To clarify the geographical distribution of scrub typhus vectors in Korea, the first survey of chigger mites was conducted from 2005 to 2007 by collecting wild small mammals twice a year (spring and autumn) at 24 sites nationwide. The two predominant mite species were Leptotrombidium pallidum (52.6%) and L. scutellare (27.1%). However, the proportions of L. scutellare in southern areas, including endemic provinces such as Chungcheongnam-Do, Jeollabuk-Do, Jeollanam-Do, and Gyeongsangnam-Do, were relatively higher than in central Korean regions where L. pallidum was predominant. In autumn, the ratio of L. scutellare increased to 42% while the ratio of L. pallidum decreased. The geographical distribution map of the L. scutellare chigger index was identical to the incidence pattern of scrub typhus, whereas those of overall mites and L. pallidum showed no relationship with case incidence patterns. Distribution mapping analysis shows an identical geographical distribution of L. scutellare and epidemic incidence of scrub typhus in South Korea. The second periodical survey was performed from 2011 to 2013. The result suggests that the distribution of L. scutellare has not been changed remarkably in comparison to the first survey.

There are over 3,000 different species of mosquitoes throughout the world; currently 56 species are reported in Korea. Mosquitoes can act as vectors for many disease-causing viruses and parasites during blood sucking. Various species of mosquitoes are estimated to transmit various types of disease to more than 700 million people annually in Africa, South America, Central America, Mexico, Russia, and much of Asia, with millions of resultant deaths. At least two million people annually die of these diseases, and the morbidity rates are many times higher still. To prevent the spread of diseases, KNIH (Korea National Institute of Health) used three categorized methods; identifying or taxonomic analysis of mosquitoes, detecting virus caring mosquitoes, and detecting malaria from Anopheline mosquitoes. We have proved that taxonomic analysis using DNA barcording method (COI gene) is useful to complement identification of mosquito species. In detecting virus, we have reported Cx. orientalis as a new potential Japanese encephalitis virus (JEV) vector. We also reported that the Anopheline mosquito species composition and Plasmodium vivax infection rates in malaria hot spot in Korea.

Aedes albopicuts, known as asian tiger mosquito or forest mosquito, is characterized by its black and white strips on legs and body. Ae. albopictus is an important vector mosquito for the transmission of many viral pathogens such as dengue fever and chikungunya fever. Unlike other mosquito, Ae. albopictus attacks people mainly during the daytime in the forest. To evaluate the diel activity of Ae. albopictus, three times mosquito collecting were conducted using BG Sentinel trap with dry ice in bamboo forest in Damyang-gun, Jeoullanam-do, in South Korea from August to September, 2009. Captured mosquitoes were counted and released every one hour during 25 hours experiment time. The result showed that Ae. albopictus activity began with sun rising at approximately 09:00, with peak in the early evening between 16:00 to 19:00, and ended with sunset at 21:00. Among the several factors affecting on the mosquito activity, light intensity seems to be a main factor. Especially, if the light intensity is over some threshold, it negatively effects on the activity of Ae. albopictus.

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.

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.

The intracellular endosymbiont bacterium Wolbachia is currently considered the most abundant in arthropods and has also been isolated from nematodes, amphipods, isopods, mites and spiders. Recently, Wolbachia-based research was focused on the control of disease vector-population, such as several mosquitoes such as genus Aedes and Anopheles which cause dengue fever and malaria, respectively. For the analysis of regional difference between vector mosquito Aedes albopictus and Wolbachia, we selected different regions and collected Ae. albopictus which were distinguished with mountain chain and waterway. Whole genomic DNA were extracted from collected specimens with 9 regions. PCR analysis and sequencing were accomplished in each specimen for Wolbachia detection and identification using WSP gene. As a results, almost mosquitoes were infected with two strain of Wolbachia both wAlbA and wAlbB. However, regional separation of vector mosquitoes, wAlbA strain of Wolbachia were showed more than 98% sequence similarity. In this study, we first reported that Wolbachia infection and type of Wolbachia in Korea and endosymbiont Wolbachia was showed highly sequence homologies.

