RPA/DAP assay that useful technique to detect Bursaphelenchus xylophilus in pinewood in the field Deok Jea Cha and Dong Soo

Deok Jea Cha,Dong Soo Kim 한국응용곤충학회 2019 한국응용곤충학회 학술대회논문집 Vol.2019 No.10

The epidemiological investigation method for Monochamus alternatus using Recombinase Polymerase Amplification (RPA)

Deok Jea Cha,Dong Soo Kim 한국응용곤충학회 2019 한국응용곤충학회 학술대회논문집 Vol.2019 No.04

Monochamus alternatus which is major vector for Bursaphelenchus xylophilus is main pest for pine trees. Moreover, their larvae mainly feed on the phloem of the host during the growth period, and this process makes host debilitate. The reason why the epidemiological investigation of that damage caused by Monochamus alternatus is difficult is that the adult and the larva remains very rarely in the host. Even if the larvae remain in the host, it is not easy to distinguish them from other cerambycidae coleopterans without expertise. For the above reason, we introduce the method of field epidemiological investigation for Monochamus alternatus using the remaining larva frass in the host with Recombinase Polymerase Amplification (RPA) method.

Verification of the ground spraying of three insecticides for Matsucoccus matsumurae

Deok Jea Cha,In Hoo Jung,Dong Soo Kim 한국응용곤충학회 2019 한국응용곤충학회 학술대회논문집 Vol.2019 No.04

Matsucoccus matsumurae is the main forest insect pest of the Pinus thunbergii and Pinus densiflora in South Korea. In this experiment, we had verified the insecticidal effects of Amitraz, deltamethrin and sulfoxaflor, using ground spraying since the trunk injection has not been greatly effective for the control recently. We divided the methods for three ways to confirm the insecticidal effects by developmental stages. Firstly, in male, only amitraz among the insecticides showed significant mortality. Secondly, in female, none of the treatments showed significant mortality but showed delay synthesize the wax filaments when the amitraz treated. In addition, sulfoxaflor treatment showed significant lower egg production rate. Lastly, in 2nd nymph, all insecticides were not shown insecticidal effect. Above the results shown that the ground spraying of three insecticides for M. matsumurae was not a proper control method.

The diagnostic detection method for Bursaphelenchus xylophilus in the pinewood using Recombinase Polymerase Amplification (RPA)

Deok Jea Cha,Dong Soo Kim 한국응용곤충학회 2018 한국응용곤충학회 학술대회논문집 Vol.2018 No.04

Bursaphelenchus xylophilus (Bx) is the main plant-parasitic nematode of the Japanese Black Pine (Pinus thunbergii), Red Pine (Pinus densiflora) and Korean Pine (Pinus koraiensis) in the South Korea. Until now, the nematode morphological classification or PCR method using specific marker of Bx were used for the diagnosis of pine wilt disease. However, both methods have a disadvantage that these take a long time to confirm the result. Thus, these methods can not be used quickly at the newly damaged regions. For above the reasons, we had been developed the diagnostic method for Bx combining direct gDNA extraction buffer (DAP) with Recombinase Polymerase Amplification (RPA). This method is able to directly use mixed lysates extracted from Bx-infected pinewood by DAP buffer as gDNA template to RPA without another process for increase gDNA yield. Together, our method is able to detect Bx within 20 mins.

