The lesser paper wasp, Parapolybia varia, belongs to large subfamily Polistinae and is distributed in Middle East, the Indo-Papuan region and East Asia. P. varia is known to become aggressive when disturbed for defending their colonies, resulting in fatal envenomation. Vespid chemotactic peptide (VCP) and vespakinin have recently been determined to be the top two genes most abundantly transcribed in venom glands of P. varia. To investigate the pharmacological and toxicological properties of VCP and vespakinin, their antitumor, antimicrobial, and cytotoxic activities were evaluated. VCP exhibited a significantly high antitumor activity against ovarian tumor cell SK-OV-3 at 100 M. VCP also showed higher hemolytic activity than vespakinin. Antimicrobial activity was only observed with VCP against yeast Candida albicans at 1 mM. Since VCP showed a relatively low hemolytic activity but a considerable level of antitumor activity, it has further merits to be exploited as a potential antitumor agent with reduced side effects on normal cells.

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.

Expression of hairpin RNA corresponding to the part of COPA transcript was done by agroinfiltration in soybean plants and was confirmed by qRT-PCR. In a pot experiment, T. urticae was infested on agroinfiltrated soybean plants and T. urticae mortality was observed and compared with control plants overtime. Significantly higher mortalities of T. urticae were observed in the COPA-agroinfiltrated soybean plants from post-infestation day 2 (15 ±5%), day 4 (50 ±10 %). At post-infestation day 6, mortality reached to (70 ± 15%). To validate the observed COPA silencing effect in T. urticae fed on the agroinfiltrated soybean plant expressing COPA hairpin RNAs, qRT-PCR analysis was performed. The transcript level of COPA gene was decreased in T. urticae fed on agroinfiltrated soybean plants expressing COPA hairpin RNA from post-infestation day 2. At post-infestation day 2, 4 and 6, COPA transcript levels were reduced by 23.8, 20.7 and 18.8 fold, respectively compared to post-infestation day 1 (control). The results obtained in this study also ruled that the plant mediated production and uptake of silencing (dsRNAs/siRNAs) is an effective way to trigger RNAi in the T. urticae.

Tetranychus urticae is extremely hard to control by conventional acaricides due to its rapid development of resistance to nearly all arrays of acaricide. As an alternative control measure of acaricide-resistant mites, RNA interference (RNAi)-based method has 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 120.3 h, respectively]. The transcript level of the COPE gene was significantly (F3,9 = 16.2, P = 0.001) reduced up to 24% following dsRNA treatment, suggesting that the toxicity was likely mediated by the RNAi of the target gene. To identify the deferentially expressed gene upon dsRNA ingestion, RNA-seq was employed to compare the transcriptional profiles between mites fed dsEGFP and dsCOPB2. Approximately 928 of genes were up- or down-regulated significantly (P < 0.05) compared to control and 182 genes were commonly responded to the treatment of both dsRNAs. Those dsRNA-responsible genes were mainly categorized into metabolic enzymes, transporters and secretory proteins. Further study would be necessary to elucidate the roles of dsRNA-responsible genes in mite’s dsRNA uptake and defense.

Human body and head lice are obligatory human ectoparasites. Although both body and head lice belong to a single species, Pediculus humanus, only body lice are known to be a vector of several bacterial diseases. The higher vector competence of body lice is assumed to be due to their weaker immune response than that of head lice. To test this hypothesis, immune reactions were compared between body and head lice following infections by two model bacteria, Staphylococcus aureus and Escherichia coli, and a human pathogen, Bartonella quintana. Following dermal or oral challenge, the number of these bacteria increased both in hemocoel and alimentary tract of body lice but not in head lice and the viability of the B. quintana was significantly higher in body louse feces, the major route of infection to human. In addition, body lice showed the lower basal/induced transcription level of major immune genes, cytotoxic reactive oxygen species and phagocytosis activity compared with head lice. These findings suggest that a reduced immune response may be responsible, in part, for the increased proliferation and excretion of viable bacteria which are associated with the high level of human infectivity seen in body versus head lice.

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.

