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.

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.

The black pine bast scale, Matsucoccus thunbergianae Miller et Park, is one of the severe pest to pine forests in South Korea. In 1963, a severe pine damage by unknown causes were reported at Goheung region and it turned out that the damage was caused by the black pine bast scale, M. thunbergianae 20 years later. The black pine bast scale have been causing serious damage to Japanese black pine forests in the east and southwestern coastal area of South Korea and its distribution has been expanded. The areas of Japanese black pine forests damaged by M. thunbergianae was 4,043ha in 41 cities or districts in 2017. In males, two nymphal instars are followed by wingless preadult, the pupa, and the adult, whereas the female adult directly emerges from the second nymphal instar. According to recent sex pheromone survey for male adult, M. thunbergianae occur mainly single generation in South Korea with possibility its phenology was advanced probably due to climate change. Until now, the most effective measure to control the black pine bast scale is to trunk injection with insecticides after thinning of a damaged pine forest.

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.

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.

Until recently, molecular detection method for Bursaphelenchus xylophilus (Bx) infesting pine trees and killing themhas been developed several ways such as RFLP, Real-time PCR and LAMP. However, these were not only time-consumingprocess but also could not confirm the result in a short time. Furthermore, these methods also need thermo cycler orthermostat which are not portable and inexpensive. For above reasons, we had been developed the detection method forBx remaining in the pinewood chips without using electronic devices. In this study, we had used DUNT buffer that isable to extract genome DNA from Bx in the pinewood within 10 min and isothermal amplification that could be amplifiedthe specific DNA fragment of Bx at 37 ℃ for 10 min. As a result, our method is able to confirm the presence or absenceof Bx in pine trees within 20 min and could be used in the field as it does not require the electronic devices.

Monochamus alternatus (M. alternatus) and Monochamus saltuarius (M. saltuarius) are major vectors for Bursaphelenchus xylophilus in South Korea. When an adult, they are easily distinguishable by several morphological classification. However, it is difficult to identification between M. alternatus and M. saltuarius when they are larvae as they have very similar morphological characters. Thus, they are not easily distinguishable without expertise about Cerambycidae taxonomy. Furthermore, during epidemiological investigation, sometimes, adults or larvae would not be founded in death pine trees. For these reasons, in this experiment, we are able to identified between M. alternatus and M. saltuarius by mitochondrial 12S rRNA gene primers that are specific to 12S rRNA gene fragment of M. alternatus using larvae tissue and frass. Moreover, we had examined whether vectors that were already escaped from dead pine tree have Bursaphelenchus xylophilus or not by multiplex PCR using larva frass that was remained in dead pine tree.

Monochamus alternatus (M. alternatus) and Monochamus saltuarius (M. saltuarius) are major vectors for Bursaphelenchus xylophilus in South Korea. When an adult, they are easily distinguishable by several morphological classification. However, it is difficult to identification between M. alternatus and M. saltuarius when they are larvae as they have very similar morphological characters. Thus, they are not easily distinguishable without expertise about Cerambycidae taxonomy. Furthermore, during epidemiological investigation, sometimes, adults or larvae would not be founded in death pine trees. For these reasons, in this experiment, we are able to identified between M. alternatus and M. saltuarius by mitochondrial 12S rRNA gene primers that are specific to 12S rRNA gene fragment of M. alternatus using larvae tissue and frass. Moreover, we had examined whether vectors that were already escaped from dead pine tree have Bursaphelenchus xylophilus or not by multiplex PCR using larva frass that was remained in dead pine tree.

