The objective of this study was to evaluate in vitro production of bovine embryos in Hanwoo. Oocytes were collected by ovum pick up (OPU) from ovaries of genetically high-value Hanwoo or by needle puncture from ovaries of slaughtered cattle. OPU was done every 3 4 days duing experimental period and collected oocytes were fertilized in vitro in both OPU and needle puncture groups. First, We compared the in vitro maturation rate in two groups (Experiment 1). 545 oocytes were recoverd from 4 females by 32 trials of OPU and then 433 oocytes were shown MⅡ stage after in vitro maturation (79.4%). In case of needle puncture group, 1905 oocytes were collected and then 1420 oocytes were matured to MⅡ stage during in vitro culture(74.5%). Second, we compared the developmental rate to blastocyst in two groups (Experiment 2). 1420 oocyte by needle puncture were fertilized with frozen-thawing semen; the cleavage rate 24 48 h after in vitro fertilization (IVF) was 88.6% and blastocyst development rate was 20.5% in needle puncture group. Even though there is lower cleavage rate after IVF in OPU group (84.8%), blastocyst development rate was higher compared with needle puncture group (26.4%). In conclusion, Blastocyst developmental rate could be increased by OPU than classical method of needle puncture. Improvement of bio-technique in collecting oocytes could be applied to understand the reproductive physiology in cattle, expecially Hanwoo. Therefore, further investigation should be done to clarify the efficiency and advantage of OPU involved in reproduction in animals and human being. This research was suppoted by Imsil-gun agricultural technology service center.

Some tissues retain extensive regeneration potential through out adult life and remain as active sites of cell production. Various cell types present in tissues are being produced through proliferation and progressive specialization from a pool of stem cells. In this regard, adult stem cells (ASCs) are multipotent progenitor cells with an ability to proliferate in vitro and undergo extensive self-renewal and differentiation into a wide range of cell types, including adipocytes, chondrocytes, osteocytes, myocytes, cardiomyocytes and neurons. In addition, recent studies showing the abilities of ASCs in generating oocytes-like cells (OLCs) present new perspectives to understand the specification and interaction during the germ cell formation and oogenesis. In the present study, ASCs were established from skin, adipose and ovarian tissues of minipigs. Isolated cells exhibited a fibroblast-like morphology with higher proliferation potential and stronger alkaline phosphatase (AP) activity. ASCs from all tissues expressed pluripotent transcriptional factors, such as Oct-3/4, Nanog and Sox-2 and phenotypic markers, including CD29, CD44, CD90 and vimentin. Further, ASCs were successfully dIfferentiated into osteocytes, adipocytes and neuron-like cells. Upon induction in oogenesis specific media, all ASCs were capable of differentiation into OLCs by exhibiting distinct morphological features. Generated OLCs expressed a range of germ cell specific markers, such as Vasa, deleted in Azoospermia-like (DAZL) factor, stella, c-kit, c-Mos, synaptonemal complex protein 3 (SCP-3), growth differentiation factor 9b (GDF- 9b), zona pellucida C (ZPC) and follicle stimulating hormone receptor (FSHR) at different time points of induction. Differentiated OLCs were also positive for the expression of Vasa and DAZL protein markers. Our findings showing that OLCs can be generated from ASCs of different tissue origin may offer pig as a suitable model for designing transgenic application strategies for reproductive tissue therapy. However, further studies are needed to understand the cellular and molecular mechanisms involved in germ cell differentiation from tissue specific stem cells.