Bacillus thuringiensis (Bt) is a Gram-positive and soil-dwelling bacterium and well known for its ability to produce insecticidal parasporal crystalline protein inclusions, which have attracted worldwide interest for effective pest management. A diseased silkworm by Bt was first discovered by Japanese scientist Ishiwata Shigetane in 1901, and in 10 years, it was re-discovered in Germany by Ernst Berliner, who isolated it as the cause of a disease called Schlaffsucht in flour moth caterpillars. The first commercial product, Bt. kurstaki HD-1 was released to a market in France in 1938. Optimization of mass production for crystal production and cost down enabled the industrialization to be successful, and now many products, such as WP, EC, SC and tablets are used worldwide. In 1976 Robert A. Zakharyan found that plasmids in Bt are involved in the production of crystal proteins and endospores. Pore formation model and signal transduction model were revealed to explain the mode of action of Bt. Works on Bt resistance included a group of receptors of crystal proteins, such as cadherin, APN and ALP. In 1996, a Bt cry gene was integrated to cotton, which successfully reached markets. AtMT technology was used to generate Bt crops. Now the area planted worldwide to genetically engineered Bt crops increased to 66 million hectares. Refuge may be particularly important in slowing the spread of insects resistant to the Bt insecticides. Researchers are trying to increase the insecticidal efficacy of integrated Bt crystal proteins using recent biotechnology.

The chigger mite, Leptotrombidium pallidum, is widely distributed throughout South Korea and is a major vector for Orientia tsutsugamushi, the causative agent of scrub typhus. In this study, the genome size of the chigger mite was estimated to determine the necessary coverage level prior to whole genome sequencing. Cloning of EF1α and RpS3 as putative single copy reference genes were conducted and their partial sequences were determined. Using the serially diluted reference genes with known amount as standard templates, the weight of a single copy of the genome was predicted by a method based on quantitative real time PCR. The average genome length estimated from the weight using two methods was 191 ± 7 Mb. When the genome size of other arthropods (Drosophila melanogster, Apis mellifera and Tetranychus urticae), with their genome analysis completed, were estimated using the same method and compared with actual values, the estimation accuracy was 79.8-98.9%, suggesting our current estimation of L. pallidum genome size is reliable. The estimated L. pallidum genome size is in a similar range to other Acariform mites, such as the dust mite and scabie mite, but appoximately 10-fold smaller compared to the deer tick, which belongs to Parasitiform. Our finding provides key information for further genome sequencing and understanding of mite genome evolution.

As a part of disease vector and possible overseas inflow caused by global warming monitoring around airport and port area, the distribution of domestic mosquitoes was investigated using BG-sentinel trap. Mosquitoes were collected during 5-days per each area in early September, using 10 traps in Cheongju (airport) and 15 traps in Pyeongtaek (port). The numbers of mosquitoes collected in Cheongju and Pyeongtaek area were 1,762 including 10 species and 1,042 including 11 species respectively. In Cheongju, Aedes albopictus (657 individuals, 37.3%) and Culex pipiens complex (415 individuals, 23.6%) were dominant species; while in Pyeongtaek, Cx. pipiens complex (806 individuals, 77.4%) was dominant followed by Anopheles spp. (90 individuals, 8.6%) and Ochlerotatus koreicus (79 individuals, 7.6%). Similar species were collected in both Choengju and Pyeongtaek area. Although most species collected in both Cheongju and Pyeongtaek area were the same, Cx. inatomii was only collected in Pyeongtaek. In case of the Genus Aedes, more number of Ae. albopictus was collected compared to Oc. koreicus in Cheongju; while Oc. koreicus was more in Pyeongtaek. In case of the genus Culex, the individuals belonging to the vishinui group were collected such as Cx. bitaeniorhynchus, Cx. tritaeniorhynchus and Cx. orientalis. This study may provide basic information for efficient prevention of vector mosquito and potential overseas inflow of diseases, also we would try to expand different area in Korea.

A surveillance of chigger mites was conducted to clarify the incidence of scrub typhus vectors by new chigger mite collecting traps instead of trapping wild rodents for surveillance from each 4 collection points of 5 regions from September to November 2012 in Korea. During the surveillance period, 2,829 chiggers were collected and 10 species of 4 genera were identified. The first appearance of chigger mite and patients was 39 week (9.19~9.25) and the density of chigger mites had the peak in 43 week (10.17~10.23) and the density of patients had the peak in 44 week (10.24~10.30), respectively. In Goryeong-gun, a total of 1,797 mites representing 4 genera and 6 species were collected and the predominant species were Leptotrombidium scutellare (86.0%), L. pallidum (6.5%) and L. palpale (4.7%) whereas, In Gurye-gun, a total of 89 mites representing 2 genera and 3 species were collected and the predominant species were L. scutellare (46.5%) and L. palpale (36.2%) in similar to the natural environment. The high collecting rates were recorded at rice field (71.8%) and waterway (11.6%). This result shows that the surveillance of scrub typhus vectors by new chigger mite collecting trap is useful as an eco-friendly method.

