We examined the adulticidal activity of 34 plant essential oils when they were sprayed on female Culex pipiens pallens adults. At concentrations of 1000 ppm, four oils, namely, cardamon, coriander, rosemary, and sandalwood, had adulticidal activities ranging from 67.8% to 98%. Sandalwood oil had the highest adulticidal activity with an LC50 value of 445ppm. Gas chromatographic (GC) and GC-mass spectrometric analyses revealed that the major constituents of cardamon were 1,8-cineole, α-terpinolene and α-terpinene; those of coriander were γ-terpinene, camphor, linalool and geranyl acetate; those of rosemary were β-pinene, 1,8-cineol, camphor and linalool; and those of sandalwood were α-santalol (69.4%) and β-santalol (15.3%). Both of the major components of sandalwood had the highest adulticidal activity of all the monoterpenes tested. When inhibitors of enzymes known to promote insecticide resistance were mixed with each of the four essential oils, a synergistic effect was only observed when sandalwood was mixed with 100 ppm PBO, which enhanced the adulticidal activity of 500 ppm sandalwood from 41% to 90%.
This study was conducted to investigate the regional difference in the degree of resistance development of the spirodiclofen 36% WP, an acaricide of tetronic acid class, against two-spotted spider mite, Tetranychus urticae occurred in apple orchard. Two-spotted spider mite was collected from every three regions in the Chungbuk and Gyeongbuk. To compare with the degree of resistance development, this pest was focused on the ovicidal activity (when treated acaricide). The managed orchard in Sancheok-myeon, Myeongseo-ri, Chungju was controlled by several different acaricides from 2001 to 2007, and spirodiclofen WP was used for the last six years. In case of managed orchard, LC50 of spirodiclofen WP was 14.0 ppm, it showed 1.6 fold (resistance ratio) that was estimated as considerably low level resistance ratio comparing to the those of susceptible strain showed in 8.7 ppm. LC50 of spirodiclofen WP against etoxazole-resistant two-spotted spider mite, gained from Chungbuk National University, was investigated in 7.5 ppm. This observation indicates that spirodiclofen WP did not occurred in cross resistance with etoxazole SC. At other regions in Chungju, the resistance ratio against spirodiclofen WP showed as 1.0 and 1.4 fold, respectively. In Gyeongbuk, one region of Yecheon and two regions of Yeongju, it showed as 0.7, 0.8 and 0.9 fold, respectively.
The pinewood nematode (PWN, Bursaphelenchus xylophilus) causes the pine wilt disease, transmitted to pinewoods by the pine sawyer beetle, Monochamus alternatus. It is very difficult to discriminate B. xylophilus from B. mucronatus. Therefore, it has been necessary to detect PWN-infected trees for the prevention of pine wilt disease transmission in a short time. The development of biomarkers such as DNA and protein is important for diagnosis of B. xylophilus. However, there have been no reports regarding biomarker identifications of B. xylophilus. In this study, polyclonal antisera were raised against whole proteins of B. xylophilus in BalbC mice and were primarily screened with ELISA. Twenty five among over 500 cell lines releasing polyclonal antisera revealed B. xylophilus-specific immuno-reactivity, which was at least three times higher than that of B. mucronatus. Three cell lines among them were secreting monoclonal antibody through further screening. These cell lines only detect about a 33-kDa protein in B. xylophilus in the western blot. These results suggest that these monoclonal antibodies will be useful for the development of diagnostic kit for the pine wilt disease.
