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.

Monitoring of Carbamate and Organophosphate Resistance Levels in Nilaparvata lugens Based on Bioassay and Quantitative Sequencing

Deok Ho Kwon,Si Woo Lee,Ji Hyun Park,Si Hyeock Lee 한국응용곤충학회 2012 한국응용곤충학회 학술대회논문집 Vol.2012 No.05

The resistance levels to carbamate (CB) and organophosphate (OP) insecticides were determined by topical application in 14 field strains of Nilaparvata lugens. The resistance levels to CB and OP were 1.3~47.5-fold and 1.4~14.4-fold higher than a susceptible strain, respectively. A quantitative sequencing (QS) protocol was established to determine the allele frequencies of four acetylcholinesterase point mutations putatively associated with CB and OP resistance. The allele frequencies of the four mutations (G119A, F/Y330S, F331H and I332L) in field strains ranged from ca. 0.0~51.7%, 0.0~88.9%, 2.5~47.7%, 5.1~56.0% and 6.7~57.3%, respectively. The F331H and I332L were tightly linked each other, suggesting these mutations may occur simultaneously. In the correlation analysis, G119A was not well correlated with actual resistance levels (r2= < 0.232), whereas F331H and I332L showed a better correlation with the resistance levels of benzofuranyl methylcarbamates (r2= 0.595). This finding indicates that F331H and I332L mutation frequencies may be used as molecular markers for detecting carbamate resistance in N. lugens. A QS protocol detecting the F331H and I332L mutation frequencies would be employed as a supportive tool for rapid monitoring of CB insecticide resistance levels in N. lugens

Monitoring of carbamate and organophosphate resistance levels based on bioassay and quantitative sequencing in Nilaparvata lugens

Deok Ho Kwon,Si Woo Lee,Ji Hyeon Park,Si Hyeock Lee 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.10

The resistance levels against carbamates (CB) and organophosphates (OP) were determined through bioassay and quantitative sequencing (QS) methods in 16 field populations of Nilaparvata lugens. The resistance levels to CB and OP by bioassay were 1.3~47.5-fold and 1.4~14.4-fold higher than a susceptible strain, respectively. The QS protocol was established to determine the allele frequencies of eight point mutations on acetylcholinesterase putatively associated CB and OP resistance. The allele frequencies of four mutations in local populations (G119A, F/S330Y, F331H and I332L) ranged from ca. 0.0~51.7%, 1.0~44.3%, 8.5~57.3% and 7.12~56.6%, respectively. The average prediction limits were –9.6±5.1~7.7±2.9%. The F330Y, F331H and I332L were tightly linked each other, suggesting these mutations may occur simultaneously. In the correlation analysis, G119A was not well correlated with both insecticides (r2= less 0.25), whereas F/S330Y, F331H and I332L showed better correlation with the resistance levels of carbamate (r2=0.590) than organophosphate (r2=0.235). This finding indicates that F/S330Y, F331H and I332L mutation frequencies are suitable for detecting carbamate resistance in N. lugens. QS will be applicable for the rapid monitoring of resistance levels to CB insecticides in N. lugens.

Various genes associated with the imidacloprid resistance in the Cotton aphid, Aphis gossypii (Hemiptera: Aphididae)

Ju Il Kim,Min Kwon,Jae Soon Kang,Si Hyeock Lee,Si Woo Lee 한국응용곤충학회 2010 한국응용곤충학회 학술대회논문집 Vol.2010 No.10

The cotton aphid, Aphis gossypii (Glover), is one of the most serious pest in seed potato and various vegetable cultivation. The imidacloprid resistant strain (IR) was over 200 fold more resistant to imidacloprid compared to the susceptible strain (S) as judged by the LC50 values and IR showed cross resistant to acetamiprid, thiamethoxam, thiacloprid, clothianidin. By using the suppression subtractive hybridization method, a imidacloprid resistant associated cDNA library was constructed in adult cotton aphid. In total 115 differentially expressed cDNA clones were obtained. Any point mutation detected in nicotinic acetylcholine receptor alpha 1~5 and beta 1 subunits in the IR. Based on IEF, the IR general esterase isozyme banding patterns were identical with that of S.

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.

Molecular approaches to insecticide resistance management and pest control

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

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

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.

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