Due to its rapid development of resistance to nearly all arrays of acaricide, Tetranychus urticae is extremely hard to control using conventional acaricides. As an alternative control measure of acaricide-resistant mites, RNA interference (RNAi)-based methods have recently been suggested. A double-stranded RNA (dsRNA) delivery method using multi-unit chambers was established and employed to screen the RNAi toxicity of 42 T. urticae genes. Among them, the dsRNA treatment of coatomer I (COPI) genes, such as coatomer subunit epsilon (COPE) and beta 2 (COPB2), resulted in high mortality [median lethal time (LT50) = 89.7 and 120.3h, respectively]. The transcript level of the COPE gene was significantly (F3,9 = 16.2, P =0.001) reduced by up to 24% following dsRNA treatment, suggesting that the toxicity was likely mediated by the RNAi of the target gene. As a toxicity enhancement strategy, the recombinant dsRNA was generated by reciprocally recombining half-divided fragments of COPE and COPB2. The two recombinant dsRNAs exhibited higher toxicity than the respective single dsRNA treatments as determined using LT50 values (79.2 and 81.5h, respectively). This finding indicates that the recombination of different genes can enhance RNAi toxicity and be utilized to generate synthetic dsRNA with improved RNAi efficacy.

Leptotrombidium pallidum is the major vector mites for Orientia tsutsugamushi, the causative agent of scrub typhus. To understand the molecular mechanism of L. pallidum, we sequenced the whole genome using Illumina sequencing technology. Totally four genomic libraries with different insert sizes ranging from 280 bp to 8 kb were used to generate 45.1 Gb of genome in the combination of paired-end and mate-pairs sequencing reads. Quality filtering and correction of paired-end reads for very small and/or bad-quality sequences yielded 26.9 Gb of high-quality sequences, which are used to estimate the genome size as 175 Mbusing kmer methods and assembled into a 193.7 Mb genomic sequence scaffolds with N50 length of 92,945 bp. Furthermore, 94% of CEGMA completeness score were obtained from genome scaffold assembly. To facilitate gene annotation, we used a combination of de novo and homology based tools to predict gene models in the chigger mite genome. A combination of evidence-based and de novo approaches predicted 15,842 high-confidence protein-coding genes with an average transcript length of 1,511 bp and 2.4 exons per gene which corresponds to about 12.4% total gene length. Bacterial endosymbiosis are very common in mite species and can range from mutualistic to pathogenic associations. Henceforth, the endosymbionts in L. pallidum were predicted using the NCBI microbial draft genomes and mitochondrial genome. Besides, this L. pallidum draft genome can be used as a significant reference for comparative genomic studies across mite species.

Transcriptome analysis was conducted for the identification of genes associated with insecticide resistance in Frankliniella occidentalis. Resistant strain (FO_RDAHC) exhibited 39.2- ~ 533-fold resistance to acrinathrin, spinosad, emmamectin benzoate and thiamethoxam compared with a susceptible FO_RDA strain. Average 7.6 million reads (± 5,068,895 reads) were obtained from the pyrosequencing and were assembled into the draft CDS database. Gene annotation was conducted by BLAST (UniProt), Pfam, FUNCAT and COG analysis. In the deferentially expressed gene (DEG) analysis, 838 genes were up-regulated and 815 genes were down-regulated over 2-fold ratio in FO_RDAHC strain. Highly up-regulated genes included genes encoding several cuticle-related proteins, cytochrome P450s, esterases and transporter genes. An autotransporter protein gene exhibited the highest up-regulation (596 fold) whereas a GMC oxido-reductase revealed the highest down-regulation (12 fold). Further study would be necessary to validate the actual transcript levels of DEGs and to investigate their functional roles in insecticide resistance.

The two-spotted spider (Tetranychus urticae) is one of the most serious pests worldwide and has developed resistance to almost all types of acaricides. Various mutations on acaricide target and detoxification genes and their duplication (including overexpression) have been identified since the completion of T. urticae genome analysis. The mutations are mainly observed in functionally important domains (i.e. transmembrane, cellular loops and catalytic triad, etc.), which likely confer acaricide resistance directly or indirectly. Gene duplication was found on major detoxification and insecticide target enzymes, such as cytochrome P450, glutathione S-transferase, ABC-transporter, UDP-glycosyltranferase and acetylcholinesterase (AChE). Interestingly, co-occurrence of both mutation and gene duplication (especially, single gene amplification) was found in AChE, which possibly explains the compensatory role of gene duplication to minimize the fitness cost mediated by point mutations. Such mutation and duplication traits associated with resistance can be utilized as molecular markers for the determination of resistance levels based on the quantitative sequencing and real-time PCR.