The nematicidal activity of Kaempferia galanga rhizome-derived materials (methanol extract, steam distillate, and powder) toward M. incognita second-stage juveniles (J2) was examined using residual contact toxicity and pot bioassays. In residual contact toxicity bioassays with J2, the active principles of K. galanga rhizome were identified as the phenylpropanoids ethyl cinnamate (EC, 1) and ethyl-p-methoxycinnamate (EMC, 2) by spectroscopic analysis. EC exhibited 95% and 49% mortality at 0.06 and 0.04 mg/ml toward J2, respectively, whereas EMC showed 98% and 63% mortality at 0.1 and 0.08 mg/ml. In pot tests with J2, K. galanga rhizome methanol extract gave 92% and 82% mortality at 100 and 20 mg/50g soil, respectively. Steam distillate gave 88% and 68% mortality at 85 and 42.5 mg/50g soil, respectively, whereas rhizome powder provided 83% and 62% mortality at 400 and 200 mg/50g soil. K. galanga rhizome-derived materials, particularly EC and EMC, merit further study as potential root-knot nematode control agents or leads because of their great activity as a nematicide.
The study was conducted to gather basic information on the occurrence of phytophagous nematodes for the establishment of nematode management strategy in major highland crop production such as chinese cabbage, carrot and potato. Soil samples were collected from five areas in 48 fields in the highland and sub-highland (425m to 1136m asl) areas in 2006. Majority (88%) of the soil samples contained the root-knot nematodes, Meloidogyne hapla. The average density of the root-knot nematodes in chinese cabbage, carrot and potato fields were 8.3, 18.0 and 25.9 nematodes/100g soil, respectively. Helicotylenchus sp., Pratylenchus sp. and Dorylaimida sp. were also detected but in low densities.Root-knot nematode damage was not detected in potato growth and harvest at 0, 2, 12, 23 nematodes/100g soil densities.
담배거세미나방에서의 반수치사농도는 선충의 계통에 관계없이 담배거세미나방 유충의 령기가 발육할 수록 감수성이 낮아졌다. 반수치사농도가 가장 낮았던 것은 GSN1(Steinernema carpocapsae GSN1) 계통으로 2령충에서 4.0마리였고, 가장 높았던 것은 GSNUH-1(Heterorhabditis sp. GSNUH-1) 계통으로 5령충에서 11.5마리였다. 담배거세미나방의 곤충병원성 선충 5계통의 병원성은 pot 실험에서 선충 계통, 처리농도 및 처리회수에 따라 차이가 있었다. GSN1 계통은 처리농도와 처리회수에 관계없이 다른 4계통의 선충보다 병원성이 높았다. 선충 처리농도에서는 처리농도가 많을수록 병원성도 유의하게 증가하였고, 선충 처리회수에서는 3회 처리가 가장 병원성이 높았으며, 다음으로 2회 처리와 1회 처리 순으로 처리회수가 많을수록 병원성도 증가하였다. 담배거세미나방 유충에 대해서 GSN1, GSNUS-14(Steinernema sp. GSNUS-14), GSNUS-10 계통을 30,000Ijs/30㎖의 농도로 1회 처리했을 때와 5계통의 선충을 90,000Ijs/30㎖의 농도로 1회 이상 처리했을 때 치사율이 80% 이상으로 나타났다. 담배거세미나방에 대한 곤충병원성 선충 5계통의 병원성은 선충 계통, 처리농도 및 엽채류 종류에 따라 차이가 있었다. 선충 계통과 처리농도에 관계없이 엽채류의 종류에 따른 병원성에서는 배추가 양배추나 케일보다 유충 치사율이 높았으며 30,000Ijs/30㎖의 농도 이상으로 처리한 것에서는 Heterorhabdtidis sp. GSNUH-1을 제외하고는 80% 이상의 유충 치사율을 보였다. 처리농도에 따른 병원성에서는 처리농도가 많을수록 유충 치사율이 증가하였다.
