콜로라도감자벌레의 살충제저항성 돌연변이의 진단을 위한 유전형 분석기법

이시혁 서울대학교 농업개발연구소 2000 농업생명과학연구 Vol.4 No.-

Three DNA-based genotyping techniques, bi-directional PCR amplification of specific allele (bi-PASA), single stranded conformational polymorphism (SSCP) and minisequencing, have been developed and compared for the detection of the S291G (insensitive acetylcholinesterase) and L1014F (insensitive sodium channel) mutations associated with azinphosmethyl and permethrin resistance, respectively, in the Colorado potato beetle (CPB). Extraction of genomic DNA from individual neonates that were hatched from the previously collected egg masses was determined to be the most efficient and reliable means to obtain suitable templates in terms of convenience, economy, speed, and DNA quality. bi-PASA, employing two allele-specific primers, was determined to be the most efficient and rapid genotyping method for the simultaneous detection of both resistant/susceptible homozygous (SS, RR) and heterozygous (RS) allele. Its resolution, however, was strongly dependent on the quality of template genomic DNA. SSCP also allowed clear genotyping, including the detection of heterozygous alleles and was less dependent on template DNA quality but required a longer processing time. Minisequencing was amenable to a 96-well microtiter plate format for the processing of a large number of samples and allowed direct detection of resistant/susceptible homozygous alleles but was not as efficient as the PASA and SSCP in detecting heterozygous alleles. In considering the advantages and disadvantages of each technique, DNA-based genotyping is best employed in combinations, with the bi-PASA as the primary method and the SSCP and minisequencing as the secondary validating methods. The availability of such DNA-based genotyping techniques, using neonate genomic DNA as templates, will allow the precise monitoring of the resistant and susceptible allele frequencies, including those of heterozygote individuals, in field populations of CPB. These methods are rugged, rapid, cost-effective and capable of resolving SS, RR and RS individual.

Current status and management of fluvalinate resistance of Varroa mites in Korea

Susie Cho,Joonhee Lee,Youngcheon Lim,Jonghyeok Lee1,Si Hyeock Lee 한국응용곤충학회 2023 한국응용곤충학회 학술대회논문집 Vol.2023 No.10

The Varroa mite, Varroa destructor is an ectoparasite that parasitizes honey bees. The widespread usage of acaricides, particularly fluvalinate, has resulted in the emergence of resistance in Varroa mite populations all over the world. The goal of this study is to track fluvalinate resistance in Varroa mite field populations in Korea using both bioassay and molecular markers. To accomplish this, a residual contact vial (RCV) bioassay for on-site resistance monitoring was developed. Early mortality evaluation in the RCV bioassay was effective for reliably separating mites with the knockdown resistance (kdr) genotype, but late mortality evaluation was useful for distinguishing mites with additional resistance factors. The RCV bioassay of 14 field mite populations collected in 2021 revealed potential resistance development in four populations. Quantitative sequencing was used as an alternate method to examine the frequency of the L925I/M mutation in the voltage-gated sodium channel (vgsc), which is related with the fluvalinate kdr phenotype. While the mutation was not present in Varroa mite populations in 2020, it appeared in 2021, rose in frequency in 2022, and was practically ubiquitous across the country by 2023. This recent emergence and rapid spread of fluvalinate resistance within a span of three years demonstrate the Varroa mite's significant potential for developing resistance. This situation emphasizes the critical necessity to replace fluvalinate with alternate acaricides, such as fenpyroximate, coumaphos, and amitraz. A few novel vgsc mutations potentially involved in resistance were identified. Potential factors driving the rapid expansion of resistance were further discussed.