The origin and evolutionary history of two insect acetylcholinesterases

Deok Jea Cha,Si Hyeock Lee 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

Acetylcholinesterase (AChE) plays a pivotal role in the synaptic transmission in the cholinergic nervous system of most animals, including insects. Insects have two different ace (ace1 and ace2) loci that encode two distinct AChEs (AChE1 and AChE2), which were originated by duplication events long before the radiation of insects. However, little is known about when the ace duplication occurred and how each duplicated ace locus has evolved to retain the original functions. In this study, we conducted phylogenetic analysis for cholinesterase genes from all the lower animals with their genome sequenced together with all known arthropod ace1 and ace2, including those from a number of insects that were newly cloned. Among several independent duplications in lower animal lineages, one duplication event found in platyhelminthes appeared to be the direct origin of arthropod ace1 and ace2. Comparison of the evolutionary distance (d) of two aces from different insect groups relative to those from common ancestors revealed that ace1 has evolved with a significantly slower rate compared to ace2, suggesting that the ace1 lineage has maintained relatively more essential functions following duplication. When the dN/dS ratio was compared between ace1 and ace2 within different insect orders, ace2 was determined to have received relatively more positive selection pressure in Diptera and Hymenoptera whereas the same was true for ace1 in Coleoptera, Hemiptera and Lepidoptera. Along with the relatively more decreased d value for ace2, such an increased dN/dS ratio for ace2 in Diptera and Hymenoptera implied the incidence of functional transition of ace1 to ace2. Our findings should provide with new insights into the evolution of two insect AChEs: when they were generated and how they retain and gain the neuronal functions.

Structural conservation and differentiation of two insect acetylcholinesterases during evolution

Deok Jea Cha,Si Hyeock Lee 한국응용곤충학회 2013 한국응용곤충학회 학술대회논문집 Vol.2013 No.10

Insects possess two distinct acetylcholinesterases (AChE1 vs. AChE2), which are encoded by two paralogous loci originated from duplication. Kinetic analyses of several insect AChEs revealed that both AChE1 and AChE2 retain common catalytic properties of AChE but subtle kinetic differences also exist between these two AChEs. To understand how selection pressure has shaped the protein structure of AChEs and affected their function during evolution, we measured and compared the nucleotide diversity (Pi) and amino acid site-specific selection pressure between AChE1 and AChE2 from various insects. Highly conserved were the majority of the amino acid residues involved in forming the essential domains, including peripheral anionic site (PAS), and little differences were revealed between AChE1 and AChE2, suggesting the presence of strong purifying selection pressure over these essential residues. Interestingly, the EF-hand like motif was mostly found in the AChE1 lineage but not in AChE2. In addition, a unique amino acid difference in the PAS (D72 vs. Y72) was highly conserved between AChE1 and AChE2. Three-dimensional modeling of insect AChEs by particularly focusing on the PAS revealed that a subtle but consistent structural alteration in the active site topology was caused by the PAS amino acid substitution. Taken together, despite the long evolutionary history and low overall sequence similarity, both insect AChE1 and AChE2 still share a extremely high degree of structural and functional conservation, indicative of a strong purifying selection pressure. Nevertheless, only a small change in the PAS, appears to be associated with a local but significant alteration of AChE2 structure, which in turn drives the functional differentiation of AChE.

Efficacy Comparison between the RNA Interference Based on Feeding Long-hairpin RNA and Double-stranded RNA in Nilaparvata lugen

Deok Jea Cha,Si Hyeock Lee 한국응용곤충학회 2012 한국응용곤충학회 학술대회논문집 Vol.2012 No.10

RNA interference (RNAi) technology based on feeding double-stranded RNA (dsRNA) has been employed for the control of insect pests. In general, strong lethal effects have been observed when feeding RNAi is applied to chewing insects. However, the efficacy of feeding RNAi for sap-sucking insects has not been reported to be limited most likely due to the reduced rate of dsRNA translocation into the plant sap. In this experiment, therefore, we tested whether the long-hairpin RNA (lhRNA) structure, which mimics the viroid, can improve its translocation within plant tissues, thereby increasing lethality of target gene, when compared with dsRNA structure. Either lhRNA of dsRNA structure (75 ng/ul) of vacuolar ATP synthase subunit A (V-ATPase) gene was delivered via rice seedling to Nilaparvata lugens, which is one of the major sucking insects on rice, and mortality was measured until 60 h post-treatment. Treatment of the lhRNA and dsRNA of V-ATPase gene caused increased mortality over time compared with eGFP-treated control, reaching the maximum level at 48 h post-treatment, and the mortality was significantly higher in lhRNA treatment than in dsRNA treatment. Gene silencing of target gene was confirmed at 24 h and 48 h post-treatment. In summary, treatment of lhRNA resulted in significantly higher mortalities than that of dsRNA, suggesting that delivery of lhRNA has an apparent advantage over dsRNA in exerting RNAi-induced lethality.