Successful pregnancy requires well-coordinated interactions between the maternal uterus and the developing embryo in pigs. In pigs, implantation begins around Day 12 of pregnancy. During this period, conceptus undergoes a dramatic morphological change and secretes various factors such as estrogens, interleukin-1 beta (IL1B), and interferons. Estrogens produced by conceptuses act as the signal for maternal recognition of pregnancy, and the mechanism of estrogen action is explained by the endocrine and exocrine theory. The uterine endometrium becomes receptive to the conceptus by changing cell adhesion molecules, polarizing epithelial cells and increasing secretory activity. Some changes of uterine activity are affected by the ovarian hormone, progesterone, but the presence of conceptus in the uterus also induces changes of endometrial functions, including most importantly maternal recognition of pregnancy. Many factors, such as hormones, cytokines, enzymes, extracellular matrix proteins, and transport proteins are reported to be present at the maternal-fetal interface and function in the establishment of pregnancy in pigs. However, understanding of the cellular and molecular events occurring in the endometrium is not complete. In recent studies we made some progress on understanding of expression and function of genes involved in maternal-fetal interaction for the establishment and maintenance of pregnancy in the uterine endometrium in pigs. Firstly, we found that lysophosphatidic acid (LPA) was present at the maternal-and fetal interface at the time of implantation and LPA receptor 3 was uniquely expressed in the endometrium during early pregnancy. Secondly, we observed that salivary lipocalin (SAL1), a lipid-binding protein, was uniquely expressed in the uterine endometrium at the time of embryo implantation, and its expression was regulated by IL1B. Furthermore, expression of IL1B receptors are regulated by estrogen and IL1B, and IL1B functions in expression of genes related to prostaglandin synthesis and transport. Thirdly, we found that calcium regulatory molecules TRPV6 and S100G were dynamically regulated in the uterine endometrium during pregnancy, suggesting that regulation of calcium ion concentration may important for the embryo implantation and the maintenance of pregnancy. Finally, we observed that an MHC class II molecule, SLA-DQ, is expressed in the uterine endometrium at the time of conceptus implantation and its expression is essential for successful pregnancy, indicating that appropriate maternal-fetal immune interaction is required for the maintenance of pregnancy. Further analysis of these molecules will provide insights into the cellular and molecular basis of maternal-and fetal interaction during pregnancy in pigs.

In the first part of this study, a novel culture device the named oil-free micro tube culture (MTC) system for in vitro culture (IVC) of murine and porcine embryos was introduced. Parthenogenetic mouse and porcine embryos were placed into 0.2-mL thinwall flat cap PCR tubes and cultured to the blastocyst stage. Conventional drop culture was used as the control. Murine embryos in MTC had a higher blastocyst formation rate and larger population of cells in the blastocysts. This was due to higher numbers of trophectoderm (TE) cells rather than inner cell mass cells. On the other hand, the 'MTC' system in the pig showed similar (in 20 μl medium volume) or lower (in 10 μl medium volume) blastocyst formation rate when compared with drop culture system. In the second part of this study, dexamethasone (DEX) and leukemia inhibitory factor (LIF), which suppress PGF2α, were directly supplemented into ET media, and transfer of the embryos to surrogate was followed. In the cattle industry, embryo transfer technology has been used to produce the most valuable cows or bulls. Numerous factors such as heat stress, mastitis, manipulating female reproductive tract may contribute to early embryonic loss through premature increases of uterine luminal concentrations of PGF2α in cows. Furthermore, addition of PGF2α to culture medium has been shown to inhibit the development and hatching of mammalian embryos. When DEX and LIF were supplemented, the pregnancy rate (6 month post-ET) was increased from 56.0% to 68.3%. In IVC experiment, DEX and LIF supplementation supported hatching of bovine embryos in the presence of PGF2α in the medium (from 16.9% to 40.6%). Additional ET experiments using alternative drugs are currently under investigation. The present work was supported by the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry and Fisheries (MIFAFF; 109020-3).

Although baculoviruses have a long history of safe use as specific, environmentally benign insect control agents, their use has been limited by several factors, especially their slow speed of action. In this study, we intended to improve the insecticidal activities of Autographa californica nucleopolyhedrovirus (AcMNPV) by expressing Kunitz-type toxin isolated from venoms of Bombus ignitus or Araneus ventricosus. For this, recombinant AcMNPVs, AcBi-KTT, AcAv-Tox1 and AcAv-Tox2 expressing Bi-KTT, Av-Tox1 and Av-Tox2, respectively, under the control of p10 gene promoter were constructed. While polyhedra produced by these recombinant viruses were identical to those of the wild-type AcMNPV in shape, their sizes were relatively smaller than those of the AcMNPV. Among recombinant viruses, AcBi-KTT and AcAv-Tox2 showed significant reduction in median lethal time (LT50) against Spodoptera exigua larvae. Especiaaly, these two viruses showed about 6.2~10-folds higher polyhedra production rate compared to that of the AcMNPV. These results suggested that Kunitz-type toxins from insect venom could be successfully applied to improve insecticidal activity of baculoviruses.