Molecular approaches to insecticide resistance management and pest control

Si Hyeock Lee 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.05

Development of a DNA microarray for species identification of quarantine aphids

Lee, Won Sun,Choi, Hwalran,Kang, JinSeok,Kim, Ji‐,Hoon,Lee, Si Hyeock,Lee, Seunghwan,Hwang, Seung Yong John Wiley Sons, Ltd 2013 Pest Management Science Vol.69 No.12

<P><B>Abstract</B></P><P><B>BACKGROUND</B></P><P><B>Aphid pests are being brought into Korea as a result of increased crop trading. Aphids exist on growth areas of plants, and thus plant growth is seriously affected by aphid pests. However, aphids are very small and have several sexual morphs and life stages, so it is difficult to identify species on the basis of morphological features. This problem was approached using DNA microarray technology</B>.</P><P><B>results</B></P><P><B>DNA targets of the cytochrome <I>c</I> oxidase subunit I gene were generated with a fluorescent dye‐labelled primer and were hybridised onto a DNA microarray consisting of specific probes. After analysing the signal intensity of the specific probes, the unique patterns from the DNA microarray, consisting of 47 species‐specific probes, were obtained to identify 23 aphid species. To confirm the accuracy of the developed DNA microarray, ten individual blind samples were used in blind trials, and the identifications were completely consistent with the sequencing data of all individual blind samples</B>.</P><P><B>CONCLUSION</B></P><P><B>A microarray has been developed to distinguish aphid species. DNA microarray technology provides a rapid, easy, cost‐effective and accurate method for identifying aphid species for pest control management. © 2013 Society of Chemical Industry</B></P>

Mutation and duplication of arthropod acetylcholinesterase: Implications for pesticide resistance and tolerance

Si Hyeock Lee,Young Ho Kim,Deok Ho Kwon 한국응용곤충학회 2017 한국응용곤충학회 학술대회논문집 Vol.2017 No.04

A series of conserved point mutations in acetylcholinesterase (AChE) confer resistance to organophosphorus and carbamate insecticides in most arthropod pests. However, the mutations associated with reduced sensitivity to insecticides usually results in the reduction of catalytic efficiency and leads to a fitness disadvantage. To compensate for the reduced catalytic activity, overexpression of neuronal AChE appears to be necessary, which is achieved by a relatively recent duplication of the AChE gene (ace) as observed in the two-spotted spider mite and other insects. Unlike the cases with overexpression of neuronal AChE, the extensive generation of soluble AChE is observed in some insects either from a distinct non-neuronal ace locus or from a single ace locus via alternative splicing. The production of soluble AChE in the fruit fly is induced by chemical stress. Soluble AChE acts as a potential bioscavenger and provides tolerance to xenobiotics, suggesting its role in chemical adaptation during evolution.

Mechanisms, diagnosis and management of pesticide resistance: Current status and future prospects

Si Hyeock Lee 한국응용곤충학회 2024 한국응용곤충학회 학술대회논문집 Vol.2024 No.04

전 세계적으로 농업해충의 약제 저항성 발달에 따른 방제 효율 저하는 농약의 과다 사용을 초래하여 농업의 생산성 저하 및 환경 파괴 문제 등을 야기하고 있다. 뿐만 아니라 위생해충의 경우에도 약제 저항성 발달이 심각한 수준으로 나타남에 따라 인류 보건에 큰 위협이 되고 있다. 해충의 약제 저항성은 자연계에 존재하는 극히 낮은 빈도의 저항성 형질이 반복적인 약제 사용에 따라 선발되는 과정을 통해 발달 되는데, 해충 약제 저항성의 효과적 관리를 위해서는 저항성 발달 기작의 규명과 신속·정확한 진단법의 확립이 필수적이다. 현재까지의 저항성 관리 는 저항성 발달 후 대응적(reactive)으로 이루어져 왔으나, 미래에는 보다 다양한 분자 마커를 활용하여 저항성 형질 빈도를 관리 가능한 수준에서 유지하는 선제적(proactive) 저항성 관리도 가능할 것으로 예상하고 있다. 본 발표에서는 해충 약제 저항성 연구의 현재 상황과 미래 전망에 대해 소개하고자 한다.