배추, 양배추 및 배추 시설재배지에서 담배거세미나방에 대한 방제효과는 선충 계통, 처리농도, 처리회수 및 엽채류의 종류에 따라 차이가 있었다. GSN1(Steinernema carpocapsae GSN1), GSNUS-14(Steinernema sp. GSNUS-14) 및 GSNUH-1(Heterorhabditis sp. GSNUH-1) 계통중에서는 GSN1 계통이 가장 방제효과가 높았고, 처리농도에서는 7, 200,000IJs의 처리가 가장 방제효과가 높았다. 그리고 처리회수간에는 3회 처리가 1회와 2회 처리보다 방제효과 높았고, 엽채류 종류에 따라서는 배추가 양배추나 케일보다 방제효과가 높았다. 배추에서의 방제효과가 가장 높았던 것은 GSN1 계통을 7,200,000Ijs의 농도로 3회 처리하였을 때 95.9%였고, GSNUH-1 계통을 800,000Ijs의 농도로 1회 처리시 64.9%로 가장 낮은 방제효과를 보였다. 양배추에서 방제효과는 GSN1 계통을 7,200,000Ijs의 농도로 3회 처리하였을 때 92.2%로서 가장 높았고, GSNUS-14 계통을 2,400,000Ijs의 농도로 1회 처리시 57.3%로 가장 낮은 방제효과를 보였다. 케일에서 방제효과가 가장 높았던 것은 GSN1 계통을 7,200,000Ijs의 농도로 3회 처리했을 때로 88.6%였고, GSNUH-1 계통을 800,000Ijs의 농도로 1회 처리시 55.3%로 가장 낮은 방제효과를 나타내었다.
Bumblebees are important pollinators in greenhouse and have colonized all parts of the World. In Korea, the value of bumblebees is increasing as pollinator. However, the more recent use of reared colonies may ultimately allow pathogens to spread to peripheral areas for bumblebees. Generally, bumblebees are hosts to a large number of parasites which are mites, flies, protozoa, fungi, virus and bacteria. For most of these, very little is known about their effects against host insect, epidemiology or evolutionary ecology. So, we report pathogenic bacteria isolated from Bombus terrestris and B. ignitus at first time in Korea. Bacillus fusiformis and Klebsiella oxytoca are isolated from B. terrestris and confirmed with 16S rRNA gene nucleotide comparison in NCBI genebank. Pathogenicity of B. fusiformis was 35~40% against B. terrestris. Pantoea dispersa and K. oxytoca are isolated from B. ignitus and confirmed with 16S rRNA gene nucleotide comparison in NCBI genebank. Pathogenicity of these species were 35~40% against B. ignitus. These pathogenicity are considered as low-level.
SAGE technique is a sequenced-based approach that identifies which genes are expressed and quantifies their level of expression. The SAGE catalog of gene expression for a given cell or tissue is defined as the 'transcriptome'. With a goal of obtaining a set of quantitative information of expressed genes of posterior silk gland (PSG) of silkworm, we have generated a SAGE tag library from the PSGat day 4 of 5th instars of Bombyx mori. In this study, atotal of 2,406 tags were identified, representing 682 unique transcripts. Of these SAGE tags, 1,982 tags were detected twice or more accounted for 82% of the total tag population, whereas 445 tags were detected only once accounted for 18% of the total tag population. Four percent (27 tags) of the unique tags were detected at least ten times each, which corresponds to a representation of more than 53% of the total tag population. In addition, we have discussed a comparative aspect of the transcript abundance between expressed sequenced tags (ESTs) and the SAGE tags.
식·약용 곤충에 주로 발생하는 진균병은 녹강병(Metarhizium anisopliae)과 백강병(Beauveria bassiana)이다. 본 실험에서는 두 병원균에 각각 특이적인 프라이머를 제작하고 동시에 검출할 수 있는 기술을 개발하였다. 녹강병 진단을 위해 M. anisopliae 유전자 염기서열 중 200여개의 프라이머를 제작하고 그 중 특이적인 프라이머를 선발하였다. 제작 프라이머를 이용한 PCR 진단 결과, 녹강병원균에서만 약 900base의 진단 band를 확인할 수 있었고, 검출 감도는 102 spores/ml 정도로서 육안으로 불가능한 농도에서도 진단이 가능하였다. 또한, 백강병 진단을 위해 B. bassiana 유전자 염기서열로부터 약 150여개의 프라이머를 제작하고 특이 프라이머를 선발하였다. PCR 진단 결과, 백강병원균에서만 약 500base의 진단 band를 확인할 수 있었고, 검출 감도는 103 spores/ml 정도 이었다. 각각 제작된 프라이머를 이용하여 duplex-PCR를 해보았다. 검출감도는 낮았지만 동시진단도 가능함을 확인하였다. 이러한 시스템은 경제적 손실을 끼치는 주요 질병들은 조기 진단할 수 있기 때문에 병 예방차원에서 매우 유용할 것으로 기대되며, 질병의 역학조사나 주변 환경 요인분석 등에도 유용하게 활용될 것이라 생각된다.
