Acquisition of a reference Tetranychusstrains that are completely susceptible to acaricides and retain identical genetic backgrounds to acaricide-resistant strains is an essential step in elucidating mechanisms of resistance. To establish both completely susceptible and acaricide-resistant strains for this purpose, I collected Tetranychus mite populations from various regions in South Korea including both heavily cultivated and remote regions. Suitability as a susceptible or resistant reference strain was tested by determining species identity as Tetranychus urticae along with baseline susceptibility to common acaricides. The majority of mite populations collected from cultivated areas belonged to a monophyletic group of the previously reported green-type T. urticae as determined by mtCOI- and ITS2-based phylogenetic analysis. These strains showed relatively reduced levels of susceptibility to the acaricides tested, suggestive of the development of resistance. Among them, the AbaR strain was classified as a minor group in the T. urticae complex. The UD strain, originally collected from a remote island region, was found to be susceptible to the acaricides tested and generated an independent phylogenetic branch. The UD strain was also considered a minor group in the T. urticae complex. Phenotypic analysis based on morphological characters confirmed that both the AbaR and UD strains were statistically undistinguishable from the major green-type T. urticae. Taken together, I propose that the UD strain be used as a susceptible reference strain as it provides baseline susceptibility to acaricides and a wild-type genetic background for the resistance studies.
Previously, we found that expression by translational fusion of the polyhedrin (Polh)-green fluorescence protein (GFP) led to the formation of granular structures and these fluorescent granules were easily precipitated by high-speed centrifugation. Here, we developed an easy, fast, and mass purification system using this baculovirus expression system (BES). An enhanced GFP (EGFP) fused with Polh gene at the N-terminus including an adaptor and enterokinase (EK) site between Polh and EGFP was expressed in Sf9 cells. The cells infected by AcPolhEKA-EGFP produced fluorescent granules. The EGFP fusion protein was purified from granule-containing cells according to three steps; cell harvest, sonication and EK digestion. Through the final enterokinase digestion, EGFP was presented mainly in the supernatant (93.3%) and the supernatant also showed a pure EGFP band. These results suggest that the combined procedure of Polh fusion expression and enterokinase digestion can used for the rapid and easy purification of other proteins.
Plasmid capture systems (PCS) facilitate cloning and manipulation of circular double-stranded DNA. We recently developed an improved PCS (PCS-LZ) to clone relatively large DNA molecules of 30-150 kb. The PCS-LZ donor consists of a mini-F replicon and a kanamycin resistance marker between Tn7 left and Tn7 right ends. Both the replicon and marker gene of the PCS-LZ donor are transferred into target plasmid DNAs by in vitro transposition, followed by replication in E. coli. Colonies are tested for lacZ expression by blue/white screening. Circular DNAs were obtained from plasmids of Bacillus thuringiensis, genome segments of Cotesia glomerata bracovirus and polymorphic genomes of Autographa californica nucleopolyhedrovirus. PCS-LZ is a powerful tool for use in genomic analysis and mutagenesis in invertebrate pathology, and we are extending its application to include vertebrate research.
The baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV), a large circular double-stranded DNA virus whose genome encodes at least 155 open reading frames (ORFs), is highly pathogenic to a number of lepidopteran insects and widely used to transduce various cells for exogenous gene expression. Although many genes of AcMNPV have been identified, the genome-wide study related to viral replication has not been well announced. In this study, to elucidate DNA replication cascade of AcMNPV, we firstly developed a novel baculovirus genome that can be maintained in Escherichia coli as a plasmid and can infect susceptible lepidopteran insect cells. This genome, named bAc-MK, contains a mini-F replicon and a kanamycin resistance marker. Using a convenient Tn7 transposon-based system, pPCS-S, which contains an ampicillin resistance gene, ORF knock-out mutants were generated by random insertion into bAc-MK genome. These mutants will be suffered DNA microarray to elucidate AcMNPV replication cascade.
The Nilaparvata lugesn(BPH) and Laodelphax striatellus(SBPH) are major insect pests of rice in Korea. These BPHs migrate from China to Korea, but we have not exactly known about the route of long-distance migration of them. Molecular markers including microsatellite will be critical to asses gene flow in relation to geographical distance. In our work several microsatellite markers were developed for basic population genetic analysis of BPH and SBPH. Among each 21 microsatellite markers in BPH and SBPH, we could select 6 markers for BPH and 2 markers for SBPH as putative markers for analysis of population genetics. Others are further investigated for the possibility as markers. Development of polymorphic microsatellite loci are final stage of our work. But for good microsatellite markers to population genetics, we should do more experiments. Our work for development of microsatellites should facilitate the study of future population genetics of BPH and SBPH, and eventually elucidate the route of BPH and SBPH migration to Korea.