The acetylcholinesterase 1 (AmAChE1) of the honey bee is known to be abundantly expressed both in the central and peripheral nervous systems. AmAChE1 exists mostly in the soluble form with little catalytic activity and has non-neuronal functions. Our preliminary observation showed that AmAChE1 expression fluctuated between the forages and nurses. A more systematic expression profiling of AmAChE1 over a year cycle on a monthly basis revealed that AmAChE1 was predominantly expressed during the winter months with being moderately expressed during the rainy summer time. However, no significant difference in AmAChE1 expression was noticed between the nurse and forager workers. Interestingly, AmAChE1 expression was inhibited when bees were allowed for brooding by placing overwintering bee hives in strawberry green houses with the supplement of pollen diets whereas it was resumed when the bee hives were removed from the green houses, thereby suppressed brooding. To confirm whether brooding status is a main determining factor for the suppression of AmAChE1 expression, active bee hives were placed in a screen tent, thereby hindering foraging, until brooding was completely suppressed, and then allowed to restore brooding by removing the screen. The AmAChE1 expression in the head was up-regulated when brooding was suppressed whereas its expression was down-regulated when brooding was resumed. These finding demonstrates that AmAChE1 expression in the central nervous system (i.e., head) is related with brooding status of honey bee. To understand the connection between the AmAChE1 expression and other pathways related with brooding, currently in progress are the analyses of head transcriptomes of honey bee workers with or without their brooding suppressed.

The small brown planthopper (SBPH), Laodelphax striatellus Fallen, is an important pest that causes severe yield losses by transmitting plant viruses to rice. For the efficient control of SBPH in Korea, the respective resistance levels in the immigrant and indigenous populations need to be discerned. The resistance levels to 10 insecticides (three carbamates, an organophosphate, four neonicotinoids, and a phenylpyrazole) were evaluated in 21 field populations collected from either SBPH-immigrating or indigenous regions during two different seasons (early spring vs. late summer). Imidacloprid resistance was most widely observed in many regional populations, followed by thiamethoxam resistance. Interestingly, the resistance level to imidacloprid was significantly higher in both immigrant and late-summer-collected populations than in indigenous and early spring-collected populations, respectively [3.3- (p = 0.018) and 2.6-fold (p = 0.026)]. Moreover, the late summer immigrant population exhibited higher imidacloprid resistance (2.4-fold) than the early spring-collected population from the same region, suggesting that the migratory SBPH that immigrated into Korea already exhibited imidacloprid resistance traits and were further selected after inhabitation. All field populations showed little resistance to fipronil (0.1- to 0.7-fold), suggesting that it is the most effective among the tested insecticides to control field populations of SBPH. The coefficient of variation of the resistance ratio (RR) among different regional populations and the correlation coefficient of RR among different insecticides have been suggested as supplementary parameters when determining appropriate insecticides as respective indicators for the dispersion status of resistance among SBPH populations and the possibility of cross resistance among tested insecticides.

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.

Acetylcholinesterase (AChE) is a hydrolase that hydrolyzes the neurotransmitter acetylcholine. Soluble form of AChE is generated via alternative splicing and functions as a bioscavenger in Dropsophila melanogaster. In this study, effects of acetic acid on the soluble AChE expression were investigated. Treatment of acetic acid resulted in over-expression of soluble AChE in the abdomen in a dose-dependent manner. The soluble AChE was determined to be expressed in the fat body. However, no apparent change in AChE expression was observed in the head. Our finding suggests that the soluble AChE is involved in chemical defense against high concentration of acetic acid, which is a common by-product in fermenting foods. The high level of acetic acid resistance in D. melanogaster, thus, appears to have been evolved via the induction mechanism of soluble AChE expression.

Vespa crabro and V. analis are social hornet species commonly found in Asia, including Korea. Mastoparan is one of the major venom peptides of these two hornets but its amino acid sequence defers substantially. To examine the differences in the potential toxicity and bioactivity of mastoparans between these two social hornets, differential toxicological and pharmacological activities of synthesized mastoparan were investigated. V. analis mastoparan showed a 7-fold higher hemolytic activity, suggesting its higher cytotoxic potential compared with V. crabro mastoparan. Mastoparans from both hornet species exhibited similar levels of antimicrobial activities against Staphylococcus aureus and Botrytis cinerea, whereas the mastoparan from V. analis showed more potent antimicrobial activities against Escherichia coli and Candida albicans. Nevertheless, the antimicrobial activities of mastoparans of V. crabro and V. analis were relatively lower compared with those of other wasps. Both mastoparans also exhibited some levels of antitumor activity but the activity was significantly higher in V. analis mastoparan. In summary, the hemolytic, antimicrobial, and antitumor activities of synthesized V. analis mastoparan were higher than those of V. crabro mastoparan. These differential bioactivities are likely due to the amino acid sequence differences in the mature peptides. In particular, the additional Lys residue present in V. analis mastoparan may contribute to the higher levels of bioactivity as proposed by secondary structure prediction.