The intracellular endosymbiont bacterium Wolbachia is currently considered the most abundant in arthropods. Many Wolbachia manipulate host reproductive systems, it lead to male-killing, cytoplasmic incompatibility, parthenogenesis and feminization of genetic males with large impact on host ecology and evolution in arthropods. In this study, we investigated the distribution of Wolbachia infection in Ae. albopictus according to geographical distribution. We selected 9 areas and collected Ae. albopictus, these areas were distinguished with mountain chain and waterway. Whole genomic DNA were extracted from collected specimens and PCR analysis were accomplished in each specimen using Wolbachia specific primers such as 16S and WSP genes. As a results, we collected 730 Ae. albopictus at least 30 specimens in each area. Wolbachia infection rate showed different patterns between geographical region. Cheonbook, Cheonnam and Yeongdong showed 100% infection rate followed by Jeju (97%), Chungchung (88%), and Gyungnam (85%) with two Wolbachia marker genes. Our results indicated that Ae. albopictus are commonly infected with Wolbachia, it is possible that Wolbachia may act as endosymbiont in Ae. albopictus regardless of geographical region. Although, low infection rate of Wolbachia, we need continuous survey for the evaluation of Wolbachia strain within Ae. albopictus as a vector of dengue fever.

Habitats for migratory birds provide good blood source for blood sucking insects including mosquitoes, which may lead to high population mosquito species. This study was intended to know mosquito fauna in habitats for migratory birds that have preference for bird’s blood. We selected 7 locations for migratory birds (Ansan: a great reedy marsh in lake of Shihwa; Cheonan, Gyeongju, and Pyeongtaek: pine forest; Seosan: a reclaimed land near bay of Cheonsu ; Ulsan: great bamboo forest around Teahwa river) and subdivided each location with four habitats (forest, swamp, cow shed and downtown area) as mosquito collecting site. We used two types of trap for mosquito collection such as CDC black light trap and BG Sentinel trap. Additionally, we use black light and dry ice as an attractive source, respectively. A total of 27,615 mosquitoes representing 9 genera and 18 species were collected. In Ansan, 9 genera and 17 species were collected and in the other locations only 11 to 12 species. Representing by habitats shows this; in forest 9 genera and 17 species, in swamp 8 genera and 16 species, in cow shed 6 genera and 12 species, in downtown 8 genera and 17 species. The dominant species was Culex pipiens (60%) followed by Aedes vexans (11%), Anopheles spp. (8%), Aedes albopictus (7%), and Armigeres subalbatus (5%).

Trombiculid mites are known to be the vector of tsutsugamushi disease by transmitting Orientia tsutsugamushi to human. Although the classification of trombiculid mites is necessary for vector surveillance, their classification by morphological observation is only possible at the larval stage and not easy because of similar shapes as well as tiny body sizes. Further the classification need the specimen production process, it takes much time and the accuracy of classification is changed according to the technology of the researcher. The internal transcribed spacers (ITS) regions of 8 trombiculid mite species were analyzed by amplification using tick common ITS primer sets. We designed molecular marker sets for the identification of five Leptotrombidium species, the lengths of marker L. orientale (1078 bp), L. pallidum (820 bp), L. palpale (1202 bp), L. scutellare (447 bp) and for L. zetum (621 bp) and three Neotrombicula species, the lengths of marker N. gardellai (264 bp), N. japonica (460 bp) and N. kwangneungensis (309 bp) based on alignment of ITS sequences. The markers will be helpful for exact classification of trombiculid mites. This study is the first report on molecular marker of ITS regions of trombiculid mites.

