Honey bees are crucial pollinators for agricultural and natural ecosystems, but are experiencing heavy mortality in Korea due to a complex suite of factors. Extreme winter losses of honey bee colonies are a major threat to beekeeping but the combinations of factors underlying colony loss remain debatable. Finding solutions involves knowing the factors associated with high loss rates. To investigate whether loss rates are related to Varroa control and climate condition, we surveyed beekeepers in korea after wintering (2021–2022 to 2022–2023). The results show an average colony loss rate of 46%(2022) and 17%(2023), but over 40% colony loss before wintering at 2022. Beekeepers attempt to manage their honey bee colonies in ways that optimize colony health. Disentangling the impact of management from other variables affecting colony health is complicated by the diversity of practices used and difficulties handling typically complex and incomplete observational datasets. We propose a method to 1) Varroa mite population Control by several methods , and 2) Many nursing bee put in hive before wintering.

We assessed the susceptibility of three fumigants on Phthorimaea operculella, which is an important pest of stored potato worldwide. 5 to 6 initial dosage of each fumigants were treated on every growth stages of P. operculella. Methyl bromide showed 100% mortality at CT 33.40mg h/ L on egg, CT 14.41mg h/L on late larvae, CT 31.89mg h/L on pupae and CT 16.01mg h/L on adult, respectively. The LCT50 of methyl bromide was 19.115mg h/L on egg, 3.934mg h/L on late larvae, 13.810mg h/L on pupae and 6.260mg h/L on adult, respectively. In case of phosphine, 98% mortality was achieved at CT 16.77mg h/L on egg, and 100% mortality was achieved at CT 16.58mg h/L on late larvae, CT 18.54mg h/L on pupae and CT 12.28mg h/L on adult, respectively. The LCT50 of phosphine was 1.457mg h/L on egg, 2.236mg h/L on late larvae, 1.282 mg h/L on pupae and 0.253mg h/L on adult, respectively. In case of ethyl formate, 100% mortality was achieved at CT 96.21mg h/L on egg, CT 101.30mg h/L on late larvae, CT 120.66mg h/L on pupae and CT 148.30mg h/L on adult, respectively. The LCT50 of ethyl formate was 23.730mg h/L on egg, 13.706 mg h/L on late larvae, 29.578mg h/L on pupae and 19.235mg h/L on adult, respectively.

Stored grain pests can cause reduction of grain quantity, quality, commercial value and germination rate. Susceptibility of three fumigants, methyl bromide, ethyl formate and phosphine, were assessed on Tribolium castaneum, which is an important stored grain pest. On susceptible insects, LCT50 of phosphine was 0.654mg h/L for egg, 0.127mg h/L for late larvae, 0.105mg h/L for pupae and 0.048mg h/L for adult stage, respectively. LCT50 of methyl bromide was 33.193mg h/L for egg, 14.585mg h/L for late larvae, 8.616mg h/L for pupae and 11.967mg h/L for adult stage, respectively. LCT50 of ethyl formate were 25.165mg h/L for egg, 80.912mg h/L for late larvae, 176.326mg h/L for pupae and 68.578mg h/L for adult stage, respectively. On resistant insects, LCT50 of phosphine were 82.325mg h/L for egg, 33.315mg h/L for late larvae, 73.546mg h/L for pupae and 55.707mg h/L for adult stage, respectively. LCT50 of methyl bromide were 19.250mg h/L for egg, 43.413mg h/L for late larvae, 76.842mg h/L for pupae and 19.387mg h/L for adult stage, respectively. LCT50 of ethyl formate were 87.552mg h/L for egg, 113.457mg h/L for late larvae, 200.122mg h/L for pupae and 85.394mg h/L for adult stage, respectively.

Carbonyl sulfide(COS) is a naturally generated gas from fermentation process of microbes and from plant root and stem. COS was firstly registered as a fumigant at 1993 to control stored product pests. To supplement environmental problems and toxicity of commercial fumigants and develop new fumigant, we have processed the susceptibility assessment of carbonyl sulfide on important agricultural pests, Myzus persicae and Tetranychus urticae. Every growth stages of two insect species were tested, and five dosages of carbonyl sulfide were treated for 4 hours, and the mortality was investigated after 24 hours of treatment. Nymphal stage of M. persicae was completely controlled at more than 20 mg/L dosage, and adult stage showed 95.8% mortality at 80 mg/L dosage. The LC50 of M. persicae was 7.314mg/L for nymph and 26.117mg/L for adult stage. Egg stage of T. urticae showed 91.2% mortality when treated with 100mg/L carbonyl sulfide, and nymph and adult stage showed 100% and 94.1% mortality at 8mg/L and 80mg/L, respectively. The LC50 of T. urticae was 73.110mg/L for egg, 2.818mg/L for nymph and 12.054mg/L for adult stage.