Potato Virus Y (PVY) (Potyviridae: potyvirus) is one of the serious emerging virus of seed potato world-wide. It affects the seed potato by transmitting non-persistently via aphids. Here, we developed a simple PVY detection method which used the boiling technique for releasing of the viral RNA from aphid such as stylet and amplification by PVY specific primers located in the viral coat protein gene which suitable for various strains. This simplified method could save the time compared to earlier detection method due to the simplified RNA extraction step. Following this procedure, we tested this one step RT-PCR based PVY detection method by using three PVY vectoring aphid species (M. persicae, A. gossypii and M. euphorbiae) as well as other sucking type insect such as thrips (F. occidentalis). This PVY detection method is rapid, easy-to-use and suitable for large-scale testing in laboratories of seed potato.

There are many other detection methods for Bursaphelenchus xylophilus. However, Baermann funnel method, PCR-based methods, which are laborious and time consuming processes that are unsuitable to rapid diagnose the Pine Wilt Disease (PWD) on the field. For these reasons, the aim of our experiment is not only to apply field diagnostic for pine wood nematode (PWN) but also to reduce total time for detection PWN in the pine trees by loop-mediated isothermal amplification (LAMP) with phosphate (Pi)-induced coloration reaction which could be yes or no answer for detection of PWN has not required UV detector but it just could be discriminated by naked eyes within 30 min. Genomic DNA was directly extracted from pine wood chips by Wood Chips Direct Lysis procedure which can be used for LAMP only after simple dilution. Our results suggest that LAMP-Pi detection method, simple and rapid method for detection of PWN, could be applied to the field diagnostic for PWD.

We had evaluated of insecticidal effect from Insecticide-treated net (ITN) that coated by deltamethrin against Monochamus alternatus which serves as a vector for Bursaphelenchus xylophilus. In this experiment, vector has been shown that rapid insecticidal effect response time within 1 hour when they were contacted with ITN. In addition, vectors were showed high mortality within 48 hours. To evaluate insecticidal persistency of ITN and whether releasing insecticide to water, ITNs were soaked in water over various time periods. Water has not shown insecticidal effect to vectors and small amount of insecticide were detected that would not be harmful to other non-target organic being such as honeybee. Also, reduction of insecticidal effect was not observed from the ITN. Taken together, our results suggest that ITN had got highly insecticidal effect to vector as well as could be applied to effectively prevent dispersal of the vector on the field.

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.

Tropilaelaps mercedesae is an ectoparasite of immature honey bees belonging to the genus Tropilaelaps (Acari: Laelapidae). T. mercedesae has become a major threat to the Western honey bee Apis mellifera in Asia, including Korea, and is expanding its geographical range to northern regions due to global warming. To establish gene resources of T. mercedesae, the whole transcriptome was analyzed by RNA sequencing. An mRNA-focused library was generated from total RNA extracted from the mixed stages using the TruSeq RNA Library Preparation kit and sequenced using the HiSeq 2000 platform. A total of 6.0 Gb reads were obtained with 85% Q30 value. Trimmed sequence data were de novo assembled using the CLC Assembly Cell v 4.2. A total of 64,868 non-duplicate contigs were finally obtained and annotated by the Blast2GO using the NCBI nr database. The most abundant species in the resulting 14,336 Blast hits (22.1%) was Metaseiulus occidentalis, a predatory mite, followed by Ixodes scapularis and Tribolium castaneum, suggesting that the T. mercedesae transcriptome matches well with closely related other arthropod species, including mites and ticks. In order to provide basic information for efficient control and monitoring of potential resistance in T. mercedesae, acaricide target genes were annotated and characterized. One voltage-sensitive sodium channel gene encoding the molecular target of fluvalinate, a pyrethroid acaricide most widely used for the control of T. mercedesae, was identified and its molecular properties were investigated. In addition, other acaricide target genes, including acetylcholinesterase and glutamate (or GABA)-gated chloride channel, were identified and characterized.