한편, 대량사육장에서의 병 예방을 위한 주요 요령을 정리하였다. 장수풍뎅이 병 예방을 위한 주요 요령은 사육준비기, 입식기, 유충기 등 실제 사육농가에서 진행되는 시기에 맞추어 정리를 하였고, 꽃무지의 경우 1, 2령 때 사육상자 사육과, 3-4세대 이상 경과시 새로운 세대로 교체, 과습 방지 등의 내용을 정리하였다.
Protease from various sources have been studied biotecnologically. For biotechnological applications, one highly preferred enzyme is protease.
There have been no reports of cloned genes encoding digestive proteases in the Laccotrephes japonenis, Ranatra unicolor, Muljarus japonicus. These insects are considered to be a predator of aquatic insects.
RT-PCR was used to amplify cDNA fragments for digestive proteases from total RNA the hole body of the insects. The flanking sequences of the 5'- and 3'- end of the these genes were characterized by RACE-PCR. Sequence analysis showed that these genes contained complete ORF. The deduced amino acid sequences of these protease showed 62% identity to the serine protease of Creontiades dilutus, 58% to Lygus loneolaris trypsin-like serine proteinase , 54% to Triatonatoma infestans salivary trypsin.
In the further study, to generate digestive protease, the DNA fragment coding for serine protease, trypsin-like serine protease were cloning into suttle vector pBACⅠ, and infected to Spodoptera frugiperda (sf9) insect cell. After that, we expect to carry out the proteolytic activity of these recombinant proteases. This is intended as a basis for future studies on the digestive protease in the insects.
We have previously shown that the larvae of swallowtail butterfly, Papilio xuthus, exhibit substantial antibacterial activity in the hemolymph, upon challenging with bacterial lipopolysaccharide (LPS). Here we report the isolation and molecular characterization of several immune inducible genes that are specifically expressed by employing annealing control primer (ACP)-based GeneFishing polymerase chain reaction (PCR) from P. xuthus the larva. Using 120 arbitrary ACPs, we identified 24 differentially expressed genes (DEGs) that are up-regulated in response to injected LPS. Sequence analysis showed that 18 DEGs revelaed a high sequence similarity to the previously characterized genes of other insects, although 6 DEGs showed no significant similarity to any known genes. Among these inducible transcripts we found 8 putative immune-related genes including cecropin and attacin. Finally, we analysed the expression profiles of potential immune-related genes by RT-PCR and found all of them were considerably increased in the mRNA levels by LPS injection.
Insect nicotinic acetylcholine receptors (nAChRs) are targets for insecticides. Despite the importance of the nAChR as a major target for insecticide action, modulators of nAChRs in insects remain unidentified. Here we describe the cloning and identification of a nAChR modulator gene in an insect. This gene was isolated by searching the firefly Pyrocoelia rufa cDNA library, and the geneitself encodes a protein 120 amino acids in length, named Pr-lynx1. Pr-lynx1 shares all the features, including a cysteine-rich consensus motif and common gene structure, of the Ly-6/neurotoxin superfamily. The recombinant Pr-lynx1, which is expressed as a 12-kDa polypeptide in baculovirus-infected insect Sf9 cells, is normally present at the cell surface asa GPI-anchored protein. Northern and Western blot analyses revealed that Pr-lynx1 is expressed in various tissues, such as the ganglion, brain, mandibular muscle, proventriculus, leg muscle, and epidermis. This expression pattern is similar to the distribution of nAChRs as assayed by α3 nAChR immunoreactivity. Co-expression of Pr-lynx1 in Xenopus oocytes expressing α3β4 nAChRs results in an increase in acetylcholine-evoked macroscopic currents, indicating a functional role of Pr-lynx1 as a protein modulator for nAChRs. This study on Pr-lynx1 is the first report of a modulator of nAChRs in an insect species.
