Grapholita molesta occur four times a year and Carposina sasakii occur twice a year, and both pests do damage on stone fruits such as peach, apple, plum, apricot, etc. Grapholita molesta is worldwide distributed in temperate and subtropical areas including South Korea. But, Carposina sasakii distributed in South Korea, Japan, China and Asia, and has been managed as an important import quarantine pest by the authorities of United States, Canada and Taiwan. Forecasting of both pests in Korea is currently done through the investigation of 1,000 fruits per 10 trees (100 fruits / tree) in designated peach orchard. However, this method is very difficult to observe the pest by bagging of peach and require too much time and labor. Therefore, we tried to carry out a new forecasting method by using of sex pheromone traps for newly standardized method as an alternative. Using sex pheromone trap, attractiveness of G. molesta was proved to be 2.5 > 1.5 > 0.5 m by the height and the border => outside > center by the position. Attractiveness of C. sasakii made no difference in height, but, more trapped at the center and border than outside in position at peach orchard.

The spotted-wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), originally distributed across a few Asian countries, including South Korea, has invaded North America and Europe, but is absent from Australia. In order to export the South Korean grape cultivar Campbell Early to Australia, its potential to serve as oviposition and development medium for SWD must first be determined. In this study, we determined the oviposition and development potential of SWD on Campbell Early, after elucidating the SWD life cycle and establishing an artificial diet-based mass-culturing system. An investigation of the life cycle under five temperature regimes (16, 19, 22, 25, and 28°C) showed that the durations of the egg, larval, and adult stages were shorten when temperature was increased from 16, 19, 22, 25, and 28°C, but pupal duration was shortest at 25°C and extended again at 28°C. A test of oviposition and development potential of SWD on Campbell Early grape clusters showed oviposition of 30.8 ± 6.8 eggs per cluster of injured grapes and 157.7 ± 16.2 eggs on a culture dish of artificial diet. However, in a similar experiment using uninjured grape clusters, only a single egg was deposited on the grape skin, which soon dried. In light of these results, newly harvested grapes left at vineyards during daily harvests are unlikely to serve as an oviposition and development medium for SWD, as long as the grapes remain uninjured.

Recently an outbreaked pest belongs to Hemiptera: Recaniidae, Ricanula sp. is greatly concerned about the outspreading throughout the South of Korea by wide range of host, including Cornelian cherry, Jujube, and peach trees. In Chungbuk province, this pest was first occurred at Jincheon and Okcheon in 2012, Cheongju in 2013 and now found out at Eumseong and Goesan in 2015. Ricanula sp. was oviposited directly into one-year twig, did damages on fruit-bearing formation and finally withered the host. This study was performed to understand the ovipositional characteristics and to develop the standardized forecasting method. Oviposition by Ricanula sp. was abundant in tree than in bush, adult laid eggs on new inner twigs and then covered with wax compound. Total no. of oviposited egg-mass was 10 to 318, and that of on new twig was 5 to 185 per tree, with different to host trees. Thickness of oviposited twigs were done within 2 ~ 5.5 mm and the height was mostly founded with range of 1 to 2 m, founded with highest height over 3 m. Oviposited no. of egg-mass within 30 cm twig was appeared differently from 2 to 7 every host. From based on this investigated result, we provide this for standardized forecasting method. This pest will need to control when egg-mass will occur over 2 at new twig, within 30 cm from the tip, set as total 25 point/ 5 plants (5 point per plant).

The aim of this study was to carry out the comparative analysis of the brushing force following various brushing techniques bytoothbrush mounted pressure sensing unit. The study group consisted of 10 dental hygienist participants. The brushing forces (on buccal area of each first molar) were monitored on 8 different kinds of brushing techniques; Fones, Bass, Rolling, Scrub, Charters, stillman, Modified bass and Modified stillman. In Bass, Charters, Fones and Scrub method, force distributions showed a small gap of maximum and minimum value (Max/Min) while a big difference was noted in Modified bass, Modified stillman, Rolling and Stillman methods. Especially, the biggest difference of Max/Min value was observed in the area of lower left first molar. In conclusion, highly delicate manual skill is needed in showing big error range of force distribution. It means that careful force needs to be focused during toothbrushing instruction when a delicate manual skill was carried out.