Leptotrombidium pallidum is the major vector mite for Orientia tsutsugamushi, the causative agent of scrub typhus, in Asian countries, including Korea. The genome size of L. pallidum was previously estimated to be 191 ± 7 Mb (Kim et al., 2014). Genomic DNA (gDNA) was extracted from a single female from a 9-generation inbred L. pallidum colony and used for whole genome amplification (WGA). The resulting amplified gDNA was used for the construction of paired-end and mate-pair libraries and sequenced using Illumina platforms (HiSeq2000 and MiSeq). An unamplified gDNA sample extracted from 20 female mites was also used for sequencing in parallel. More than 45Gb sequence reads from both paired-end and mate-pair libraries of the WGA gDNA were trimmed and then de novo assembled using the CLC Asembly Cell v.4.0 for contig assembly and SSPACE for scaffolding. The assembly generated approximately 6,545 scaffolds with N50 value of 92,945 and total size of ~193Mb, which was in a good agreement with our previous estimation. Repeat analysis showed that about 30% of genome (~58Mb) was masked as repeats, most of which were unclassified novel elements. For gene predictions, generated were the PASA models based on genomic alignments of RNA-seq reads from 4 different chigger mite samples (i.e. male, female, larva, and protonymph) and the GeneWise models based on genomic alignments of protein sequences from 4 closely related species with chigger mite. Independently, ab initio gene predictions were performed with AUGUSTUS and FgeneSH with custom trained matrices optimized for L. pallidum and GENEID with pre-trained matrix for Acyrthopsiphon pisum. By combining all together, 15,842 genes were predicted finally. Manual curation is in progress for various groups of genes, including chemosensory receptor genes, immune-related genes, acaricide target genes, etc.

An easy and rapid resistance detection protocol for the Western flower thrips Frankliniella occidentalis was established based on the residual contact vial bioassay (RCV), in which insecticide resistance levels can be estimated at 8 h-post treatment of diagnostic doses. The RDA strain was used as a reference susceptible strain, which has been reared under laboratory conditions over 10 years without exposure to any insecticides. Seven insecticides were tested for the determination of diagnostic dose. Among them, five insecticides (chlorfenapyr, acrinathrin, spinosad, emmamectin benzoate and thiamethoxam, ranged as 0.03 ~ 0.42 μg-1cm2) were applicable to the RCV. However, two insecticides (omethoate and imidacloprid) were not able to be used for the RCV because the treated inner surface of glass vials by these insecticides were too viscous, causing non-specific mortality. The RCV detection kit was employed for the estimation of resistance levels for the five insecticides in five local populations. Almost field-collected populations revealed high levels of resistance to the four insecticides (acrinathrin, spinosad, emmamectin benzoate and thiamethoxam) by showing less than 50% mortality. The baseline resistance detection by RCV method will facilitate the selection of proper insecticides for farmers to manage insecticide resistant-populations of F. occidentalis.

Nilaparvata lugens Stål is one of the important migratory pests of rice paddy fields in Korea. Resistance levels to nine insecticides were monitored in 12 local strains and correlation analysis was conducted to determine cross-resistance relationships among the tested insecticides. The local strains revealed 1.3- to 28.0-, 1.6- to 6.0-, 2.8- to 237.0-, 0.6- to 0.9-, and 0.7- to 1.3-fold resistance to carbamates, organophosphates, neonicotinoids, fipronil and etofenprox, respectively. Organophosphates revealed moderate correlations with benzofuranyl methylcarbamate (r = 0.566 - 0.614, p > 0.01). Three neonicotinoids were not correlated each other, but imidacloprid and clothianidin were moderately correlated with several benzofuranyl methylcarbamate and organophosphate insecticides (r = 0.590 - 0.705, p > 0.05), indicating that unknown common factors (such as detoxification enzymes) might contribute to resistance to both insecticides. Fipronil and etofenprox exhibited low levels of resistance and cross-resistance with other insecticides, suggesting their potential as an effective insecticide for field application. Resistance level monitoring and correlation analysis would be valuable for the selection of appropriate insecticides to control insecticide-resistant N. lugenes, a typical migratory pest in Korea.