Rice stripe virus (RSV), the type member of the genus Tenuivirus, causes rice stripe disease and the viral transmission is mediated through the sucking by small brown planthopper, Laodelphax striatellus. Considerations have been mainly focused on the protection of rice from RSV and/or the planthopper, rather than the interaction between RSV and the insect. To clarify the interaction, in this work, mRNA was extracted from RSV-viruliferous planthopper with non-viruliferous control, and expressed sequence tag (EST) databases were generated based on 454 GS-FLX pyrosequencing technology for comparative analysis. RSV-viruliferous planthopper had ca. 2500 isotigs, which included genes on biological process (19%), cellular component (13%), molecular function (22%) and no hits (46%) from gene ontology (GO) analysis; this structure was similar to the control. However, in the viruliferous planthopper, 109 isotigs were up-regulated and 660 isotigs were down-regulated, compared to the non-viruliferous control. These RSV-dependently regulated genes may have important function in the behavior of planthopper or the transmission of RSV.

Plasmids from Bacillus thuringiensis have been implicated in pathogenicity as they carry the genes responsible for different types of diseases that in mammals and insects. A novel serogroup (H3a3b3d), B. thuringiensis strain K4 which showed mosquitocidal activity against Anopheles sinensis and Culex pipiens pallens, was isolated from fallen leaves in Mungyeong city, Republic of Korea. In contrast to the complicated plasmid profiles of B. thuringiensis H3 serotype strains, the strain K4 (designated as serovar mogi) had only one large plasmid (>200kb) on which the toxin genes were occasionally located. A 454 pyrosequencing was used for the complete sequencing of the large plasmid. The sequence analysis showed that k4 plasmid had at least seven putative cry genes, ending up to showing 84%, 75%, 73%, 58%, 84%, 39% and 75% homology with Cry27Aa, Cry19Ba, Cry20-like, Cry56Aa, Cry39ORF2, Cry8Ba and Cry40ORF2 toxins in amino acids, respectively. This novel 3a3b3d type strain, B. thuringiensis serovar mogi, can be used as a good resource for studying unknown mosquitocidal cry genes. The E. coli-B. thuringiensis shuttle vector, pHT1K was used to clone these cry genes for characterization. In each clone, the level of transcription and production of crystal proteins will be investigated in near the future.

The green peach aphid (Myzus persicae) is a cosmopolitan pest of agricultural and horticultural crops and causes serious economic damages. M. persica has rapidly developed resistance to a wide variety of insecticides, including pyrethroids. Target site insensitivity mechanism mediated by two mutations (L1014F and M918T) on the para-type voltage-sensitive sodium channel (vssc) is mainly responsible for pyrethroid resistance. To predict the vssc resistance allele frequency, quantitative sequencing (QS) protocol was established. Frequency prediction equations generated from the plots of signal ratios and amplification critical time showed a high correlation coefficient (r2>0.993), indicating its high accuracy in prediction. QS results revealed that the kdr-type L1014F mutation is only present in Pyeongchang strain. No field strains of M. persicae possessed the super-kdr type M918T mutation. However, a novel M918L mutation was found by genotyping approach. The allele frequencies of M918L and L1014F were 0% to 53% in populations examined, and the level of M918L mutation frequency was closely related with pyrethroid resistance. Therefore, QS-based detection of M918L mutation frequency should faciltate the monitoring of pyrethroid resistance in the field.

The green peach aphid (Myzus persicae) is a serious pest of agricultural and horticultural crops all over the world. M. persica has rapidly developed resistance to a wide variety of insecticides, including carbamates. The E4/FE4 carboxylesterase is known to be involved in carbamate resistance. To compare the E4/FE4 carboxylesterase gene copy number, as a genetic resistance marker, between seven field strains, quantitative real-time PCR (qPCR) was performed. In addition, quantitative sequencing (QS) was employed to predict the frequencies of acetylcholinesterase (AChE) mutations (A301S and S431F) that are associated with target site insensitivity. All M. persica strains examined possessed the S431F mutation in the heterozygous state except for a susceptible strain, implying the possibility of AChE duplication. In contrast, no A301S mutation was found. Frequency prediction equation was generated from the plots of signal ratios and amplification critical time, which showed a high correlation (r2>0.996). QS analysis of M. persicae populations revealed that the allele frequency of S431F ranged 4% to 63%. Taken together, the AChE resistance allele frequencies determined by QS and the E4/FE4 gene copy number by qPCR should facilitate the detection and monitoring of carbamate resistance in M. persicae in the field.