Because mealybugs are one of the most economically damaging groups of insects on food crops and ornamental plants, some are regulated-species in quarantine with the foreign trade of agricultural products. However, the absence of morphological characteristics enabling the discrimination of early life stages often causes a significant delay or the rejection of a shipment when fruit is discovered containing them, causing much economic loss. A PCR-based method for species identification was developed for six mealybug species known from Korean pears including two regulated insects, Planococcus kraunhiae (Kuwana) and Crisicoccus matsumotoi (Siraiwa). Six sets of species-specific primers were designed based on the sequence comparison of the internal transcribed spacer 1 and 2 regions. Efficiency tests against many mealybug samples showed that this method could effectively discriminate different mealybug species regardless of their developmental stages. Blind tests against 11 field collected mealybug nymph samples indicated that a single PCR is enough to discriminate unidentified mealybugs collected on Korean pears. This new method will be useful in quarantine as well as pest monitoring by providing an easy, accurate way of identifying any life stage of these mealybugs.
Recently, hundreds thousands of CO1 barcodes have been produced in many insect groups however, relatively little is known in Hemiptera. In this study, we newly determine 1173 sequences for 374 species distributed among 190 genera and 33 families collected mostly from North America. Average age of the specimen was 12.2 years and about 70% of the specimens produce barcode over 500 bp among examined 1700 specimens. Con-generic divergency calculated by K2P distance was 9.99 which was 11fold higher than the mean intraspecific variation. A steady increase of genetic variation through increasing taxonomic levels was also observed. We found several taxon groups with deep divergency (over 2.0 K2P distance) which were morphologically identified as a same species. Some mixed clusters with several species were also found and suggested more detailed study for these clusters through both morphological and molecular biological method.
The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The genome of CSFV is a positive single-stranded RNA molecule 12.3 kb and contains a single large open reading frame (ORF). The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. To determine the characteristics of the CSFV, LOM strain, we investigated the nucleotide sequence of the glycoprotein E0, E1 and E2. Comparison of the LOM with the other strains revealed nucleotide sequence identity ranging from 97 to 98%. Expression of the glycoprotein E2 was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies in Sf21 cells. The expression levels of glycoprotein E2 were observed from day 3 and 5 days maximum. In addition, its expression efficiency by media and cell line was investigated. The result showed that High-Five cells and Grace’s insect media for Sf21 were the best conditions for the expression of the glycoprotein E2.
To determine differential gene expression profiles in the venom gland and sac (gland/sac) of a solitary hunting wasp species, Eumenes pomiformis Fabricius (1781), a subtractive cDNA library was constructed by suppression subtractive hybridization. A total of 541 expressed sequence tags (EST) were clustered and assembled into 102 contigs. In total, 37 genes were found from the library by BLASTx search and manual analysis. A eumenitin-like venom peptide, EpVP1, occupied ca. 26% of the library. A novel venom peptide, EpDTX, shared sequence similarity with trypsin inhibitors and dendrotoxin-like venom peptides known as K+ channel blockers, implying it could be responsible for the paralysis of prey. As well as phospholipase A2 and hyaluronidase known to be main components of wasp venoms, several contigs encoding enzymes, including metalloendopeptidases and a decarboxylase likely involved in the processing and activation of venomous proteins, peptides, and neurotransmitters, were also isolated from the library. The presence of a gene encoding insulin-like growth factor binding protein suggests that solitary hunting wasps might control the prey to stay in larval stage by their venom. The abundance of these venom components in the venom gland/sac and alimentary canal was confirmed by quantitative real-time PCR.
Recently, the genome of Spodoptera litura granulovirus (SlGV) which encodes 133 putative open reading frames (ORFs) was completely sequenced. In this study, to screen novel insecticidal genes of SlGV, we first constructed an advanced plasmid capture system, pPCS-TPI, which contains not only pUC19 ori and ampicillin resistance gene but also Autographa californica nucleopolyhedrovirus (AcMNPV) ORF603 and ORF1629 homologous region between Tn7L and Tn7R. In order to introduce genomic segments of SlGV into the genome of AcMNPV, genomic DNA of SlGV was digested with EcoRI and self-ligated. These self-ligated segments were in vitro transposed with the pPCS-TPI donor by the help of TnsABC* transposase. By this, 10 EcoRI-digested genomic segments of the SlGV were cloned, and these clones were co-transfected with the bApGOZA DNA into sf9 cells to generate corresponding recombinant virus, respectively. The resulting recombinant viruses harboring genomic segments of the SlGV could be used to investigate the insecticidal activity and/or other functions originated from the introduced genomic segments of the SlGV.