The hornets Vespa crabro and V. analis are widely distributed in Asia and are known to be aggressive when disturbed, resulting in frequent stinging accidents. To investigate the differences in venom properties and toxicities between these two hornets, the transcriptomic profiles of venom glands, in conjunction with the venom components, were analyzed and compared. A total of 35 venom-specific genes were identified in both venom gland transcriptomes, but their transcriptional profiles were different between V. crabro and V. analis. In addition, the major venom components were identified and confirmed by mass spectroscopy. Prepromastoparan, vespid chemotactic precursor and vespakinin were the top three genes most prevalently transcribed in the venom gland of V. crabro, and their transcription rates were 112-, 16- and 161-fold higher, respectively, compared with those in V. analis, as judged by FPKM values. In the venom gland of V. analis, however, vespid chemotactic precursor was the most abundantly transcribed gene, followed by premastoparan and vespakinin. In general, most major venom genes were more abundantly expressed in V. crabro, whereas some minor venom genes exhibited higher transcription rates in V. analis, including muscle LIM protein, troponin, paramyosin, calponin, etc. Our findings reveal that the overall venom components of V. crabro and V. analis are similar, but that their expression profiles and levels are considerably different. The comparison of venom gland transcriptomes suggests that V. crabro likely produces venom with more highly enriched major venom components, which has potentially higher toxicity compared with V. analis venom.

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.

Vespa crabro is a cosmopolitan social wasp species whereas Vespa analis is commonly found in Asia. Both species are widely distributed in Korea and known to be aggressive when disturbed, resulting in frequent sting accidents. Although major venom components of well known Vespa wasps have been reported, no comparative transcriptomic analysis of venom gland between V. crabro and V. analis has been conducted to date. To investigate the differences in venom properties between these two wasps, total RNA was extracted from each venom gland and used for RNA-sequencing. A total of 31 venom-specific genes were identified in both venom gland transcriptomes but their expression profiles were different between V. crabro and V. analis. Venom allergen 5, premastoparan A and phospholipase A were the top three genes that were most prevalently transcribed in the venom gland of V. crabro, and their transcription rates were 902-, 112- and 4164-fold higher compared with V. analis, respectively, as judged by FPKM values. Their differential transcription profiles were confirmed by quantitative real-time PCR. In the venom gland of V. analis, however, premastoparan A was most abundantly transcribed gene, followed by calponin and tropomysin. In general, most venom-specific genes were more abundantly expressed in V. crabro but some genes exhibited higher transcription rates in V. analis, including muscle LIM protein, troponin, paramyosin, calponin, etc. Our findings suggest that V. crabro produce venom with much more enriched venom components, thereby with higher toxicity compared with V. analis.

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.

Recently, the expression of acetylcholinesterase1 (AChE1) in honeybee worker has been found to be seasonally fluctuated. Seasonal investigation on the AChE1 expression profiles revealed that it is abundantly expressed in January but its expression was completely abolished in February in both head and abdomen. In an attempt to predict the physiological function of seasonally expressed AChE1, proteomic analysis of honeybee worker was conducted using the samples collected in January and February. Total protein samples separately extracted from the head and abdomen of honeybee forager were compared by 2-D electrophoresis (2-DE). More than 2-fold differences in expression patterns between the two different samples were observed in 50 and 85 protein spots in the head and abdomen, respectively. Among them, 20 protein spots showing >17-fold differences in expression between the two different samples of the head were identified by mass spectrometry. Most of the proteins were identified to be the major royal jelly protein (MRJP) families (e.g., MRJP, MRJP2 and MRJP3), which are known to be expressed in nurse bees during brooding season, and their expression was significantly higher in January than in February. This result was unexpected because brooding usually began in the study site apiary during February and the worker bees used for analysis were assumed to be foragers (old workers). Thus, current findings suggest, though speculative, that the workers collected in January may function as nurses despite their old ages in January or that MRJPs may have other not-yet-characterized functions, which is apart from the conventionally known roles. Finally, possible association of MRJPs with AChE1 was discussed.

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.