We investigated the prevalence of severe fever with thrombocytopenia- syndrome virus (SFTSV) and geographical distribution of ixodid ticks from 25 regions of the Korea during 2011-2012. We collected 25,940 ixodid ticks with 3 genera and 7 species using dry ice bait trap and flagging. Among them, Genus Haemaphysalis, Ixodes and Amblyomma counted 25,821 (99.5%), 118 (0.5%) and 1 (<0.0%). Genus Haemaphysalis consisted of 4 species, H. longicornis (90.4%), H. flava (9.2%), H. formosensis and H. hystricis (<0.1%). Genus Ixodes consisted of 2 species, I. nippoenesis (0.4%) and I. persulcatus (0.1%). Only one Amblyomma testudinarium was collected. The highly infested sites were grassland (50.1%) and mixed stand forest (20.3%). Minimum infection rate (MIR) of SFTSV in H. longicornis was 0.5% in Korea and this rate was similar to that in China (0.46%). Our results showed that H. longicornis is a major vector of SFTSV and its surveillance will be needed at high risk area.

We conducted a investigation for dispersal ability of Aedes albopictus, a dengue vector mosquito, using mark-release-recapture method in Korea. A certain number of fluorescence marked Ae. albopictus was released in four different habitats – urban (Cheonan), suburban (Jochiwon), port (Pyeongtaek), and airport (Cheongju), and sticky traps and BG-traps were used to recapture the mosquito in designated time period. In case of urban and suburban areas, 100 sticky traps were used in 3-week period for both regions, and 19 BG-traps and 15 BG-traps were used in 36-hour period for urban and suburban areas respectively. Using sticky trap, 21 from 1,300 released mosquitoes (1.62%) were recaptured in urban area; while in suburban area, only 2 individuals (0.1%) were recaptured. Most of them were recaptured within 100m from releasing point and about 40% were within 20m distance. When BG-traps were used, 35 from 900 released mosquitoes (3.8%) were recaptured in urban area; while 26 mosquitoes (2.8%) were recaptured in suburban area. The maximum distance of dispersal was 200m in urban and 100m in suburban area, while most of them were recaptured in 50 ~ 80m range. In case of port and airport area, 15 and 10 BG-traps were used respectively to recapture 1,000 released mosquitoes in 5-day period. In port area, 23 mosquitoes (2.3%) were recaptured; while 166 mosquitoes (16.6%) were recaptured in airport area. The maximum distance of dispersal was 130m in both areas; while most of them were recaptured in 50m range. In conclusion, Ae. albopictus has a narrow dispersal range compared to other mosquitoes, a typical characteristic of genus Aedes, and their dispersal ability is greatly affected by various environmental factors. These results can provide basic information for effective prevention of Ae. albopictus and overseas inflow of dengue fever.

Repellency of 20 plant essential oils to malaria main vector in the Republic of Korea (ROK), Anopheles kleini, was evaluated using skin direct contact bioassay. Anopheles kleini showed the highest repellency to Pelargonium graveolens with EC50 value of 0.244 mg/cm2, followed by Pinus sylvestris and Cinnamomum camphora with EC50 values of 0.484 mg/cm2 and 0.862 mg/cm2. The lowest repellency of An. kleini was revealed from Clary sage oil with EC50 value of 4.665 mg/cm2. Anopheles kleini did not demonstrated any repellency to Lemon, Orange, Neem, Coconut and Olive oil over 20 mg/cm2. Major repellent constituents of Geranium, Pine and Camphora oil were analyzed and identified using Mass-data, GC and GC-Mass. Major constituent of Geranium were β-citronellol (37.0%) and Camphora, 1,8-cineole (35.8%) and Pine, α-terpineol (39.5%). Anopheles kleini showed higher repellency to β-citronellol and 1,8-cineole than to DEET and IR3535 and did not showed any repellency to sabinene and γ-eudesmol over 20 mg/cm2. Residual repellent time of 1,8-cineole and β-citronellol were 26 and 41 min, respectively and DEET, 84 min and IR3535, 102 min. In the light of global efforts to reduce the level of highly toxic synthetic repellents, the three essential oils and their major constituents described merit further study as potential biorepellents for the control of An. kleini populations