A thioredoxin peroxidase (TPx) gene was cloned from the bumblebee, Bumbus ignitus. The B. ignitus TPx (BiTPx) contains an open reading frame of 585 bp encoding 195 amino acid residues and possesses two cysteine residues that are characteristic of 2-Cys subgroup of peroxiredoxin family. The deduced amino acid sequence of the BiTPx cDNA showed 90% identity to Apis melifera (AmTPx-1), 80% to Aedes aegypti (AaTPx), and 78% - 47% to other insect 2-Cys TPx. Northern blot analysis revealed the presence of BiTPx transcripts in all tissues examined. Western blot analysis showed the presence of the BiTPx in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting the BiTPx is not secretable. The cDNA encoding BiTPx was expressed as a 27-kDa polypeptide in baculovirus-infected insect Sf9 cells. The purified recombinant BiTPx was shown to reduce H2O2 in the presence of electrons donated by dithiothreitol and shown to be active in the presence of thioredoxin as electron donor.
As a fundamental study for chronic test (definitive test), the range finding test must have been done prior to conducting chronic test. The aims of this study were 1) to search the effective concentration range of two different heavy metals (cadmium and nickel) to indigenous collembolan species (Paranura rosea) 2) to compare the toxicity of cadmium and nickel depending on exposure durations. Toxicity tests were conducted in accordance with the standardized toxicity test proposed by OECD. In this study, there were no mortality occurred at all test concentrations for 7-days exposure. In contrast to 7-days exposure, there were slight increases in mortalities were observed at the concentration higher than 400 mg/kg for both heavy metals for 14-days exposure. The estimated LC50s for cadmium and nickel were 3980.65 (2036.08-64668.61) and 1597.85 (1087.87-3817.61) mg/kg, respectively. The results of this study may be used as a fundamental toxicity data for two heavy metals to proceed in chronic test in the future study.
Arsenic is a naturally occurring element in the earth's crust and is found throughout the environment, but compared with other toxic elements, little toxicological work has been conducted on arsenic. In this study, we tested the effect of two different arsenic compounds (arsenite (NaAsO2) and arsenate (Na2HAsO4)) on the reproduction of soil inhabiting collembolan species (Paronychiurus kimi). The toxicity tests for each arsenic compound were conducted in accordance with the ISO 11267. The estimated EC50s for reproduction of each arsenic compound were 20.43 (16.97-24.60) and 32.86 (25.64-42.13) mg/kg, respectively. There were statistically differences in the toxicity of two different arsenic compounds based on overlapping 95 % confidence intervals. The relative toxicity ratio of arsenite to arsenate (approx. 0.62) as well as the concentration-dependent decrease in reproduction of P. kimi was steeper at arsenite than that of arsenate. These results show that arsenite was more toxic to P. kimi than arsenate.
We examined the effects of cadmium exposure and various temperature stress on the expression of Pardosa astrigera heat shock protein 70 (HSP70). To do this, P. astrigera HSP70 gene was cloned and its sequence determined. Female spiders collected from non-contaminated region were exposed to 40mM CdCl2 for 2, 4 and 6 weeks by dietary uptake. At the end of every 2, 4 and 6 weeks of exposure, a batch of 5 spiders was collected and total RNA was extracted from each batch of whole bodies. Female spiders were also exposed to different temperatures (20, 25, 30 and 35℃) for 3h and RNA extracted likewise. Transcription profiles of HSP70 in response to cadmium and temperature were determined by quantitative real-time PCR using 18S rRNA as reference gene for data normalization. HSP70 transcription gradually increased during 2,4 and 6 weeks of exposure to cadmium. In particular, the expression level at 6-week exposure was 3.4-fold higher than that of untreated control. In the temperature response, an increased expression of HSP70 was also observed as temperature increased up to 30℃ and then slightly decreased at 35℃. The expression level at 30℃ was 2.3-fold higher than that of 25℃. Taken together, HSP70 gene appears to be up-regulated by general stress factors including cadmium exposure and temperature increase.
