A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection and differentiation among Nosema apis and Nosema ceranae in honeybee. Three sets of primers were selected from different genomic sequences to specifically amplify a 831 bp amplicon within the SSU rRNA gene, specific for both N. apis and N. ceranae (MSSR primer); a 375 bp amplicon within the SSU rRNA gene, specific for N. apis (NA primer); and a 1,131 bp amplicon with in SSU rRNA gene, specific for N. ceranae (NC primer). Using the primers in conjunction (multiplex PCR) we were able to N. apis and N. ceranae and to differentiate between them. The sensitivity of this PCR assay was approximately 102spores per milliliter. We proposed that the multiplex PCR was sensitive, specific and rapid tool that can serve as a useful differential diagnostic tool for detecting N. apis and N. ceranae in honeybee.

Bacillus thuringiensis 1-3 (Bt 1-3), isolated from Korean soil sample, showed high insecticidal activity against Plutella xylostella. Recently, we improved plasmid capture system donor-s (PCS-S) by inserting attB sites including lacZ between transposable elements (designated as pTroy), to reduce background and construct E. coli-Bt shuttle vector. Through in vitro transposition with total plasmid DNA of Bt 1-3, at least 6 different size plasmids of Bt 1-3 were cloned. Among them, 47 clones which have approximately 10 kb plasmid in size were sequenced and 5 contigs were assembled. These contigs showed partial similarity with two known plasmids, pGI3 or pBMB175, separately. These cloned plasmids will acquire erythromycin resistance by BP recombination reaction with pDonrattPEm vector. After transformation into Bt cells, final erythromycin resistant Bt cell might contain novel E. coli-Bt shuttle vector. This scheme proposes that pTroy and pDonr-attPEm system can easily construct new shuttle vector by in vitro transposition, BP reaction, and erythromycin selection with any Bt plasmids.

Recently, we constructed a novel recombinant baculovirus genome, bEasyBac, enabling easy and fast generation of pure recombinant baculovirus without any purification step. In the bEasyBac, bacteriophage lambda site-specific attachment (att) sites were introduced to facilitate the generation of recombinant viral genome by in vitro transposition. Moreover, extracellular RNase gene from Bacillus amyloliquefaciens, barnase, was expressed under the control of Cotesia plutellae bracovirus (CpBV) ORF3005 early promoter to negatively select against non-recombinant background. The bEasyBac could replicate in host insect cells only when the barnase gene was replaced to gene of interest by in vitro transposition. When the bEasyBac was transposed with pDualBac-EGFP and the EGFP expression efficiency along passage was investigated, the resulting recombinant virus, EasyBac-EGFP, showed comparable level of EGFP expression efficiency with the plaque-purified recombinant virus, AcEGFP, which was constructed using bAcGOZA system, whereas, the non-purified AcEGFP showed quite reduced level of EGFP along passages. Moreover, no non-recombinant backgrounds were detected from unpurified EasyBac-EGFP stocks. Based on these results, high-throughput condition for generation of multiple recombinant viruses in a time was established. These results suggest that the bEasyBac has an effective benefit enabling for high-throughput baculovirus expression vector without purifying recombinant virus.

To determine the characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), CA, which was isolated from the serum of an infected pig in 2006, we investigated the nucleotide sequence and expression of the structural ORFs (ORFs 2 to 7) using the bApGOZA system. We found that the structural ORFs 2 to 7 of CA consisted of 3188 nucleotides that were the same as those formed from VR-2332. Comparison of the CA with the other strains revealed nucleotide sequence identity ranging from 89.8 to 99.5%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The CA strain was closely related to the other North American genotype strains but formed a distinct branch with high bootstrap support. Additionally, expression levels of the PRRSV proteins in Sf21 cells were strong or partially weak. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.