Insecticide resistance and activation of the metabolic detoxification enzymes of female Culex pipiens pallens by the blood meal were assessed using a micro-application bioassay and micro-plate enzyme activity assays. Four group of Cx. pipiens pallens were used, a susceptible non-engorging group at seven days after emerging, SNE7 Cp; a resistant non-engorging group at seven days after emerging, RNE7 Cp; a resistant engorged group at one day after blood feeding and a resistant engorged group at seven days after blood feeding, REG7 Cp. Insecticide resistance of Cx. pipiens pallans was increased by the blood feeding. Based on LC50 values, SNE7 Cp demonstrated >50 fold of higher susceptibility to all tested insecticides when compared with RNE7 Cp. RNE7 Cp showed higher susceptibility to all tested insecticides than REG1 Cp and REG7 Cp with a relative susceptibility LC50 (SRLC50) of 25.8 to 50.0 and 25.0 to 48.8. In micro-plate enzyme assays, the metabolic detoxification enzyme activity of Cx. pipiens pallans adult females was increased by the blood feeding. Activation of non-specific esterases (EST), glutathione-S-transferase (GST) and mixed function oxidase (MFO) in RNE7 were higher than in SNE7 and the all tested enzymes in REG1 and REG7 demonstrated significantly higher enzyme activation than RNE7, except for activation of GST in REG1. Activation of MFO in REG1 and REG7 were 209.4- and 74.6- fold higher than in REG7, respectively. Non-specific esterases (EST) and glutathione-S-transferase exhibited < 10 fold of higher Rr values. These results may be significant in terms of the criteria that are used to evaluate resistance, because blood fed female mosquitoes may show enhanced expression of the resistance phenotype, possibly allowing for earlier detection of insecticide resistance.

Culex orientalis is belonging to the mimeticus group of the genus Culex and shows various patterns of white pale spots on the wing and wing venation which cause an ambiguity to identify as this species. To confirm whether or not these variations are limited within the species, we observed 230 Cx. orientalis specimens collected in Korea and divided them into 51 variations according to their wing spots patterns. To compare a molecular similarity between the variations, the ITS2 regions of five major variations were analyzed. The results showed that there are more than 97% nucleotide sequence similarity between the variations as well as within a variation. This results suggest that the wing variations of Cx. orientalis are limited a within-species divergence. To further confirm, ITS2 regions of other species (Cx. mimeticus and Cx. jacksoni) in the mimetiucs group will be analyzed and compared with those of Cx. orientalis variations.

Although baculoviruses have a long history of safe use as specific, environmentally benign insect control agents, their use has been limited by several factors, especially their slow speed of action. In this study, we intended to improve the insecticidal activities of Autographa californica nucleopolyhedrovirus (AcMNPV) by expressing Kunitz-type toxin isolated from venoms of Bombus ignitus or Araneus ventricosus. For this, recombinant AcMNPVs, AcBi-KTT, AcAv-Tox1 and AcAv-Tox2 expressing Bi-KTT, Av-Tox1 and Av-Tox2, respectively, under the control of p10 gene promoter were constructed. While polyhedra produced by these recombinant viruses were identical to those of the wild-type AcMNPV in shape, their sizes were relatively smaller than those of the AcMNPV. Among recombinant viruses, AcBi-KTT and AcAv-Tox2 showed significant reduction in median lethal time (LT50) against Spodoptera exigua larvae. Especiaaly, these two viruses showed about 6.2~10-folds higher polyhedra production rate compared to that of the AcMNPV. These results suggested that Kunitz-type toxins from insect venom could be successfully applied to improve insecticidal activity of baculoviruses.

Bee venom contains a variety of peptides and enzymes, including serine proteases. While the presence of serine proteases in bee venom has been demonstrated, the role of these proteins in bee venom has not been elucidated. Furthermore, there is currently no information available regarding the melanization response or the fibrin(ogen)olytic activity of bee venom serine protease, and the molecular mechanism of its action remains unknown. Here we show that bee venom serine protease (Bi-VSP) is a multifunctional enzyme. In insects, Bi-VSP acts as an arthropod prophenoloxidase (proPO)-activating factor (PPAF), thereby triggering the phenoloxidase (PO) cascade. Bi-VSP injected through the stinger induces a lethal melanization response in target insects by modulating the innate immune response. In mammals, Bi-VSP acts similarly to snake venom serine protease, which exhibits fibrin(ogen)olytic activity. Bi-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products, defining roles forBi-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings provide a novel view of the mechanism of bee venom in which the bee venom serine protease kills target insects via a melanization strategy and exhibits fibrin(ogen)olytic activity.