Entomopathogenic fungus Aschersonia aleyrodis naturally occurred on citrus whitefly, Dialeurodes citri nymph was often observed in organic citrus orchards, Jeju. The genus Aschersonia is also known to be toxic against scale insects and other pests. However, little is known about artificial media for mass production of spores of Aschersonia species. Grains are excellent sources of media for mass conidia production of various entomopathogenic fungi. The yeast extract, which converts carbohydrates to carbon dioxides and alcohols, contains a large amount of vitamin B complexes which facilitate the carbohydrate metabolism. The more yeast extract content the more conidia production on artificial medium made from commercial corn flour and corn gluten feed. The number of conidium produced on oat, millet, sorghum, and unhulled barley medium containing 1% yeast extract were 1.8, 1.8, 1.6, and 2.1×1010/plate (90mm × 15mm), respectively. However, the greatest yeast effect among four media showed appeared on sorghum medium, which produced 25 times higher spore production than sorghum alone. Furthermore, the conidia from solid sorghum medium could be easily harvested with cell scraper.

Aschersonia aleyrodis was well-known to be a biological control agent for citrus whitefly, Dialeurodes citri. This entomopathogenic fungus is naturally occurred in organic farming citrus orchards in Jeju. Both lime-sulfur and Bordeaux mixtures are extensively used today to control citrus diseases like citrus melanose and citrus scab, especially in organic farming pest management program. The high concentrated lime-sulfur is also used for pest control such as pink citrus rust mite and scale insects. This study was focused to test the conidial germination and sporulation of Aschersonia aleyrodis on potato dextrose agar (PDA) medium containing different concentrations of two fungicides. The conidia of Aschersonia aleyrodis grown on PDA mixed with commercial bordeaux mixtures, CM150-505, at dilution rate 1:200 (water : bordeaux mixture) were well-germinated but not sporulated at all. On the other hand, Aschersonia aleyrodis did not sporulate and germinate on lime sulfur treated PDA medium even at extremely low dilution rate 1:2,048,000 (water : lime sulfur). However, when the mycelial cells grown at 25o C for at least 7 days at soluble starch-tryptone medium were added to PDA, they were well-sporulated even at high dilution rate 1:100 (water : lime sulfur). This result suggested that the spore mixtures of Aschersonia aleyrodis should be applied to field quite long after lime-sulfur spray.

The nematicidal activity of Phellodendron amurense rhizome-derived materials (methanol extract) toward Meloidogyne Spp. second-stage juveniles (J2) and these effects on Cucumis sativus and Cucumis melo. Results were compared with these of fosthiazate. J2 was examined using 24-well plate tests, pot bioassays (C. sativa and C. melo) and Field trials (C. melo). In 24-well plate test with J2, methanol extract of P. amurense exhibited 98.7% and 69.8% mortality at 0.25 and 0.125 mg/ml toward J2, respectively, whereas Fosthiazate showed 100% mortality at 1 mg/ml. In pot bioassays with J2, P. amurense rhizome methanol extract gave 79.5% and 57.4% mortality at 2ℓ/m2(1,000x) and 2ℓ/m2(2,000x)/3kg soil from C. sativa and 73.7% and 53.3% mortality at 2ℓ/m2(1,000x) and 2ℓ/m2(2,000x)/3kg soil from C. melo, respectively. In Field test at C. melo in greenhouse showed 55.1% and 26.9% mortality at 2ℓ/m2(1,000x) and 2ℓ/m2(2,000x) applied soil. P. amurense rhizome-derived materials, merit further study as potential root-knot nematode control agents because of their nematicidal activity.

Among 154 putative ORFs of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), ac78 and ac79 are highly conserved genes in baculovirus, but their functions in the virus life cycle have been unknown so far. To determine their roles in AcMNPV replication, knockout mutants, ac78KO and ac79KO, were constructed using the plasmid capture system (PCS). Real-Time PCR analysis showed that both of ac78 and ac79 transcripts were first detected at 6 hours post-infection, and accumulated to maximum at 24 hours post-infection, suggesting that both of ac78 and ac79 are belong to late gene. When the genomic DNA of ac78KO was transfected into Sf9 cells, viral replication was restricted to a single cell infection. These results demonstrated that the ac78 play an important role in BV production, and therefore is essential for AcMNPV to mount a successful infection. Whereas Sf9 cells infected with the ac79KO showed normal viral symptoms such as rounding and swelling, OBs were not observed from majority of infected cells. These results suggested that the ac79 might play an important role in OB production.