Bacillus thuringiensis 1-3 (Bt 1-3), belonging to subsp. aizawai (H7), showed different characteristics in plasmid profiles from type strain and had cry2A gene in addition to cry1Aa, cry1Ab, cry1C and cry1D. To clone its plasmids and construct E.coli-Bt shuttle vector, we constructed the plasmid capture system (PCS) by inserting attB sites including lacZ between transposable elements (designated as pPCS-Troy). Through in vitro transposition with total plasmids DNA of Bt 1-3, 53 clones were acquired and their sizes were approximately 10 kb. Based on the sequence analysis, they were classified in four groups showing similarities with four known Bt plasmids, pGI3, pBMB175, pGI1 and pGI2, respectively. One of pGI3-like clones, named as pBt1-3, was fully sequenced and its putative open reading frames (ORFs), Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified. The structure of pBt1-3 showed high similarity with pGI3 which is one of rolling-circle replication (RCR) group VI family. As a donor for construction of shuttle vector, pDonr-attPEm vector harboring erythromycin resistant gene between attP sites was constructed. Through BP recombination with pPCS-Troy-cloned Bt plasmids and pDonr-attPEm, erythromycin resistant gene was transposed to Bt plasmids. This scheme proposes that in vitro transposition using pPCS-Troy and BP recombination using pDonr-attPEm can easily clone Bt plasmids and construct novel shuttle vectors.
Inflammatory bowel disease (IBD) is a group of chronic disorders of unknown etiology characterized by inflammation of the gastrointestinal tract. Recent data showed that the development of IBD is associated with the interplay of genetic, bacterial, and environmental factors and dysregulation of the intestinal immune system. We investigated how the gut cells were repaired after injury in Drosophila melanogaster. In this study we made D. melanogaster intestine damage model by oral feeding with variety IBD inducer such as pathogenic bacteria Serratia marcescens, Dextran Sulfate Sodium (DSS) and bleomycin, because its function is very similar with human, even though D. melanogaster has relatively simple organism. We repeated oral feeding with variety IBD inducer and got the survival rate and 50% lethal dose (LD50). After feeding with IBD inducer, we investigated the change of the intestinal stem cells, innate immune-related gene expression, and apoptosis in D. melanogaster gut. We examined the Delta, stem cell marker, staining image in the gut after feeding with DSS and S. marcescens with LD50 concentration. The Delta positive cells greatly increased in gut cells damaged by DSS or S. marcescens. This result supports the idea that intestinal gut stem cells are increased after gut cell damage and play very important role in damaged cell repair. Expression level of antimicrobial peptides was dramatically up-regulation after gut damage. As a result of TUNEL (terminal deoxynucleotidyl transferase mediated X-dUTP nick end labeling) assay, we confirmed that cell death by apoptosis was very increased in DSS feeding flies. Accordingly, we suggest that D. melanogaster is a proper IBD model organism to study how intestine damage can be repaired.
곤충생태 조사 및 학습관찰효과를 접목한 자동개폐식 곤충 야간채집트랩과 지하부 서식곤충 사육용기를 개발하였다.
곤충트랩은 자동개폐식으로 원하는 시간대별로 채집이 가능하며, 안전채집방식으로 곤충을 살아있는 채로 채집 및 관찰할 수 있는 것이 가장 큰 특징이다. 세부적으로는 관찰부가 설치되고, 타이머의 설정된 시간에 의해 동력전달수단의 작동에 따라 개폐기가 하부 방향으로 이송되면서 개방부가 열리게되는 것과 동시에 관찰부에 구비된 유인등이 발광(빛)되면서 수납부에 놓여 있는 유인물질의 냄새를 맡은 딱정벌레류 등을 유인하여 채집율을 향상시킬 수 있는 학습관찰용 야간채집트랩이다.