Leptotrombidium pallidum and Leptotrombidium scutellare are the major vector mites for Orientia tsutsugamushi, the causative agent of scrub typhus. Before these organisms can be subjected to whole-genome sequencing, the genome sizes of L. pallidum and L. scutellare were estimated by a method based on quantitative real-time PCR. In addition, k-mer analysis of the genome sequences obtained from Illumina sequencing was conducted to verify the mutual compatibility and reliability of results. The genome sizes estimated by qPCR were 191.3±7 Mb for L. pallidum and 262.1±13 Mb for L. scutellare. The estimated genome sizes based on k-mer analysis were 175.5 Mb for L. pallidum and 286.6 Mb for L. scutellare. The estimates from two independent methods were mutually complementary and in a similar range to those of other Acariform mites. The relatively small genome size would facilitate genome analysis, which could contribute to understanding Arachnida genome evolution and mite vector competence and provide key information for scrub typhus prevention.

Establishment of rapid resistance level detection system is essential step to adopt the adaptive management for the control of various kinds of resistant pest population. Here, we established acaricides resistance detection methods based on residual contact vial bioassay (RCV) and quantitative sequencing methods (QS), and applied to determine the resistance levels from several populations in two-spotted spider mite, Tetranychus urticae, which has been considered as major notorious pest in rose cultivation area in worldwide. 12 acaricides were applicable to the RCV among 19 representative acaricides by showing the dose-dependent mortality within 8 hr, suggesting the acaricide suitability for the RCV might be varied by toxicity mechanism in each acaricides. The QS regression was established for 10 point mutations associated with five number of acaricides resistance such as organophosphate, pyrethroid, abamectin, bifenazate and etoxazol. The 95% prediction level was ranged from 10.8±5.4∼92.2±3.2%. The resistance levels were determined by above two detection methods from a total 12 strains. The laboratory-reared populations were revealed high susceptibility with low resistance allele frequencies to some acaricides, suggesting the several acaricides would be chosen for the control of those populations. However, the field-collected populations were exhibited a severe cross resistance with low susceptibility and high resistance allele frequency to almost tested acaricides, suggesting the current acaricides resistance levels are serious in rose cultivation area in Korea. The RCV and QS methods would be useful for the rapid and accurate collection of valuable information associated with acaricide resistance.

The common bed bug, Cimex lectularius L. (Hemiptera: Cimicidae), is an ectoparasitic pest that feeds on humans as well as other mammals. We investigate that point mutations on the voltage-sensitive sodium channel are associated with the resistance to pyrethroids. Two point mutations (V419L and L925I) in the voltage-sensitive sodium channel (VSSC) α-subunit gene have been identified in deltamethrin-resistant bed bugs. L925I, located the intracellular loop between IIS4 and IIS5, has been previously found in a highly pyrethroid-resistant populations of whitefly. V419L, located in the IS6 transmembrane segment, is a novel mutation. To establish a population-based genotyping method as a molecular resistance monitoring tool, a quantitative sequencing (QS) protocol was developed. Frequency prediction equations were generated from the plots by linear regression, and the signal ratios were shown to highly correlate with resistance allele frequencies (r2 > 0.993). In addition to QS, the filter contact vial bioassay (FCVB) method was established and used to determine the baseline susceptibility and resistance of bed bugs to pyrethroids. A pyrethroid-resistant strain showed > 9375- and 6990-fold resistance to deltamethrin and λ-cyhalothrin, respectively. Resistance allele frequencies in different bed bug populations predicted by QS correlated well with the FCVB results, confirming the roles of the two mutations in pyrethroid resistance. Taken together, employment of QS in conjunction with FCVB method should greatly facilitate the detection and monitoring of pyrethroid resistant bed bugs in the field.

Metcalfa pruinosa has been spreading in Korea since 2005, which its first report at Gimhae in Kyeongnam province. It has been harmful to grape and major forest crops by direct sucking and indirect transmitting sooty mold disease causing economical loss. It is necessary to study its tracing route and movement conditions for the further efficient population management and prevention of its re-invasion. A total of 23 haplotype were observed in the analysis of nucleotide polymorphisms on mitochondrial cytochrome c oxidase I from total 124 voucher specimens among five countries. Only two haplotypes were exist in Korea and HAP1 was accorded with its of some European individuals. Moreover, the analysis of FST and AMOVA, the Korean population was relatively nearer with Spanish and Italian population than American populations, suggesting the Korean population might be originated from some European countries. Eight microsatellite loci were developed and characterized to facilitate more delegate population genetic analysis from 468 individual in five countries. The average character of each or overall population was revealed 18 average individual number, six alleles and 0.676 heterozygosity. The genetic distance (FST) was a little bit high among each populations ranged as – 0.010~0.245. In the tracing route analysis, the originating country of Korean population was also shown as migrated from some European countries. The population genetic analysis using genetic markers will be useful to trace the origin of pest and prevent from re-invasion fro efficient pest management.