A new gregarine parasite (Apicomplexa: Protozoa) was observed in the population of the striped cabbage flea-beetle, Phyllotreta striolata (Coleoptera: Chrysomelidae) in Hoengseong-gun. Gregarines are well known species-specific parasites of various Arthropoda. Based on 954bp of 18S rDNA sequence, this gregarine species was grouped in eugregarine and also this new gregarine showed 85% homology that of the reported Gregarina sp. from daikon leaf beetle, Phaedon brassicae (FJ481523). These two coleopteran hosts were generally found coexist in highland cabbage fields. However, after gut dissection (n>50), each species confirmed has their own gregarine parasite species. These results were identical in surveyed three local populations (Pyeongchang-gun, Hoengseong-gun and Inje-gun). Therefore, we suggested that the gregarines and their coleopteran hosts has species specific interaction via co-evolution.

PVY (Potyviridae: potyvirus) is one of the most important potato virus affecting seed potato production and also it is transmitted non-persistently via aphids. For healthy seed potato production, a virus detection system is highly important in addition to aphid monitoring and control. To achieve this detection method, it need to fast and easy to use. About two decades ago RT-PCR based PVY detection method was developed. However that was very time consuming and has low sensitivity. Here, we developed an advanced PVY detection method which a uses the boiling extraction of the viral RNA from aphid stylet and amplification by specific primers located in the viral capsid protein gene. Therefore, it could directly synthesize cDNA of PVY viral capsid gene from extracted RNA of PVY using one-step RT-PCR method in very short time compared to previous methods due to the omission of RNA extraction step. We confirmed this PVY detection method using the two aphid species (Macrosiphum euphorbiae and Aphis gossypii) that known as PVY vectors. The efficiency of this PVY detection method was 60% to 80% from two the aphid species. Hence, this method could be potentially applied to virus free seed potato production programs.

The genus Diadegma is a well known parasitoid group and some are known to have symbiotic virus, PDV. A novel IV was discovered from the calyx of D. fenestrale female. This virus was named as D. fenestrale Ichnovirus (DfIV). The encapsidated DfIV genome contains 65 circular DNA segments with an aggregate size of 247,191 bp. Based on BLAST analysis, a total of 120 ORFs were predicted as follows: rep; 48, cys-motif; 11, vinnexin; 10, vankyrin; 9, PRRP; 3 and other unassigned genes (39). These viral genes were expressed in lepidopteran hosts (Phthorimaea operculella and Plutella xylostella) after parasitization which means DfIV genome segments were integrated into lepidopteran hosts. This study was focused on this result. Based on gene expression profile, candidate promoter and integration motifs were selected and then, fused with eGFP as a reporter gene. Modified DfIV genome segment was ligated to a commercial containing f1 ori and Ampr gene to propagate in E. coli. We have named this fusion vector as pIN. The construction methodology of pIN and its application would be further discussed in detail.

The cotton aphid, Aphis gossypii (Glover), is one of the main pests in various vegetable crops due to insecticide resistance in Korea. Some insect pests noticed neonicotinoid insecticide resistance such as Nilaparvata lugens, Bemisia tabaci and Myzus persicae etc. and the major player which contributed for developing the resistance was over-expression of P450, particularly CPY6 family. However, A. gossypii was a unique case that they developed non-P450 dependent resistant mechanism. Previously we reported that two point mutations (RtoT in nicotinic acetylcholine receptor, nAChR, beta 1 subunit and RtoT with LtoS in a transcript variant) contribute to develop the imidacolprid resistance in A. gossypii. Moreover we surveyed the mutation(s) in various local field populations. Based on the 3D modeling, we hypothesize that RtoT mutation can reduce the imidacloprid sensitivity. A stretch of 33 amino acid was deleted in the N-terminal region of original transcript of nAChR beta 1 subunit that contained RtoT with LtoS mutations in resistant strain. Among the two transcripts, only original transcript differently expressed between imidacloprid susceptible and resistant strain (resistant ratio = 3,800). Six alpha subunit (1∼5, 7) transcript levels were not significantly different between two strains. Therefore mutation and down-regulation of nAChR beta 1 subunit is also associated with imidacloprid resistance in the A. gossypii.

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.

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.