The porcine reproductive and respiratory syndrome virus (PRRSV) has six structural proteins which encoded by ORFs 2 to 7 are designated as GP2, 3, 4, 5, M and N, repectively. In this study, we determined the expression of each protein using novel transfer vector, pBmKSK4 which has the polyhedrin promoter of BmNPV and 6xHis tag. The recombinant transfer vector was co-transfected into Bm5 cells along with bBpGOZA DNA. Recombinant virus was purified by plaque assay and amplified in Bm5 cells. Expression of each protein was identified by SDS-PAGE and Western blot analysis using anti-6xHis monoclonal antibody. The expression levels of the structural proteins in Bm5 cells were stronger than the expression system using pBacPAK9 transfer vector in Sf21 cells. As expected, GP5 was expressed at low levels from its structural properties and its toxicity for cells. In addition, each recombinant protein was purified using Ni-NTA spin columns. The ability to produce each protein in the baculovirus system indicates that these could be major candidates for the development of a vaccine against PRRSV.

The nucleotide sequence of the Spodoptera litura granulovirus (SlGV) genome was determined and analysed. It was 124,121 bp long, with a 61.2% A+T content and contained 133 putative open reading frames (ORFs) of 150 nucleotides or larger. The 133 putative ORFs covered 86.3% of the genome. Among these, 29 ORFs were conserved in most completely sequenced baculovirus genomes, 44 were granuloviruses (GVs)-specific, 4 were nucleopolyhedroviruses (NPVs)-specific, and 56 were present in some NPVs and/or GVs. Especially, we proved that there were 9 SlGV-specific ORFs in 44 GV-specific ORFs by RT-PCR. Chitinase and cathepsin genes involved in the liquefaction of the infected hostwere not found in the SlGV genome, which explains why SlGV-infected insects do not degrade in a typical manner. When the phylogenic relationship was analyzed using the nucleotide sequence of granulin gene, SlGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestia c-nigrum granulovirus (XcGV) which were belonged to TypeI granulovirus.

Transferrin and ferritin are iron-binding proteins involved in transport and storage of iron as part of iron metabolism. Here, we describe the cDNA cloning and characterization of transferrin (Bi-Tf) and the ferritin heavy chain subunit (Bi-FerHCH), from the bumblebee Bombus ignitus. Bi-Tf cDNA spans 2,340 bp and encodes a protein of 706 amino acids and Bi-FerHCH cDNA spans 1,393 bp and encodes a protein of 217 amino acids. Comparative analysis revealed that Bi-Tf appears to have residues comprising iron-binding sites in the N-terminal lobe, and Bi-FerHCH contains a 5’UTR iron-responsive element and seven conserved amino acid residues associated with a ferroxidase center. The Bi-Tf and Bi-FerHCH cDNAs were expressed as 79 kDa and 27 kDa polypeptides, respectively, in baculovirus-infected insect Sf9 cells. Northern blot analysis revealed that Bi-Tf exhibits fat body-specific expression and Bi-FerHCH shows ubiquitous expression. The expression profiles of the Bi-Tf and Bi-FerHCH in the fat body of B. ignitus worker bees revealed that Bi-Tf and Bi-FerHCH are differentially induced in a time-dependent manner in a single insect by wounding, bacterial challenge, and iron overload.

Metamorphosis is a development process involving the programmed cell death of obsolete larval organs. Aspartic proteinase cathepsin D (BmCatD) is involved in the silkworm Bombyx mori metamorphosis. Here we show a novel functional role of cysteine proteinase cathepsin B during B. mori metamorphosis. The B. mori cathepsin B (BmCatB) was expressed in the fat body, epidermis, ovary, testis, and hemocyte of the larval and pupal stages. The BmCatB was ecdysoneinduced, expressed in the fat body of the molting, the final larval instar and pupal stages, and its expression led to programmed cell death. RNA interference (RNAi)-mediated BmCatB knock-down inhibited the programmed cell death of larval and pupal fat body, resulting in the arrest of larval-pupal transformation. BmCatB RNAi is up-regulated the expression of BmCatD. Based on these results we concluded that BmCatB is critically involved in the histolysis of the larval and pupal fat body, indicating that BmCatB and BmCatD are mutally regulated during silkworm metamorphosis.