Emergence of resistant two-spotted spider mite (TSSM) can induce the over usage of standard amount of acaricides and result in various side effects. Rapid resistance monitoring is essential step for the efficient management of resistant populations by enabling the selection of appropriate acaricides. Here, we evaluated the 19 acaricides to determine its suitability for residual contact vial bioassay (RCV) by using PyriF strain as a reference. Twelve acaricides (Amitraz Abamectin, Bifenthrin, Bifenazate, Chlorfenapyr, Cyenopyrafen, Cyflumetofen, Endosulfan, Fenothiocarb, Monocrotophos, Omethoate and Tebufenpyrad) revealed the dose-dependent mortality within 8 h, whereas other remaining acaricides (Dicofol, Etoxazole, Fenbutatin oxide, Fenpyroxymate, Flufenoxuron, Spiromesifen and Pyridaben) did not. This finding suggests that the application of RCV method is limited depending on the mode of action and physicochemical properties of each acaricide. Resistance levels to 12 acaricides were determined for four field populations of TSSM by using RCV diagnostic kit. All TSSM populations showed the highest sensitivity to cyflumetofen, indicating that it would be most effective in controling field populations. RCV diagnostic kit would enable to provide crucial information for choosing the most appropriate acaricides in the field.

Abamectin, soil-born bacterial acaricides, has been intensively used to control Tetranychus urticae. Target site insensitivity and enhanced activity of detoxification enzymes have been considered as major resistance mechanisms. Here, we identified the differentially expressed genes after feeding sublethal dose of abamectin for 36 h by RNA-seq analysis. About 4.9 million reads (± 2,630,543 reads) were assembled into T. urticae. Among a total of 75 genes showing differential transcription, 18 and 14 genes were up- and down-regulated over two fold ratio, respectively. In the validation analysis, the calculated fold change of each genes between RNA-seq and qPCR were moderately correlated (r 2 = 0.661). A cuticular protein was shown as the highest up-regulated gene (192.3-fold) and a chitinase was revealed to be the highest down-regulated (-16.4-fold). Further study would be necessary to validate their roles in T. urticae adaptation to acaricides.

Most insects possess two different acetylcholinesterases (AChEs) (i.e., AChE1 and AChE2). It has been recently reported that both AChEs are equally active in a damselfly species, providing a unique example of the incomplete specialization of one AChE function after duplication (Kim and Lee, 2013). In this study, we investigated the tissue distribution patterns and the molecular and inhibitory properties of two AChEs (i.e., VgAChE1 and VgAChE2) from the Vestalis gracilis damselfly. VgAChEs exhibited almost identical catalytic activity and were expressed in the central nervous system (CNS). The most predominant molecular form of both VgAChEs was a disulfide-bridged dimer, which is associated with the cell membrane via a glycosylphosphatidylinositol anchor. In an inhibition assay, however, VgAChE1 and VgAChE2 exhibited different sensitivities to organophosphate and carbamate insecticides depending on the structure of the inhibitors. These findings suggest that both VgAChEs have neuronal functions. In addition, soluble monomeric and cleaved molecular forms were detected in both the CNS and peripheral nervous system tissues by an AChE2-specific antibody, implying that VgAChE2 probably shares both neuronal and non-neuronal physiological functions in V. gracilis. Our results support the notion that both VgAChEs, paralogous of each other, are involved in synaptic transmission, with VgAChE2 being in the early stage of acquiring non-neuronal functions.

Emergence of resistant two-spotted spider mite (TSSM) can induce the over usage of standard amount of acaricides and result in various side effects. Rapid resistance monitoring is essential step for the efficient management of resistant populations by enabling the selection of appropriate acaricides. Here, we evaluated the 10 acaricides to determine its suitability on residual contact vial bioassay (RCV) by using PyriF strain as standard. Five acaricides (Abamectin, monocrotophos, tebufenpyrad, bifenazate and cyflumetofen) revealed the dose-dependent mortality within 8 h, whereas other remaining acaricides (Fenbutatin oxide, fenpyroxymate, flufenoxuron, spiromesifen and etoxazole) did not. This finding suggests that the application of RCV method is limited depending on the mode of action and physicochemical properties of each acaricide. Resistance levels to five acaricides (Abamectin, monocrotophos, tebufenpyrad, bifenazate and cyflumetofen) were determined for four field populations of TSSM by using RCV diagnostic kit. All TSSM populations showed the highest sensitivity to cyflumetofen, indicating that it would be most effective in controling field populations. RCV diagnostic kit would enable to provide crucial information for choosing the most appropriate acaricides in the field.