학습용 사육용기는 비단길앞잡이 등과 같이 지하부에 은닉하면서 먹이곤충을 포식하는 곤충들에 대하여 학습 및 사육이 가능한 것이 가장 큰 특징이다. 세부적으로는 하우징, 방수용 필름, 덮개로 구성되며 하단부 서식부를 흙으로 서식처를 형성함으로써, 일반적으로 흙속에서 은닉하여 생활하는 곤충의 생태적 특성을 최대한 고려함은 물론, 곤충이 지하 공간까지 자연스럽게 굴을 파고 들어가 생활할 수 있는 공간을 제공하며, 또한 서식처는 흙 외에도 젤(Gel)과 같은 투명하면서 고정된 물질을 이용하면 곤충이 굴을 파고들어가거나 먹이를 갱도속에서 포식하는 장면 등을 직접 관찰할 수 있어 먹이사슬에 대한 학습효과를 높일 수 있는 이점이 있다. 또한 사육용기의 안을 소비자가 직접 꾸미는 등 실내 장식이 가능하도록 고려한 이점도 있다.
2009년 4월부터 8월까지 쭈쭈가무시병의 다발생지역으로 알려진 중부지방의 충남 아산시, 경기 평택시와 화성군 3개 지역에서 털진드기의 개체군 밀도를 조사하였다. 채집된 들쥐는 총 48마리였는데 이 중 등줄쥐(A. agrararius)가 45마리로 93.8%를 차지하여 절대 우점종이었고, 갈밭쥐(Microtis fortis)가 1마리(2.0%)였으며, 땃쥐(Crocidura laisura)가 2마리(4.2%)였다. 조사 기간 중 전 지역에서 채집한 털진드기는 총 290개체였다. 채집된 털진드기는 1속 4종이었는데 이중 대잎털진드기(Leptotrombidum pallidum)가 234개체로 80.7%를 차지하여 우점종이었고, 동양털진드기(L. oriental)가 30개체(10.3%), 수염털진드기(L. palpale)가 10개체(3.4%), 반도털진드기(L. zetum)가 16개체(5.5%)였다. 대잎털진드기는 3개 지역(충남, 평택, 화성)에서 절대 우점종으로 나타났다. 충남 아산지역에서는 대잎털진드기, 수염털진드기, 동양털진드기, 반도털진드기 4종이 서식하는 것으로 나타났으며, 경기 평택지역에서는 대잎털진드기, 동양털진드기, 반도털진드기 3종이 서식하며, 경기 화성지역은 대잎털진드기, 수염털진드기 2종만이 서식하는 것으로 나타났다.
논산시에 소재한 11개의 저장곡물 취급소 및 창고의 가공 후 부산물속 저장곡물 응애류를 채집 후 조사하던 중에 진드기아강 무기문진드기목 가루진드기과 Suidasia속 1 미기록 종, Suidasia medanensis (Oudemans, 1924), 고리뿌리응애(신칭)를 발견하였기에 보고한다.
Recently, ambrosia beetles have become very important pest of 2~5 year old apple trees with M9 dwarf rootstocks in South Korea. The beetles have killed the branches and stems of the young trees, especially, frozen damage trees in winter or drought stressed tree in spring. By the increase in planting area and weaken property in winter of M9 dwarf rootstock, ambrosia beetles are becoming a key pest in Korean apple orchards using M9 rootstock. According to the survey of damaged apple trees by ambrosia beetles, Xylosandrus germanus Blandford, Xyleborus apicalis Blandford and Xyleborinus saxeseni (Ratzeburg).
These insects are hosts of the ambrosia fungi. ITS region of rDNA has shown to be a useful source for phylogenetic studies and identifying speices in previous published articles. We analyzed the nucleotide sequences of ITS1, 5.8S and ITS2 region of ambrosia fungi isolated from three ambrosia beetles, in order to observe molecular variation among the fungi strains and to reveal phylogenetic relationships.
한방에서 봉침요법으로 여드름 치료 및 개선의 목적으로 사용하는 봉독을 봉독 채집장치를 사용하여 국내산 꿀벌로부터 대량 분리, 정제하여 여드름 원인균인 P. acnes와 내성균인 P. acnes, 피부 상재균인 S. aureus, S. epidermidis 및 S. pyogenes 균에 대한 성장 억제 효과를 조사하여 다음과 같은 결론을 얻었다.
1. 액체배지 희석법에 의한 최저성장억제농도는 P. acnes에 대해서는 MIC, 내성균인 P. acnes, 의 경우 이며, 피부 상재균인 S. aureus, S. epidermidis 및 S. pyogenes 균에 대한 MIC는 각각 으로 성장 억제 효과가 우수하였다.