The existing ethyl formate fumigant is carbon dioxide (CO2) mixed liquified gas in metal cylinder, but this product type costs a lot to manufacture, translate and maintain cylinder. To supplement these problems, we have developed a new ethyl formate fumigation technique with nitrogen (N2) carrier. We assessed the susceptibility of mealy bugs, the most frequently detected pests in imported banana, and phytotoxicity of banana fruits. Ethyl formate and nitrogen were concurrently treated on citrus mealybug, one of the most resistant mealybug to fumigant, and ethyl formate was treated with LC50 product of independent treatment dosage. Nitrogen was treated with 7 dosages from 79% to 95% concentration. Phytotoxicity of banana was assessed by treating EF 35 mg/L with N2 79% for 14 days, and color, sugar contents and loss of weight were measured. EF with N2 treatment showed more than 50% of mortality on every growth stages, and there was no significant difference between control and treatment banana fruits. These results indicate that concurrent treatment of EF and N2 can be used to control mealybug in banana fruits.

Recently, phosphine resistance of Sitophilus oryzae has been reported from China, India, Brazil and Australia. In this study, susceptibility of three fumigants were assesses on phosphine resistant and susceptible S. oryzae to investigate domestic phosphine resistance level and to use base data for resistance control. On susceptible insects, LCT50 of phosphine was 0.440mg h/L for egg, 0.602mg h/L for early larvae, 3.901mg h/L for late larvae, 6.171mg h/L for pupae and 0.295mg h/L for adult stage, respectively. LCT50 of methyl bromide was 9.997mg h/ L for egg, 12.113mg h/L for early larvae, 18.952mg h/L for late larvae, 21.104mg h/L for pupae and 17.824mg h/L for adult stage, respectively. LCT50 of ethyl formate was 75.795mg h/L for egg, 60.110mg h/L for early larvae, 160.491mg h/L for late larvae, 255.797mg h/L for pupae and 77.711mg h/L for adult stage, respectively. On resistant insects, LCT50 of phosphine was 6.959mg h/L for egg, 28.456mg h/L for early larvae, 48.170mg h/L for late larvae, 29.106mg h/L for pupae and 16.550mg h/L for adult stage, respectively. LCT50 product of methyl bromide was 17.842mg h/L for egg, 14.900mg h/L for early larvae, 25.840mg h/L for late larvae, 43.520mg h/L for pupae and 16.397mg h/ L for adult stage, respectively. LCT50 of ethyl formate was 60.034mg h/L for egg, 64.450mg h/L for early larvae, 149.028mg h/L for late larvae, 140.408mg h/L for pupae and 66.043mg h/L for adult stage, respectively. Domestic resistant S. oryzae showed 4 to 56 times higher resistance rate than susceptible insects.

The susceptibility of three fumigants, methyl bromide, ethyl formate and phosphine, and concurrent treatment of ethyl formate and phosphine were tested on Lasioderma serricorne. Susceptibility assessment were performed by treating 5 to 6 initial dosage on every growth stages of L. serricorne. The LCT50 of methyl bromide was 13.896mg h/L for egg, 36.038mg h/L for late larvae, 25.172mg h/L for pupae and 21.758mg h/ L for adult, respectively. The LCT50 of phosphine was 0.317mg h/L for egg, 0.649mg h/L for late larvae, 3.748mg h/L for pupae and 0.703mg h/L for adult, respectively. In case of ethyl formate, the LCT50 was 43.657mg h/L for egg, 137.606mg h/L for late larvae, 72.676mg h/L for pupae and 52.951mg h/L for adult, respectively. Concurrent treatment of ethyl formate and phosphine was performed by treating 5 to 6 initial dosage of ethyl formate with 0.5 mg/L phosphine for 4 hours on every growth stages of L. serricorne. The LCT50 of ethyl formate concurrent treatment was 13.746mg h/L for egg, 8.156mg h/L for late larvae, 27.087mg h/L for pupae and 11.353mg h/L for adult, respectively, and these results indicate that concurrent treatment can control pest with lower dosage and shorter period. Sorption rates and ventilation periods of each fumigants were also calculated for efficacy and safety

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.