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

The screening of effective lethal genes was conducted via the systemic delivery of dsRNA for the RNA interference (RNAi)-based management of Tetranychus urticae. Four candidate genes (coatomer protein complex, T-COP; M1 metalloprotease, T-M1MP; Ribosomal protein S4, T-RPS4; A subunit of V-ATPase, T-VATPase) with a reference genes (EGFP) were tested for the experiment. Translocated dsRNA to the leaf disc (ca. 30 mm diameter) was detected at 12 h post-treatment, indicating that dsRNA could move through leaf veins. In the evaluation of mortality, the T-COP, T-RPS4 and T-VATPase exerted moderate levels of toxicity at 72 h post-treatment among four dsRNA tested. Knockdown of the target genes were confirmed by quantitative PCR, suggesting that RNAi of target genes was accomplished by feeding dsRNA. In summary, the dsRNA delivery via leaf disc was effective in screening lethal genes, and some genes, such as T-COP, T-M1MP, T-RPS4 and T-VATPase, can be applicable for establishing a RNAi-based control system against T. urticae.

We analyzed molecular and enzymatic properties of three cholinesterases (ChEs; ClAChE1, ClAChE2 and ClSChE) from Cimex lectularius. The ClAChE1 and ClAChE2 were generally present as a membrane-anchored dimeric insoluble form in the brain and ganglia. In the case of ClSChE, monomeric and dimeric soluble forms were observed. To investigate enzymatic properties, three ChEs were functionally expressed using baculovirus expression system. ClAChE1 revealed a significantly higher activity than ClAChE2 to acetylthiocholine iodide (ATChI) substrate. Kinetic analysis using two choline substrates (ATChI and butyrylthiocholine iodide) demonstrated that ClAChE2 had higher catalytic efficiency but lower substrate specificity than ClAChE1. Inhibition assay was conducted by using three inhibitors (BW284C51, eserine, Iso-OMPA) and two insecticides (chlorpyrifos-methyl and carbaryl). Two ClAChEs revealed high sensitivities to BW284C51, eserine, chlorpyrifos-methyl and carbaryl, but were not sensitive to Iso-OMPA. This inhibition profile confirmed that both ClAChEs are categorized as ChEs. Interestingly, the salivary specific cholinesterase did not show any measurable activities to choline substrates, confirming its non-synaptic function in C. lectularius

Metcalfa pruinosa (Say), native to North America, is spreading rapidly in the Korean Peninsula, causing serious damages on many deciduous forest trees, ornamental trees, and agricultural crops. Before the first report from Korea in 2005, M. pruinosa has not been reported from any other countries in the Eastern Palaearctic, while it has been record in Italy in 1979, and rapidly spreading into many European countries. To trace the invasion route of this species, we analyzed haplotype analysis of the mitochondrial cytochrome c oxidase subunit-I (mtCOI, 577bp), and developed microsatellite markers. In the haplotype analysis, a total of fourteen haplotypes were found from 69 individuals in 18 populations: 12 haplotypes (native region), 4 haplotypes (European region) and 2 haplotypes (Korea). Interestingly, Korean populations were clustered with some European populations. Eight polymorphic microsatellite loci were developed, and population structures were analyzed from 145 individuals in 8 populations. The origin and invasion route of M. pruinosa are under investigation.

The green peach aphid (Myzus persicae) is a cosmopolitan pest of agricultural and horticultural crops and causes serious economic damages. M. persica has rapidly developed resistance to a wide variety of insecticides, including pyrethroids. Target site insensitivity mechanism mediated by two mutations (L1014F and M918T) on the para-type voltage-sensitive sodium channel (vssc) is mainly responsible for pyrethroid resistance. To predict the vssc resistance allele frequency, quantitative sequencing (QS) protocol was established. Frequency prediction equations generated from the plots of signal ratios and amplification critical time showed a high correlation coefficient (r2>0.993), indicating its high accuracy in prediction. QS results revealed that the kdr-type L1014F mutation is only present in Pyeongchang strain. No field strains of M. persicae possessed the super-kdr type M918T mutation. However, a novel M918L mutation was found by genotyping approach. The allele frequencies of M918L and L1014F were 0% to 53% in populations examined, and the level of M918L mutation frequency was closely related with pyrethroid resistance. Therefore, QS-based detection of M918L mutation frequency should faciltate the monitoring of pyrethroid resistance in the field.