The pine sawyer beetle, Monochamus alternatus, transmits the pinewood nematode (PWN, Bursaphelenchus xylophilus), causing the pine wilt disease (PWD), which gives rise to enormously economic as well as forest damage. However, PWN has been identified as a pathogen of PWD, it is very difficult to discriminate B. xylophilus from B. mucronatus in a short time, which are genetically and morphologically very similar. Therefore, it has been necessary to detect and eliminate PWN-infected trees in the forest area for the prevention of PWD transmission. Up to date, there is no report on biomarkers such as DNA and protein for the diagnosis of B. xylophilus. In this study, we produced a B. xylophilus monoclonal antiserum (D9-F10) from BalbC mice and screened its specificity with various proteins extracts. Western blot analysis revealed that the D9-F10 is only reactive with B. xylophilus protein extract among other tested protein extracts, indicating that the D9-F10 is specific for a B. xylophilus protein. Furthermore, two-dimensional electrophoresis showed the D9-F10 detects a very highly acidic protein, pI≒3.5. These results suggest that the D9-F10 monoclonal antibody is useful for the development of a diagnostic kit for the pine wilt disease.

The toxicity of perilla-chinese Basil, Perilla frutescens whole plant-derived materials to third-instar of larva Plutella xylostella was examined using that of four insecticides and 5 constituents of P. frutescens from other research materials. The active principle of P. frutescens was identified as the sesquiterpenoids α-farnesene by spectroscopic analysis. In leaf-dipping bioassay, α-farnesene (LD50, 36.9) was 3.9 times more toxic than β-farnesene (LD50, 145.2) against P. xylostella larva, based on 48h LD50 values. This compound was less toxic than insecticides. Naturally occurring α-farnesene merit further study as potential diamond back moth control agent.

Recently, Ussur brown katydid outbreaks caused a serious pest problem in areas of Yeongdong, Chungbuk. This study was performed to control the pest with environment-friendly method. Trap was made of PET plastic bottles that easily found near farmhouse. Attractant materials such as oak (Quercus acutissima) leaf, fruits (peach, apple, grapefruit and pear) sarcocarp or its juices, rice wine (makgeolli) and fish meal were directly applied into the manufactured trap and investigated for the attraction efficacy compared with the funnel trap. During one day, manufactured trap (fish trap) attracted the Ussur brown katydid more than funnel trap. The efficacy of attractant materials were as follows: peach juice (32.7 adults)> rice wine+fish meal (31.3 adults) > rice wine (27.0 adults) > pear juice (19.0 adults) > apple juice (17.2 adults) > fish meal (16.7 adults) > grapefruit juice (14.4 adults) > oak leaf (2.3 adults). The attractive efficacy of fruit juices to ussur brown katydid was more than fruit carcocarps, and the trap hangover 1m in height more than that on ground. The composition of rice wine and fish meal prolonged its efficacy when treated with disinfectant.

Tobacco whitefly-Bemisia tabaci is considered one of the most important pests in tropical and subtropical agriculture, as well as in production systems in glasshouses in temperate zones. Principle research on the identity of B. tabaci began with the recognition of more than one biotype differing in life history parameters, host plant associations, plant-related damage and insecticide resistance. Our laboratory strains of B. tabaci were identified and classified as biotype B and Q, through mtCOI PCR. Also, they were tested for their host plant preference and reaction to different insecticide. Biotype Q prefers to feed on red pepper and tomato, was less susceptible to tested insecticides, for instance acetamipirid, spinosad and thiamethoxam, than the biotype B (feed on tomato alone). There has been a report on the presence of gut bacteria in B. argentifolii (= B. tabaci biotype B) and its influence on the host insect processes. Hence, as a further pursuit, we examined our laboratory B. tabaci biotypes B and Q for their gut bacteria, whether these two biotypes are differed with each other. Gut bacterial strains isolated by standard surface sterilization method was identified through 16S rRNA gene sequence. Gut bacterial strains of B. tabaci biotypes B and Q and their close relatives retrieved from the public database (NCBI) indicated that the biotype B was less diversified only with four genera viz., Bacillus, Micrococcus, Pseudomonas and Staphylococcus, whereas the biotype Q diversified with six such as Bacillus, Janibacter, Micrococcus, Staphylococcus, Stenotrophomonas, and Streptomyces. Results of the present investigation suggesting that there may be a relationship with gut bacterial strains and susceptibility to insecticides and host plant preference of B. tabaci biotype B and Q.