The spotted-wing drosophila (SWD), Drosophila suzukii (Diptera: Drosophilidae), was originally observed in a few Asian countries, but is now found even in North America and Europe. Genetic information on geographic variation and relationship may broaden our understanding of origin and migration. As a first step, in this study, a portion of mitochondrial COI gene was sequenced to understand genetic relationship and diversity in Korea. Sequencing of 104 individuals provided 57 haplotypes, with the maximum sequence divergence of 1.5%, suggesting high haplotype diversity and moderate sequence divergence. Comparison to GenBankregistered D. suzukii haplotypes (possibly from Spain, Portugal, USA) has shown 100% sequence identity to most of the haplotypes found in this study, but two USA sequences were found to be independent haplotypes, with the sequence divergence ranging from 0.5% ~ 1.4% from our samples in the 553-bp comparison. Phylogenetically, no separable group was found, but, population genetically, the only Chinese population, Sandong, was significantly differentiated (p < 0.05) from all Korean populations, without sharing any haplotype. Among 28 pairwise comparisons of Korean populations only two comparisons showed a significant genetic differentiation, indicating that no population in Korea is completely isolated. Geographically, one haplotype (SWDBA06) was relatively widespread (five among nine localities) and a few haplotypes were found in more than one locality, but most haplotypes were restricted in a locality as a single individual. Overall, high rate of per generation female migration (Nm = 0.75 ~ infinite) and low level of geographic separation (FST=0~0.40) among localities were characteristic. Current data is limited mainly to Korean localities, thus, an expanded study may provide further scrutinized analysis for the fly.

We newly sequenced mitogenomes of five skippers belonging to Lepidoptera to obtain further insight into characteristics of butterfly mitogenomes and performed phylogenetic reconstruction using all available gene sequences (PCGs, rRNAs, and tRNAs) from 85 species in 19 families in eight superfamilies. The general genomic features found in the butterflies also were found in the five skippers: a high A/T composition (79.3% - 80.9%), dominant usage of TAA stop codon, similar skewness pattern in various levels, consistently long intergenic spacer sequence between tRNAGln - ND2 (64-87 bp), the ATACTAA motif betweent RNASer(UCN) and ND1, and characteristic features of the A+T-rich region (the motif ATAGA, varying length of poly-T stretch, and poly-A stretch). The start codon for COI was CGA in four skippers as typical, but Lobocla bifasciatus evidently possessed canonical ATG as start codon. Phylogenetic analyses mainly yielded the consensus superfamilial relationships ((((((Bombycoidea + Noctuoidea + Geometroidea) + Pyraloidea) + Papilionoidea) + Tortricoidea) + Yponomeutoidea) + Hepialoidea) with a high support for most nodes, confirming the validity of Macroheterocera and its sister relationship to Pyraloidea. Within Rhopalocera the familial relationships (Papilionidae + (Hesperiidae + (Pieridae + ((Lycaenidae + Riodinidae) + Nymphalidae))) were strongly supported, confirming invalidity of the superfamily Hesperioidea. On the other hand, superfamilial relationships among Noctuoidea, Geometroidea, and Bombycoidea and the familial relationships among Saturniidae, Sphingidae, and Bombycidae were dubious, requiring further representative taxon sampling.