An ambrosia beetle, Platypus koryoensis, is a vector of Raffaelea quercimongolicae that is known to cause Korean Oak Wilt (KOW), one of the serious threats to forest healthy in Korea. To manage P. koryoensis properly, it is necessary to clarify flight period of the adult. This experiment was conducted to elucidate the relationship between temperature and the flight period based on field observation in three forests consisted of Quercus mongolica from 2007 to 2009 except winter season. Date of flight period for 50% (FP50) was estimated by the cumulative Weibull distribution model based on cumulative proportion of the adult density and air temperature. Relationship between site temperature and the date of FP50 of P. koryoensis was the most significant when temperatures below 6.5℃ were excluded, suggesting lower threshold temperature for the flight period based on the site temperature. The pooling cumulative proportion of flight period against degree days was well described by the degree-day model, which has explanatory power for the 89% of year and site variation in the flight period and predicted accurately the flight pattern in 2011.

A total of 5,000 ethanolic and methanolic extracts of different plant species from 23 nations including Costa Rica, Vietnam, Philippines, India, South Africa, Pakistan and Peru were evaluated for their larvicidal activities against Aedes aegypti, the major vector of dangue, dangue hemorhagic fever and yellow fever. The larval mortalities were observed 24h after treating the larvae to the extracts. At 500 ppm, 179 extracts showed >80% larval mortality in the 24h exposure. Among the extracts tested, the highest larval mortality was observed in the extracts of Piper guianense, Piper nigrum, Piper mocropodum, Piper sem-immersum, Piper magen and Piper pubicatulum. The LC50 value of extract P. guianense, P. nigrum, P. mocropodum, P. sem-immersum, P. magen and P. pubicatulum were 8.84, 11.48, 8.84, 13.86, 9.48 and 10.12 ppm against Ae. aegypti. It is suggested that P. guianense, P. nigrum, P. mocropodum, P. sem-immersum, P. magen and P. pubicatulum can be developed as potent larvicidal agents against Ae. aegypti.

In DNA barcoding, the DNA degradation of old museum specimens has been limited full-length (658bp) sequencing. The challenges associated with the retrieval and authentication of degraded DNA extracts from fossil and old museum specimens were principally limited to analyze the relatively short sequences (<300 bp). Furthermore, almost protocols in other to analyzed the degraded DNA contained the cloning process after PCR causing the time-consuming and the rising costs. To overcome these problematic circumstances, we tried a modified method to analyze full-length of DNA barcoding region in 30~60 year-old butterfly specimens (225 samples in 28 species), using direct sequencing after PCR with species-specific overlapping primer sets per each species. As a result, all of 28 species have been successfully analyzed, although 178 samples (79%) are completely generated barcoding sequences ranged from 640 to 658 bp and 47 samples (21%) are partially sequenced ranged from 100 to 500 bp. Thus, the result showed that the direct PCR sequencing using the overlapping primer sets per species appears to have great potential efficiency for analysis of degraded DNA without incorrect sequences.

Rhynchium brunneum is a widely distributed wasp species in South Eastern Asia. R. brunneum females were collected from rural provinces of Cambodia, and their total RNA and venom were extracted on site. To search for novel substances in venom, a subtracted cDNA library specific to the venom gland and sac was constructed. A total of 1118 expressed sequenced sequence tags (ESTs) were sequenced and assembled into 349 contigs (107 multiple sequences and 242 singletons). In this result, we found the putative neurotoxin (DTX protein precursor), antimicrobial peptides (teratocyte-specific caboxylesterase) together with typical major components of wasp venom (venom hyaluronidase, arginine kinase, phospholipase A2, serine/theonine protein phosphatase). Additional in-depth annotation would be required for further characterization of many unidentified genes found in the EST library.

Vollenhovia emeryi chosenica (Wheeler) (Hymenoptera: Myrmicinae) is an ant species frequently found in forests. In nature, two phenotypically distinct forms are found e.g. long winged and short winged. Unlike other hymenopteran insects, the ant is unique in its mode of reproduction. In this species, queens are clonally reproduced from unfertilized eggs. On the other hand, workers develop from fertilized eggs. Strikingly, haploid males are reproduced from fertilized eggs after destroying the maternal half of the genome e.g. maternal genome loss (MGL) consequently only with the paternal half of the genome. We collected the ant colonies nationwide in 2011. In this study, we demonstrate that the ant is infected with Wolbachia, the bacterial reproductive manipulator in various insects. Interestingly, only the long winged morphs seem to be infected. Furthermore, most colonies are mulitple-infected except two colonies collected from Chuncheon and Mt. Deogyu. We will discuss potential interactions among the Wolbachia infection polymorphism and wing morphology, and evolution of clonal reproduction and MGL.