• Parasites, Hosts and Diseases
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• v.58 no.5
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• pp.551-558
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• 2020

The flaviviruses are small single-stranded RNA viruses that are typically transmitted by mosquitoes or tick vectors and are etiological agents of acute zoonotic infections. The viruses are found around the world and account for significant cases of human diseases. We investigated population of culicine mosquitoes in central region of Korean Peninsula, Incheon Metropolitan City and Hwaseong-si. Aedes vexans nipponii was the most frequently collected mosquitoes (56.5%), followed by Ochlerotatus dorsalis (23.6%), Anopheles spp. (10.9%), and Culex pipiens complex (5.9%). In rural regions of Hwaseong, Aedes vexans nipponii was the highest population (62.9%), followed by Ochlerotatus dorsalis (23.9%) and Anopheles spp. (12.0%). In another rural region of Incheon (habitat of migratory birds), Culex pipiens complex was the highest population (31.4%), followed by Ochlerotatus dorsalis (30.5%), and Aedes vexans vexans (27.5%). Culex pipiens complex was the predominant species in the urban region (84.7%). Culicine mosquitoes were identified at the species level, pooled up to 30 mosquitoes each, and tested for flaviviral RNA using the SYBR Green-based RT-PCR and confirmed by cDNA sequencing. Three of the assayed 2,683 pools (989 pools without Anopheles spp.) were positive for Culex flaviviruses, an insect-specific virus, from Culex pipiens pallens collected at the habitats for migratory birds in Incheon. The maximum likelihood estimation (the estimated number) for Culex pipiens pallens positive for Culex flavivirus was 25. Although viruses responsible for mosquito-borne diseases were not identified, we encourage intensified monitoring and long-term surveillance of both vector and viruses in the interest of global public health.

• Korean journal of applied entomology
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• v.59 no.3
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• pp.209-215
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• 2020

Debates over the ecological and public health impacts of aerial pesticide sprays are increasing. This is particularly true for controlling Monochamus beetles, which are vector insects of pinewood nematodes. In 2017, adult female orb-web spiders, Trichonephila clavata, were sampled from pine forests in Yangsan-si, Gyeongsangnam-do, Korea, where the aerial pesticide spray, fenitrothion or thiacloprid, was used for several decades. The biological traits of the spiders (body weight, body length, carapace width, and total hind leg length) were compared among treatment sites (no-spray, sprayed three times, and sprayed five times), and differences were observed. The body length, carapace width, and total hind leg length of the spiders in the sprayed areas were significantly shorter than in the no-spray area, but there were no differences between the area sprayed three or five times. These results indicate that repeated exposures to an aerial pesticide spray can alter morphological parameters, which influences population-level fitness. Future studies should monitor the spider long-term responses to pesticides (a direct effect) and prey availability (an indirect effect).

• Molecules and Cells
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• v.37 no.10
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• pp.727-733
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• 2014

Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-Omethylated rhamnose which are derived from S-adenosyl-methionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was $244mgL^{-1}$ and $129mgL^{-1}$, which was 4.88-fold and 4.77-fold higher than that in the wild-type ($50mgL^{-1}$ and $27mgL^{-1}$), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong $ermE^*$ promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Here-with, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to $372/217mgL^{-1}$ that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.

• Journal of Pharmaceutical Investigation
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• v.31 no.3
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• pp.185-190
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• 2001

Human papillomavirus (HPV) infection is known to cause cervical cancers. Human papillomavirus-like particles (VLP) have been studied as preventive vaccines of cervical cancers. To develop VLP as a therapeutic gene carrier, we studied the method to encapsulate cytokine genes in virus-like particles. HPV type 16 capsid L1 genes were amplified by polymerase chain reaction and cloned into T vector. L1 gene was then inserted into baculovirus transfer vector. The clone of baculovirus encoding L1 gene was isolated and used to express L1 protein in Sf 21 insect cells. VLP were purified by CsCl density gradient and ultracentrifugation. VLP were disassembled to capsomer units by treatment of a reducing agent. Given that interleukin-2 (IL-2) genes have been used in anticancer gene therapy and as a molecular adjuvant, IL-2 cytokine plasmids were chosen as a model gene. IL-2 plasmids were incubated with the disassembled capsomer suspension. To reassemble the particles, the mixture of capsomers and cytokine plasmids was dialyzed. The disassembly and reassembly of VLP were confirmed by transmission electron microscopy. The entrapment of cytokine plasmids in reassembled VLP was tested by the stability of plasmids against DNase I. After treatment of reassembled virus-like particles with DNase I, discrete IL-2 DNA band was observed. Our results indicate that IL-2 cytokine plasmid (3.5 kb size) can be encapsulated in the virus-like particles, suggesting the potential of VLP as a gene delivery system. Moreover, VLP containing the adjuvant cytokine plasmids might function as more effective subunit vaccines.

• Journal of Sericultural and Entomological Science
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• v.41 no.2
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• pp.102-107
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• 1999

To analysis a promoter strength of Atographa californica nucler polyhedrosis virus (AcNPV) IE1 gene, an immediate viral gene, ${\beta}$-glactosidase gene as a reporter gene was introduced under the control of the IE1 promoter. The restriction fragment containing IE1 promoter and ${\beta}$-galctosidase gene from pAcIE1-gal were inserter into pBacPAK9 to yield transfer vector pAcNPV-IE1-gal. The pAcNPV-IE1-gal was cotransfected with AcNPV genomic DNA BacPAK6 into Sf9 cells to produce recombinant baculovirus AcNPV-IE1-gal. In addition, recombinant bacvulovirus AcNPV-gal, which express ${\beta}$-galac-tosidase under the control of the polyhedrin promoter, was constrer, was constructed to compared with AcNPV-IE1-gal. The recombinant viruses were respectively infected into Sf9 cells and characterized by the virus titer and expression of ${\beta}$-galactoxidase in Sf9 cells. The promoter strength of IE1 and polyhedrin promoters was determined by the amount of ${\beta}$-galactosidase secreted into medium by viral infection. The titer of AcNPV-IE1-Gal determined by plaque assays in Sf9 cells was similar to that of AcNPV-gal. However, expression level of ${\beta}$-galactosidase by AcNPV-IE1-gal was significantly lower than that by AcNPV-gal. In conclusion, promoter strength of IE1 was approximately 25-fold lower than that of polyhedrin.