Glyphodes quadrimaculalis (Lepidoptera: Crambidae) feed on a root tuber of Cynanchum wilfordii (Asclepiadaceae) that is one of the most famous traditional medicines in Korea. The genus Glyphodes includes ~130 species distributed worldwide, so the complete mitochondrial genome (mitogenome) would be helpful for bio-identification, biogeographic studies, and multigene-based phylogeny. The 15,255-bp long G. quadrimaculalis genome comprises 37 typical genes and one large non-coding region, with the typical arrangement found in Lepidoptera. Of the 13 protein coding genes (PCGs), 12 begin with typical start codons found in insect mitochondrial PCGs, but the COI gene starts with atypical CGA. One of the noteworthy features of the genome includes the presence of a 51-bp long non-coding space sequence located between tRNAGln and ND2 that reveals high sequence homology (71.4%) to the neighboring ND2 gene, indicating the origin of the region by partial duplication of the ND2 gene.

For more than two decades, the intracytoplasmic sperm injection (ICSI) technique has been used as a valuable tool to provide opportunities for studying fertilization, treating human infertility, and producing transgenic animals. Not only in facilitating fertilization but also in propagating mammalian species, ICSI has enhanced the potential of assisted reproductive technologies in human. Polyspermic fertilization has been one of major problems in pig reproduction, but the ICSI helped to solve the problem, and used widely to generate transgenic piglets. Although the ICSI technique is considered to be a very useful tool in assisted reproductive technologies, including generation of transgenic animals, there are some disadvantages using the technique. In this review, we describe the ICSI technique and its application in animal production and human infertility, and discuss advantage and disadvantage of the technique in mammals.

Bladder cancer is a common cancer in smoking men and may correlate with mechanosensitive potassium channels because the urinary bladder is a stretch sensing organ. Two-pore K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to play a critical role in both cell apoptosis and tumorigenesis. Of the channels, TREK1 can be activated by many physiological stimuli, including polyunsaturated fatty acids, and intracellular pH, hypoxia, and neurotransmitters. Here we attempted to determine whether TREK1 is functionally expressed in bladder cancer 253J cells. K2P channels, including TREK1, TREK2, TASK1, TASK3, and TWIK1, were quantified in cultured human bladder cancer 253J cells using real time quantitative RT-PCR (qRT-PCR) analysis. Among them, TREK1-like channel was recorded at a single channel level using the patch-clamp technique. The TREKl-like channel, with single-channel conductance of ~90 pS at −80 mV, was recorded in symmetrical 150 mM KCl using an excised inside-out patch configuration. The current- voltage relationships were linear and were insensitive to tetraethylammonium. The channel was activated by membrane stretch, free fatty acids, and intracellular acidosis. These results with electrophysiological properties resemble to those of K2P channel, for instance, TREK1. Therefore, we conclude that TREK1 channel is functionally present in bladder cancer 253J cells.

Hipparchia autonoe belongs to the family Nymphalidae (Lepidoptera) and is designated as an endangered insect and national monument in Korea. It only inhabits a very restricted area on Mt. Halla but is widely distributed in several Asian countries including Mongolia. A previous study conducted to understand the genetic relationship between Mt. Halla and Mongolian H. autonoe for conservation purposes suffered from a limited number of samples. Therefore, we sequenced the DNA barcode region of an additional 36 H. autonoe individuals, combined them with previous data from 19 individuals, and performed phylogenetic and population genetic analyses. Furthermore, the internal transcribed spacer 2 (ITS2) region was also sequenced from the 36 samples as a nuclear DNA marker. The existence of independent haplotypes, sequence types, and significant FST estimates (p < 0.05) between Mt. Halla and Mongolian populations indicated hampered gene flow between the populations. Nevertheless, an absence of a reciprocal monophyletic group in Mt. Halla and Mongolian populations by cytochrome oxidase subunit I gene- and ITS2-based phylogeny suggests that the genetic isolation of the Mt. Halla population from the Mongolian populations seemed not large enough to consider them independent genetic entities.