This study was done to evaluate the susceptibility, systemic effect, residual effect and control effect in each developmental stages of biotype Q of sweetpotato whitefly against insecticides, acetamiprid+spinetoram SC and dinotefuran SG. Two insecticides were showed similar activity against the eggs, and showed higher activity in acetamiprid+spinetoram SC against the nymph and adult. In systemic effect, two insecticides have a similar activity. It was showed higher activity in root zone systemic application than leaf zone systemic application. Residual effect was showed higher in acetamiprid+spinetoram SC (92%) than dinotefuran SG (44 %) at seven days after treatment. Control effect was showed all over 90 % activity at tree- and seven days after treatment. Therefore, these insecticides are expected to control the sweetpotato whitefly effectively.

This study was performed to investigate the feeding behavior of sweetpotato whitefly, Bemisia tabaci using the DC-EPG and the each developmental periods of biotype Q against nine varieties of red pepper. The result was showed that biotype Q did not feed and develop in the Nokkwang variety. In order to analyze the antifeedant, three feeding-preferred varieties (Cheongpungdaegun, Cheonyang, and Sannedeul) and non-preferred variety (Nokkwang) was tested for their sugar compounds using HPLC (ELSD Detector). The detected sugars were erythritol, xylose, xylitol, fructose, glucose, mannitol, and sucrose. In feeding-preferred varieties, the sugar such as erythritol, xylose, and xylitol were present at higher quantity than Nokkwang variety, however, fructose was existed lower quantity than Nokkwang variety. Subsequent bioassay for antifeedant activity, only xylitol showed repelled at lower concentration; however, attracted at higher concentration. Therefore, it is believed that xylitol may play a key role in the variety choice of red pepper by Q biotype of sweetpotato whitefly.

Biotype Q of Sweetpotato whitefly, Bemisia tabaci (Homoptera: Aleyrodidae) was raised in seven tomatoes and eight red pepper varieties; however, biotype B did not grow in red pepper varieties. Rokkusanmaru variety of tomato and Cheongpungdaegun variety of red pepper showed the highest susceptibility to biotype B and Q. HPLC (ELSD Detector) analysis showed that the presence of sugars such as erythritol, xylose, xylitol, fructose, glucose, mannitol, and sucrose in red pepper varieties; erythritol, xylose, fructose, glucose, and mannitol was in tomato varieties. Tomato varieties lacks xylitol and sucrose, which were present in the red pepper varieties. Subsequent bioassay with these two sugars, sucrose did not show significant difference between two biotypes; however, xylitol was showed only repellent effect against B biotype. Therefore, it seems that xylitol may play a key role in the selection of host plant by biotype B of sweetpotato whitefly.

This study was performed to investigate the toxicity of 39 registered insecticides to the susceptibility, systemic effect, and residual effect and control effect against Pine sawyer beetle, Monochamus saltuarius. Eleven kinds of chemicals such as fenitrothion, fenthion, phenthoate, phosphamidon, dinotefuran, actamiprid, clothianidin, thiacloprid, thiamethoxam, esfenvalerate + fenitrothion, and fipronil were showed 100% insecticidal activity both in body spray and twig dipping bioassay. Among these chemicals, fenitrothion and fenthion were showed 100 % insecticidal activity when sprayed at 4000 times diluted solutions, and phenthoate, thiacloprid, thiamethoxam and fipronil were showed 100% insecticidal activity when sprayed at 2000 times diluted solution. Root systemic effect was showed 100% mortality in phosphamidon, clothianidin, thiamethoxam, and 77.7% in thiacloprid. In residual effect, fenitrothion and thiamethoxam were showed 80% mortality fifteen days after treatment (DAT), and fenthion, phosphamidon, clothianidin were showed 80% mortality ten DAT, fenitrothion, thiamethoxam, fipronil showed 100% mortality in seven DAT, thiacloprid was showed 100% mortality in three DAT. Fenthion and phenthoate were showed 100% mortality one DAT. In the control effect, 6 kinds of chemicals were showed 100% mortality one DAT and all chemicals showed 100% mortality three DAT.