• Korean Journal of Plant Resources
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• v.19 no.4
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• pp.488-490
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• 2006

In double cropping fields, there has been a risk of increase rice stripe viral disease outbreaking as the vector, small brown planthopper that overwinter in barley fields increases. Especially rice cultivation in Honam region has two types of cropping system such as rice only cropping in a season and double cropping both of rice and barley. In double cropping system, harvesting of barley could induce migration of the vector from barley to rice field. We found that the vector's density was different in rice fields by distance from barley field. The vector's density in rice field was highest 63 numbers within one meter from the neighbor barley field, and the numbers reduced greatly as the distance from barley field increases. The disease incidence according to rice cultivation methods was different, Donjin 1 was appeared to be a susceptible variety showing 2.4% and 2.5% of disease incidence in direct and transplanting cultivation, respectively. The small brown planthopper in a nearby rice field reach peak 3 days after the barley field was harvested.

• Korean journal of applied entomology
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• v.54 no.1
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• pp.7-15
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• 2015

CpBV (Cotesia plutellae bracovirus) is a polydnavirus and encodes a cystatin (CpBV-CST1) gene. Its overexpression suppresses insect immunity and alters insect developmental processes. This study aimed to construct a genetically modified (GM) tobacco to further explore the physiological function of the viral cystatin and to apply to control insect pests. To this end, the transgenic tobacco lines were screened in expression of the target gene and assessed in insecticidal activity. A recombinant vector (pBI121-CST) was prepared and used to transform a bacterium, Agrobacterium tumefasciens. The transformed bacteria were used to generate transgenic tobacco lines, which were induced to grow callus and resulted in about 92% of shoot regeneration. The regenerated plants were screened by PCR analysis to confirm the insertion of the target gene in the plant genome. In addition, the expression of the target gene was assessed in the regenerated plants by quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis showed that the transgenic line plant expressed the target gene about 17 times more than the control tobacco, indicating a stable insertion and expression of the target gene in the transgenic tobacco line. The insecticidal activity was then analyzed using the screened transgenic tobacco lines against the teneral 1st instar larvae of the oriental tobacco budworm, Helicoverpa assulta. Though there was a variation in the insecticidal efficacy among transgenic lines, T9 and T12 lines exhibited more than 95% mortality at 7 days after feeding treatment. These results suggest that CpBV-CST1 is a useful genetic resource to be used to generate GM crop against insect pests.

• International Journal of Industrial Entomology and Biomaterials
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• v.18 no.2
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• pp.125-129
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• 2009

Entomopathogenic fungi were isolated directly from a cadaver of adult Monochamus alternatus supporting fungal sporulation, using a semi-selective medium and then screened several fungal colonies. The pathogenicity of each fungus was tested using oak longicorn beetle, Moechotypa diphysis, as substitutive insect. As the result, only one of them showed high pathogenicity against M. diphysis, with up to 100% mortality within 21 days of inoculation. Selected fungus was named as MaW1 and identified by Beauveria bassiana using microscopic examination and DNA analysis. Pathogenicity was also evaluated to M. alternatus.

• International Journal of Industrial Entomology and Biomaterials
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• v.25 no.2
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• pp.187-193
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• 2012

Serine proteases are major insect enzymes involved in the digestion of dietary proteins and in the process of blood meal digestion. In this study, cDNA was constructed using the whole body of Laccotrephes japonensis. The flanking sequences of the 5- and 3- end of this gene were characterized by RACE-PCR. Sequence analysis showed that this gene contained a 963-bp ORF encoding 320 amino acids. The deduced amino acid sequence showed 62% identity with the Creontiades dilutus serine protease, 58% with the Lygus lineolaris trypsin precursor, and 54% with the Triatoma infestans salivary trypsin. To assess the expression of the L. japonensis serine protease (JGsp), the JGsp gene was cloned into a baculovirus transfer vector, pBac-1, and expressed in Sf9 cells (Spodoptera frugiperda). SDS-PAGE and western blot analysis have shown that the JGsp recombinant protein was a monomer with a molecular weight of about 32 kDa. Recombinant JGsp has shown activity in the protease enzyme assay using gelatin as a substrate.

• The Plant Pathology Journal
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• v.33 no.6
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• pp.554-560
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• 2017

After 20 years of steady increase, kiwifruit industry faced a severe arrest due to the pandemic spread of the bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa). The bacterium penetrates the host plant primarily via natural openings or wounds, and its spread is mainly mediated by atmospheric events and cultural activities. Since the role of sucking insects as vectors of bacterial pathogens is widely documented, we investigated the ability of Metcalfa pruinosa Say (1830), one of the most common kiwifruit pests, to transmit Psa to healthy plants in laboratory conditions. Psa could be isolated both from insects feeding over experimentally inoculated plants, and from insects captured in Psa-infected orchards. Furthermore, insects were able to transmit Psa from experimentally inoculated plants to healthy ones. In conclusion, the control of M. pruinosa is recommended in the framework of protection strategies against Psa.