Lepidoptera is one of the largest insect orders, but the phylogenetic relationships within this order, have yet to be completely described. One of the unresolved relationships includes the monophyly of Papilionoidea in relationship with the monotypic superfamily Hesperioidea. We newly sequenced five hesperid mitochondrial genomes (mitogenomes), representing four subfamilies: Pyrginae (Daimio tethys and Lobocla bifasciatus), Coeliadinae (Choaspes benjaminii), and Hesperiinae (Potanthus flavus), and Heteropterinae (Carterocephalus silvicola). Along with these newly sequenced hesperid genomes phylogenetic analysis was conducted with all available lepidopteran mitogenomes including three reported species of Hesperiidae that consisted of ~70 species in ten lepidopteran superfamilies. The test for the effect of optimization schemes, such as exclusion and inclusion of third codon position of 13 PCGs, other genes (22 tRNAs and two rRNAs), and with and without partitions also was performed. Majority of datasets consistently placed the monophyletic Hesperiidae the sister to ((Pieridae + Lycaenidae) + Nymphalidae), placing another true butterfly family Papilionidae as the basal lineage of this group, presenting the relationships (Papilionidae + (Hesperiidae + ((Pieridae + Lycaenidae) + Nymphalidae))). Consistent to previous result, Pyraloidea was placed as the sister to ((Bombycoidea + Geometroidea) + Noctuoidea), placing the Macrolepidoptera as non-monophyletic group.

In the present study, the 17,694-bp long complete mitochondrial genome (mitogenome) of the dwarf honey bee, Apis florea (Hymenoptera: Apidae), is described with an emphasis on the noteworthy triplicated tRNAser(AGN) region and an extraordinary long A+T-rich region with repeat regions. The gene arrangement of A. florea mitogenome is identical to that of A. mellifera, but has triplicated tRNASer(AGN), each of which contains the precedent 44 bp-long and following another 64 bp-long repeats plus one complete first repeat abutting to tRNAMet. A total of 1,610-bp long two repeat regions in 1,987 bp-long A+T-rich region is composed of nearly identical 141 ~ 219-bp long five tandem repeats and 50 ~ 52-bp long 12 tandem repeats that are encompassed by three non-repeat sequences. One of the common interpretations for such repeat sequence is slipped-strand mispairing and unequal crossing-over events during DNA replication.

The objective of this study was to investigate the effects of NEAA and leptin supplemented to in vitro culture medium on the developmental competence of porcine embryos after intracytoplasmic sperm injection (ICSI), and to modify the culture condition to improve the quality and the development of ICSI-derived porcine embryos in vitro. After ICSI, the putative zygotes were then cultured in PZM-3 medium with/without NEAA or leptin. The proportion of embryos that developed to the blastocyst stage significantly increased when 1% NEAA (24.62%) was added to the medium compared with 2% NEAA and no NEAA group (17.24% and 20.24%, respectively, p<0.05). The effect of different concentration of leptin (0, 10, 100, 500 ng/ml) was evaluated on the development of porcine ICSI embryos cultured in vitro. In case of blastocyst formation, 100 ng/ml group (27.05%) showed significantly higher rate than 10, 500 ng/ml, and control group (23.45%, 17.99%, and 19.68%, respectively, p<0.05). We also evaluated the effects of different NEAA and leptin treatment time on the development of porcine embryos after ICSI. Among groups of embryos cultured in the presence of NEAA or leptin for whole 7 days (D 1-7), first 4 days (D 1-4), the subsequent 3 days (D 5-7), both NEAA (27.13%, 21.17 %, and 17.56%, respectively, p<0.05) and leptin (25.60%, 20.61%, and 16.53%, respectively, p<0.05) showed that supplementation for whole 7 days significantly increased the blastocyst formation rate compared with the other groups of D1-4 and D5-7. We further evaluated the combination effect of 1% NEAA and 100 ng/ml leptin compared with the effect of each supplementation with 1% NEAA or 100 ng/ml leptin or no supplementation on development of embryos. For blastocyst formation, combination group of NEAA and leptin (24.78%) showed significantly higher rate than other three groups (18.37%, 20.44 %, and 13.27%, respectively, p<0.05). We further evaluated the expression of proapoptosis genes such as BAX and BAK and anti-apoptosis genes, BCL-XL and BCL-2 in blastocysts cultured in the presence of 100 ng/ml leptin. RT-PCR analysis revealed that leptin supplementation significantly decreased the expression of pro-apoptosis genes as well as increased the expression of anti-apoptosis genes. These results of present study demonstrate that NEAA and leptin could improve the in vitro development of ICSI- derived porcine embryos with optimal concentration of each reagent. Furthermore, the optimal culture condition could increase the quality of ICSI-derived embryos in vitro.