Evolutionary Origin and Status of two Insect Acetylcholinesterases and Their Structural Conservation and Differentiation

Deok Jea Cha,Si Hyeock Lee 한국응용곤충학회 2014 한국응용곤충학회 학술대회논문집 Vol.2014 No.04

Acetylcholinesterase (AChE) plays a pivotal role in the synaptic transmission in the cholinergic nervous system of most animals, including insects. Most insects possess two AChEs (i.e., AChE1 vs. AChE2), which are encoded by two paralogous loci originated from the duplication that occurred before the radiation of insects. The phylogenetic analysis suggested that the last common ancestor of ace1 and ace2 shared its origin with those of Platyhelminthes. In addition, ace1 lineage showed a lower evolutionary rate (d and dN/dS ratio) compared to ace2 lineage, suggesting that the ace1 lineage has maintained relatively more essential functions following duplication. Furthermore, structural modeling of AChEs revealed that consistent structural alteration in their active-site gorge topology was caused by amino acid substitution, likely leads to functional differentiation between two AChEs. The functional transition of ace in some hymenopteran insects appears to have occurred by only a few mutations resulting in dramatic alteration of AChE activity. Taken together, our findings provide basic information on when the ace duplication occurred and what structural features have been associated with the differentiation of two AChEs during evolution.

The attraction effect verification of sex pheromone trap for the Matsucoccus thunbergianae male

Deok Jea Cha,Dong Soo Kim 한국응용곤충학회 2018 한국응용곤충학회 학술대회논문집 Vol.2018 No.04

Matsucoccus thunbergianae is the main forest insect pest of the Japanese Black Pine (Pinus thunbergii) in the west, south and east coast of South Korea. The main damage pattern appears to be the cumulative damage over many years by the second instar fixed under the pine bark. The adult male has a pair of wings and is attracted sex pheromone emanating from the adult female so that during the mating period (Generally, middle period of March) adult male is attracted to the adult female which has no wing. Using this mating behavior from the adult male, we developed sex pheromone trap to reduce mating success rate by reducing the density of the adult male in the field. In this study, In the indoor induction experiments and the field experiments, sex pheromone trap showed high induction effect on the adult male. Thus, we expect to reduce the damage to the Japanese Black Pine by Matsucoccus thunbergianae.

Quick detection method of Pine Wood Nematode (Bursaphelenchus xylophilus) by direct pine wood chips lysis with multiplex PCR

Deok Jea Cha,Jeongmin Kim,Dong Soo Kim,Sang Gil Lee,Si Hyeock Lee,Yong Bae Park 한국응용곤충학회 2016 한국응용곤충학회 학술대회논문집 Vol.2016 No.04

It is difficult to identification between Bursaphelenchus spp. and Pine Wood Nematode (PWN) by morphological characteristics without expertise about nematode taxonomy. Furthermore, Baermann funnel method, which is nematode extraction method from wood chips or soil, requires at least 24 hours to extract nematode that is unsuitable to rapid diagnose the Pine Wilt Disease (PWD). For these reasons, the aim of this experiment is not only to improve accuracy of a PCR based method but also to reduce total experiment time for detection Bursaphelenchus spp. and PWN in the wood chips of PWD infected pine tree. In this experiment, we had been employed two PCR primer sets, which were originated from PWN specific Internal Transcribed Spacer (ITS) sequence region and Bursaphenchus spp. universal mitochondrial Cytocrome Oxidase subunit I (mtCOI) sequence region in order to discrimination between Bursaphelenchus spp. and PWN at the same PCR reaction. This experimental procedure was able to reduce experiment time and cost as well as to improve accuracy of detection than previous PCR based detecting method by not using Baermann funnel method and commercial genomic DNA extraction kit but using direct pine wood chips lysis method.