We determined the complete mitochondrial genome (mitogenome) of the jewel beetle, Chrysochroa fulgidissima (Coleoptera: Buprestidae) from two overlapping fragments and subsequent sub fragments. The 15,592-bp long C. fulgidissima mitogenome contains gene arrangement and content identical to the most common arrangement found in insects. Most individual C. fulgidissima mitochondrial (mt) genes were well within the range found in the respective genes of other insects. The 875-bp A+T-rich region is shortest among the coleopteran mitogenomes sequenced in their entirety. The region is interesting in that it contains several stem-and-loop structures and tRNA-like structure found in the A+T-rich regions of other insect mitogenomes. As seen in other insect motogenomes the start codon of C. fulgidissima COI gene also is unusual because no typical start codon is available. Three of the 13 protein-coding genes have incomplete termination codon T or TA. All tRNA formed stable stem-and-loop structure, except for tRNASer(AGN), the DHU arm of which formed a simple loop as seen in many other metazoan mt tRNASer(AGN).
We determined the complete mitogenome of the oriental mayfly, Ephemera orientalis (Ephemeroptera: Ephemeridae) and the dragonfly Davidius lunatus (Odonata: Gomphidae). The 16,463-bp long E. orientalis and the 15,912 bp long D. lunatus mitogenome contains gene arrangement and content identical to the most common type found in a diverse insect order. Most individual E. orientalis and D. lunatus mt genes were well within the size found in the respective genes of other insects. The initiation codon for the D. lunatus COI gene was typical as ATA, whereas no typical start codon was found in the start region of E. orientalis COI gene. The A+T-rich regions of both mitogenomes have a few unusual feature. The A+T-rich region of E. orientalis contains a tandem repeat composed of two identical copies of 55 bp long, whereas that of D. lunatus contains a tandem repeat composed of duplicated identical 261-bp copies and one partial copy of the repeat. Also, the A+T-rich region of E. orientalis contains a single sequence and that of D. lunatus contains nine sequences, along with the tandem triplicate sequences, that has the potential to form stem-and-loop structures, flanked by the conserved sequences, “TA(A)TA” at the 5’ end and “G(A)nT’ at the 3’ end. Furthermore, the A+T-rich region of D. lunatus contains two tRNA-like structures, tRNALeu(UUR)-like sequence and tRNATyr-like sequence that have proper anticodon TAA and clover-leaf structure that were previously found in the hymenopteran insects.
We determined the complete mitochondrial genome (mitogenome) of the Japanese Oak Silkmoth, Antheraea yamamai (Lepidoptera: Saturniidae) from two overlapping fragments and subsequent shotgun sequencing. The 15,601-bp long A. yamamai mitogenome contains gene arrangement and content identical to the most common arrangement found in lepidopteran insects. Most individual A. yamamai mitochondrial (mt) genes were well within the range found in the respective genes of other insects, except for small ribosomal RNA (1,037 bp). The 336-bp A+T-rich region is relatively smaller than that of other lepidopteran insects. The region is interesting in that it contains tRNA-like structures as found in the A+T-rich regions of other insect mitogenomes. The start codon of A. yamamai COI gene is unusual in that no typical one (ATN) is available. Three of the 13 protein-coding genes have incomplete termination codon T or TA. All tRNA formed stable stem-and-loop structure, except for tRNASer(AGN), the DHU arm of which formed a simple loop as seen in many other metazoan mt tRNASer(AGN).