Increased oxidative bioactivation is responsible for highly selective coumaphos toxicity to varroa mites, Varroa destructor

Si Hyeock Lee 한국응용곤충학회 2022 한국응용곤충학회 학술대회논문집 Vol.2022 No.10

Feeding amount and time course of plant subcellular fractions of the four sucking pest species

Si Hyeock Lee,Kyungmun Kim,Min Ju Kim,Seung Hee Han,Sang Hyeon Kim,Yue Gao 한국응용곤충학회 2018 한국응용곤충학회 학술대회논문집 Vol.2018 No.10

RNA interference (RNAi)-based strategy has been developed to control various phytophagous chewing pests. However, only a few cases of RNAi-based control success have been reported for sucking pests, suggesting that sucking pests likely ingest less amount of transgenic subcellular hairpin RNA (or dsRNA). In this study, as the basic information for the establishment of ingestion RNAi against sucking pests, feeding amount and time course of plant subcellular fractions of the four sucking pest species (Frankliniella occidentalis, Frankliniella intonsa, Tetranychus urticae and Nilaparvata lugans) were determined by quantitative PCR (qPCR). Adults of the four species were starved for 24 h and then fed with kidney bean leaf (F. occidentalis, F. intonsa, T. urticae) or rice leaf (N. lugens) for 48 h. The leaf-fed adults were collected every 6-h interval and their genomic DNA was extracted. The ingested fractions of chloroplast and nuclear were quantified using rubisco and 50s rRNA as marker genes, respectively. The ingested amount of rubisco and 50s rRNA genes in F. occidentalis, F. intonsa and T. urticae showed rapid increasing pattern after feeding and then slightly reduced over time. In contrast, N. lugens neither ingest nuclear nor showed any distinct feeding pattern of chloroplast. These results demonstrate that F. occidentalis, F. intonsa and T. urticae ingest both chloroplasts and nucleus along with cytosol as cell-feeders but N. lugens, a phloem sap feeder, does not ingest nucleus during sucking. Our findings further suggest that ingestion RNAi-based control strategy would work better for cell-feeding sucking pests compared to phloem sap-feeding sucking pests.

Complete mitochondrial genome of brown marmorated stink bug Halyomorpha halys (Hemiptera: Pentatomidae), and phylogenetic relationships of hemipteran suborders

Lee, Wonhoon,Kang, Joongnam,Jung, Chansik,Hoelmer, Kim,Lee, Si Hyeock,Lee, Seunghwan Springer-Verlag 2009 Molecules and cells Vol.28 No.3

Genomics of Body/Head Lice and Its Application

Si Hyeock Lee,Ju Hyeon Kim,J. Marshall Clark,Barry B. Pittendrigh 한국응용곤충학회 2014 한국응용곤충학회 학술대회논문집 Vol.2014 No.04

Both the body louse (Pediculus humanus humanus) and the head louse (P. humanus capitis) are obligatory human ectoparasites. The body louse is a serious public health threat because it transmits a variety of human diseases whereas the head lice causes one of the most prevalent human infestations, pediculosis. Recent genome analysis revealed that both body and head lice have one of the smallest insect genomes (~108 Mb). Comparison of transcriptional profiles uncovered that almost the same number of genes was annotated both in the head louse (10,770 genes) and the body louse (10,771 genes) among 10,775 protein-coding genes predicted from the body louse genome. Despite their similar genetic background, body and head lice have several differences in their biological features, such as choice of habitat on human host, body size and vector competence. Annotation of major detoxification genes revealed that they are dramatically reduced in human lice compared to other insects except for the honey bee and that, despite the overall reduction in number, human lice retain at least a minimum repertoire of genes known to confer metabolic or toxicokinetic resistance to insecticides, suggesting their high potential for resistance development. Comparison of insecticide target site gene sequences and transcription levels of detoxification genes enabled the identification of toxicodynamic and metabolic factors of insecticide resistance and further allowed the development of molecular markers for resistance detection. Transcriptional profiling during tolerance was used to identify ivermectinmetabolizing detoxification genes, indicating that such an approach may allow proactive resistance management. Comparison of genomes and transcriptomes between body and head lice suggested that vector competence difference is not attributed to the difference in the composition of immune related genes but rather to their transcriptional regulation and/or not-yet-identified epigenetic factors.