS2 sequences compared with other insect ITS sequences. 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.

Eumenis autonoe, a member of the lepidopteran family Nymphalidae (superfamily Papilionoidea) is an endangered species, and is found only on one isolated remote island, Jeju in South Korea, on Halla Mt., at altitudes higher than 1,400 meters. In this study, we report the complete mitochondrial genome (mitogenome) of E. autonoe. The 15,489-bp long E. autonoe genome evidenced the typical gene content found in animal mitogenomes, and harbors the gene arrangement identical to all other sequenced lepidopteran insects, which differs from the most common type found in insects, due to the movement of tRNAMet to a position 5’-upstream of tRNAIle. As has been observed in many other lepidopteran insects, no typical ATN codon for the COI gene is available. Thus, we also designated the CGA (arginine) found at the beginning of the COI gene as a lepidopteran COI starter, in accordance with previous suggestions. The 678-bp long A+T-rich region, which is second longest in sequenced lepidopteran insects, harbors 10 identical 27-bp long tandem repeats plus one 13-bp long incomplete final repeat. Such a repeat sequence has been, thus far, only rarely detected in lepidopteran mitogenomes. The E. autonoe A+T-rich region harbors a poly-T stretch of 19 bp and a conserved ATAGA motif located at the end of the region, which have been suggested to function as structural signals for minor-strand mtDNA replication.

A full genomic DNA microarray technique was employed to investigate the effects of Dongchunghacho on aortal and hepatic gene expression in apolipoprotein E knockout mice fed a high-fat/high-cholesterol diet. Male 8- week - old ApoE-/- mice were randomly divided into two groups, control(high cholesterol group; HC) and supplementation of Dongchunghacho (SD). All of the mice were fed a high-fet/high cholesterol diet with or without Dongchunghacho supplemented by 1% for 6 weeks. At first, lipid profile of the Dongchunghacho was measured by biochemical analysis. No differences were observed in serum triglyceride and total cholesterol levels between the two groups. Antigenotoxic effect of the Dongchunghacho was measured by the single cell gel electrophoresis assay (Comet assay) and quantified as % fluorescence in tail. Dongchunghacho supplementation decreased significantly leukocytic DNA damage and also there was a tendency of reduction in hepatic DNA damage in Dongchunghacho group compared with the control group. In up regulated genes in liver and aorta of the mice, genes with 0 to 2- fold difference in expression level between the two group (HD and SD) was very much more in liver than in aorta, on the contrary, those with 2-fold to 16-flod difference increased greatly rather in aorta than in liver. Also, almost the same results were observed in down regulated genes in liver and aorta between the two groups. These results suggested that supplementation of Dongchunghacho might be helpful in preventing leukocytic DNA damage induced by high fat diet, and has a more crucial roles in aortal gene expression.

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.

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.

The bumblebees, Bombus species are valuable natural resources being utilized for greenhouse pollination. Low level of genetic variation of Bombus species has been reported previously. In this study, we sequenced complete internal transcribed spacer 2 (ITS2) of the nuclear rDNA from 100 individuals of B. ardens collected from seven localities in Korean peninsula. The ITS2 sequence of B. ardens is longest, ranging in size from 1,940 bp-1,954 bp among known in insects, which ranges approximately from 241 bp-1,728 bp. The ITS2 sequences have ~51% of C+C content and contain each two 27 bp repeats, 20 bp repeats, 33 bp repeats, and 34 bp repeats at the beginning. Such repeats were not found in other insects. Uncorrected pairwise distance among 96 sequences that were obtained from 100 individuals revealed a maximum sequence divergence of 1.03%. Genetic diversity (π) of each population ranged from 0.007801 to 0.009627, and the lowest diversity was obtained from islet population of Ulleungdo, indicating possibly small, isolation of the population. Significant level of genetic distance was only found when Ulleungdo population was compared to two other mainland populations. Except for this, overall, a very high rate of per generation migration ratio (Nm=7.1-infinite) and a very low level of genetic fixation (FST=0-0.06546) were detected between pairs of localities. Analysis of hierarchical relationships among localities consistently revealed no statistically significant structure among populations. Taken these together, the B. ardens populations on the Korean peninsula are panmictic this is consistent with our understanding of the dispersal capability.

To understand geographic genetic variation of the species and relationships among populations of the bumble bee, Bombus ardens, we sequenced a portion of mitochondrial COI gene, which corresponds to "DNA Barcode" region (658 bp) from 101 B. ardens individuals collected over 11 localities in Korea. The sequence data were used to investigate genetic diversity within populations and species, geographic variation within species, phylogeographic relationship among populations, and phylogenetic relationship among haplotypes. A total of nine haplortpes were found, but they very close to each other (a maximum sequence divergence of 0. 304%). Summarized, overall moderate to low genetic diversity within populations and species was characteristic, concordant with the high potential to disperse of B. ardens in Korea. There was no clear regional subdivision was observed and relatively high rate of gene flow among localities and low FST was characteristic.

Al though the changes in tooth morphology and hardness by hydrogen peroxide(H20 z) have been r‘epor‘.ted .‘ the pαr。o야t뻐ec야tive role of heme oxygenase-l(HO-l) against the cytotoxic effects of H202 has not been clarifïed i n human pulp cells ln this st udy. we investigated whether HO-l is involved in Hz0 2-induced cytoLox icity a nd examined the production 0 1' dentin sia lophosphoprotein(DSPP) and other mineralization markers‘ in hllman pu lp cells H202 decreased cell viabi lity, but increased HO-l and DSPP expression in a concentra tion and time dependent manner . Inhibitors of gllanylate cyclase. PI3K, ERK. and p38 MAP kinase blocked H202-indllced cytotoxicity and the expression of HO-l and DSPP mRNAs in pulp cells. These data suggest that the induction of HO-l by H202 in plllp cells plays a protective role against the cytotoxic effects 0 1' HzOz and stimulates DSPP expression‘ reslllting in prematllre odontoblast dilTerentiation throllgh pathways that involve cGMP‘ p38. and ERK.