Pine sawyer beetle, Monochamus saltuarius preferred the feed on fresh twig of Korean white pine trees (Pinus koraiensis) than one-year old and two-year twig. It means that the current twig is appropriate to increase the lifespan and reproduction of this insect. In a bioassay after fractionated the hexane layer from the water layer, the hexane layer did not show feeding response; however, the water layer was preferred in the order of fresh twig>one-year twig>two-year old twig. By HPLC analysis, we identified three kinds of sugar, namely, fructose, sucrose, glucose. The quantity of fructose was the highest in fresh twig followed by one-year and two-year old twig. In the preference test using each standard sample, the order was fructose>sucrose>glucose at 10, 30 μg/filterpaper, and fructose>sucrose >glucose at 50μg/filterpaper. From the above result, fructose plays an important role as a feeding stimulant for pine sawyer adult.

Pine wilt is the most important disease of pine trees in Korea, Japan and China. The pathogen causing this disease, the pinewood nematode (Bursaphelenchus xylophylus), is transmitted vectored by adults of some cerambycid beetle species and the Japanese pine sawyer, Monochamus alternatus, is the major vector species in Korea. Although chemical insecticides have been used to kill vector insect and thus prevent transmission of the pathogen, the efficacy is not good. In Japan, to control this insect, an entomopathogenic fungus was studied and developed as an insecticide. This is thought to be the convenient and effective method to control M. alternatus. Recently, there are several reports about the pinewood nematode is vectored by also the pine sawyer, M. saltuarius, in Korea. The objective of this study, therefore, was to isolate and identify entomopathogenic fungi from M. saltuarius cadaver to control it. We collected the cadaver of M. saltuarius and then screened several fungi colonies. The pathogenicity of each fungus was tested using oak longicorn beetle, Moechotypa diphysis, as substitutive insect. M. diphysis is also serious pest to various trees in forest. As the result, only one of them showed high pathogenicity against M. diphysis. Selected fungus was identified by microscopic examination and DNA analysis. Pathogenicity was also evaluated to M. saltuarius.

Root zone application of several systemic insecticides was tested for control of the brown planthopper, Nilaparvata lugens (Stål), in Vietnam and Korea. In Vietnam, the results indicated that carbofuran showed the highest nymphal mortality in all experiments, followed by imidacloprid and carbosulfan. When the insecticides were applied on 10-day old rice, carbofuran was shown almost 100% N. lugens mortality at six days after treatment and the efficacy was extended to twelve days after application. In Korea, various root-zone application methods were tested with carbofuran and carbosulfan. The results showed that carbofuran was the most active in reducing the egg hatching rates. When root-zone treated on 40-50 day-old rice in a greenhouse, no nymphs were hatched in carbofuran treated pots, while average of 20 nymphs were emerged in carbofuran broadcasting pots. Especially the number of nymphs emerged in carbosulfan foliar spray was 54 nymphs per pot even at the eight day after application, which was higher than in control pots. This is the first study ever demonstrated the high egg mortality of N. lugens on rice due to the root-zone application of insecticides.

Bt crystal proteins, encoded by cry genes, are a group of insecticidal proteins unique in the Gram-positive and spore-forming bacterium, Bacillus thuringiensis. These cry genes are widely applied as one of the most successful candidates for constructing transgenic crops resistant to pest insects. In our previous report, we found Cry1-5 had high insecticidal activity against Spodoptera larvae although its amino acid sequences showed high similarity (95.6%) to those of Cry1Ac which had low activity. In comparison with Cry1Ac, Cry1-5 had 12 different residues on domain I and II. In order to convert these residues to Cry1-5 randomly, 10 mutagenic primers were designed. Through multi site-directed mutagenesis, we mutated the modified cry1Ac gene by plant codon usage in pOBI-Modcry1Ac based on cry1-5 and constructed 63 mutant cry genes. Among them, 10 mutant cry genes on domain II were selected and their recombinant proteins were expressed by baculovirus expression system. From bioassay results to P. xylostella and S. exigua, we found some mutants have high insecticidal activities to be applicable to transgenic crops.