Chicken Insulin-like Growth Factor-1 (cIGF-1), one of the most important hormone for regulating physiological function includes body growth, muscle volume, bone density, chicken cell development and metabolism. In order to find in vitro Knokdown expression of cIGF-1, this study introduced tetracycline inducible RNA interference expression system (TetRNAi system). Tet system can inductively control high expression of extrinsic genes and expression of intrinsic genes. So it has advantages such as minimized physiological side-effects any cell and low cytotoxicity. RNAi system is proving to be a powerful experimental tool for inhibition of gene expression and post-transcriptional mechanism of gene silencing. RNAi is mediated by small interfering RNA (siRNA) consisting of 19- to 23- nucleotide double-stranded RNA duplexes that promote specific endonucleolytic cleavage of mRNA targets through an RNA-induced silencing. Then, this study RNAi-based gene knockdown can be achieved by retroviral-based expression systems. Stable integration of our inducible siRNA vector allowed the production of siRNA on doxycycline induction, followed by specific down regulation of chicken IGF-1 gene. Analyses of Real-time PCR to determine expression of the cIGF-1 gene showed successful from chicken embronic fibroblast (CEF) cells with the reduced rate of an approximately 92%. Our results demonstrate the successful regulation of cIGF-1 knockdown expression in CEF cells and support the application of an tetracycline inducible RNAi expression system in transgenic Mini chicken production. This research was supported by Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

The aim of this study was to examine the effect of acteoside (the cyclin-dependent kinase inhibitor) on the SCNT efficiency with adult fibroblasts in dog. Canine adult fibroblasts were obtained from muscle and cell cycle of fibroblasts was synchronized by culturing to confluency, serum starvation and treating with 30 μM acteoside for 48 h. Cell cycle stages, cell cytotoxicity (apoptosis) and, prduction of reactive oxygen species (ROS) were analyzed using flow cytometry. The canine cells, prepared by confluent-cell culture or treating with 30 μM acteoside for 48 h, were injected into enucleated in vivo matured oocytes, the couplets were electrical fused and activated by calcium ionomycin. SCNT embryos using acteoside-treated fibroblasts were surgically transferred into oviducts of estrus cycle synchronized recipient dogs. In cell cycle synchronization (G0/G1), there was no significant difference between serum starvations (83.9%) and acteoside treated groups (81.3%) that were higher than confluent group (78.5%). In production of apoptosis, confluent and acteoside treated groups (4.3 and 4.5%, respectively) were generated less than serum starvation group (21.8%). In case of ROS, serum starvation group was induced a significantly higher than other groups. After synchronization of the donor cell cycle, either confluent or acteoside treated, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into naturally estrus-synchronized surrogates. Fusion and cleavage rate of acteoside treated group were 64.1 and 41.5%, which were higher than those of confluent group (53.9 and 20.6%, respectively). The reconstructed embryo development rates to 4-cell and 8-cell in acteoside treated group were 29.5 and 14.8%, respectively, while confluent group showed 11.1 and 3.2%, respectively. Total 54 SCNT embryos using acteoside-treated fibroblasts were transferred into oviducts of 2 recipient dogs and one recipient finally delivered one puppy, whereas din`t detected pregnancy on transfer of cloned embryos reconstructed with confluent cells in 6 surrogate dogs. In conclusion, the results of the current study demonstrated that canine fibroblasts could be successfully arrested at the G0/G1 stage with reduced the formation of ROS and apoptosis after acteoside treatment. This results may contribute to improve the effi-ciency of canine SCNT. * This research was supported by iPET (Grants 110056-3), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.