2. well diffusion법에 의한 성장억제 효과 P. acnes의 경우 2.5mg의 봉독 양으로도 억제환이 관찰되었으며, 내성균인 P. acnes, 피부 상재균인 S. aureus, S. epidermidis 및 S. pyogenes 균에서도 각각 5mg의 봉독으로도 세균 증식 억제 환을 볼 수 있었다.
3. 봉독 투여 후 시간별로 P. acnes와 S. aureus에 대한 항균작용이 어떻게 변하는지 측정하기 위하여 time-kill curve에서도 짧은 시간 내에도 항균효과를 보이며, 처리시간이 증가함에 따라 억제 효과가 증가하였다.
봉독은 여드름 유발균과 피부 상재균에 강한 억제 작용을 하며, 여드름 특히 염증성 여드름 치료에 효과가 뛰어나 향후 여드름 치료제의 개발 가능성이 높은 것으로 생각된다.
The Wolbachia bacterium, one of the most prevalent endosymbiotic bacteria, is known to induce reproductive anomalies such as cytoplasmic incompatibility, feminization, male killing and parthenogenesis in various arthropod species. The bacterium is considered to have had huge impacts on hosts' reproductive biology, immunity, evolution, and molecular machineries. Infection surveys on the bacterium have rather been limited to specific taxa that are mainly of economical importance or conducted with randomly collected organisms. Here we investigated infection frequency of Wolbachia in 206 Coleopteran insects collected from Korea. Among them 28 species (13.59%) across families proved to harbor Wolbachia. The phylogenetic trees based on the partial 16s rRNA gene and the partial Wolbachia surface protein (wsp) gene of Wolbachia show that all the Wolbachia strains belong to either Supergroup A or B and Wolbachia evolved independently from its hosts. In addition, the cophylogenetic analysis of the 16s rRNA gene and wsp gene implies that there have been horizontal DNA transfers and recombination events within and between divergent Wolbachia supergroups.
토양에서 분리한 B.thuringensis 균주 들 중 Spodoptera litura에 활성이 높은 CAB133을 선발하였다. H serotype을 동정한 결과 혈청형이 3abc인 kurstaki로 동정되었다. 전형적인 이중 피라미드형의 결정성 내독소 단백질과 형태적인 유사성을 보였고, S. litura 유충에 대한 생물활성 결과 kurstak HD-1균주에 비해 높은 활성을 보였다.
혈청학적으로 동일한 균주일지라도 각각의 균주가 생산하는 결정성 독소단백질의 유전자가 다르기 때문에 해충의 살충성에는 차이가 나타나므로 분자생물학적인 특성을 알아보았다. 내독소 단백질 특성을 비교하기 위하여 SDS-PAGE를 수행한 결과, kurstak HD-1은 130kDa과 약 60kDa의 주요 단백질 밴드를 나타내었으나 CAB133균주는 130kDa의 주요 단백질 밴드만을 가지고 있었다. 균주의 전체 plasmid profile을 분석한 결과 kurstak HD-1균주는 7개, CAB133균주는 10개의 plasmid를 포함하고 있었으며 crystal을 형성하지 못하는 Cry-균주를 배양하여 확인한 결과 Cry+와 하나의 밴드가 없는 9개의 plasmid를 가지고 있음을 확인하였다. 균주의 crystal toxin을 확인하기 위해 usual primer로 PCR을 수행한 결과 kurstak HD-1은 cry1Aa,cry1Ab, cry1Ac, cry1I, cry2 gene을 CAB133은 cry1Aa, cry1Ab, cry1Ac, cry1C, cry1D, cry2 gene이 있음을 확인하였다.
The traditional use of insects as food continues to be widespread in tropical and subtropical countries and to provide significant nutritional, economic and ecological benefits for rural communities. Specially, Bee brood serves as a food source to humans in many countries although limited data exists concerning its nutrient composition. Bee brood (pupa and larvae) were analyzed for Carbohydrate, Saturated fatty acid, Cholesterol, protein, fat, fiber, minerals, and vitamins. Bee brood was high in protein(46.4%~46.73%), fat(18.84%~ 20.75%),carbohydrate(24.66 %~35.79 %), Folic acid(222.30 ㎍/100g), and vitamins. Differentially, folic acid had been contained by high density in pupa of drone. While low in iron, bee brood was a good source of folic acid, and carbohydrate. The fat was composed mostly of saturated and mono-unsaturated fatty acids. The present data suggest bee brood to be an excellent source of many valuable nutrients including energy, amino acids, many essential minerals, and B-vitamins. These data suggest bee brood could be a valuable source of nutrients to various populations.