Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne zoonosis and SFTS virus is transmitted to humans by tick bites. The aim of this study was to monitor tick population and its SFTS virus infection. During March 2014 to October 2015, ticks were monthly sampled using dry-ice bait trap and flag method from 4 collecting points in Boeun-gun. A total of 16,500 ticks, including 14,646 Haemaphysalis longicornis (88.7%), 1,825 H. flava (11.1%), and 29 Ixodes nipponensis (0.2%), were collected. Ticks were pooled (5,156 ticks in 456 pools) and tested by RT-PCR and nested PCR and the minimum infection rate of ticks was 0.27% (14 pools) consisting of 0.13% H. longicornis (6 pools), 1.19% H. flava (8 pools). This annual surveillance study will be needed for long-term monitoring of ixodid ticks in Korea.

A surveillance of chigger mites was performed to monitor the incidence of scrub typhus vectors at 5 environmental collection points of 3 locations (Muju, Namwon and Gimge) from April 2016 in Jeollabuk-Do, Korea. During the surveillance period, 3,292 chigger mites were collected and the predominant species were Leptotrombidium pallidum (42.9%). The high environmental collecting rates were recorded at reservoir bank (31.6%) and grassfield (29.7%). We detected Orientia tsutsugamushi from the samples collected in Jeollabuk-Do using nested polymerase chain reaction. The specific DNA of O. tsutsugamushi were detected in 11 pools among total 119 pools and its minimum positive rate was 0.35%. In order to clarify the relationship between chigger mites and scrub typhus cases, further survey will be needed.

Ixodid ticks are notorious as the vector of severe fever with thrombocytopenia syndrome virus (SFTSV) in Korea. To confirm the transovarial transmission of SFTSV, we collected blood-fed adult ticks from animals in SFTS case reported area of Jeju island in 2015. A total of 476 ticks were collected from four host animal species, horse (81.3%), dog (9.0%), roe deer (7.1%) and wild boar (2.5%). They were identified as 1 genus and 2 species, Haemaphysalis longicornis (87.8%) and H. flava (12.2%). Among them, 131 adults hatched their eggs successfully. The body weight of females was highly correlated with the number of egg masses laid (P < 0.05). We confirmed that one adult H. flava was infected with SFTSV and its infection rate was 0.77%. The eggs of the SFTSV-infected adults also were infected with SFTSV and its transovarial transmission was confirmed. This result suggests that the parent generation of H. flava could transmit SFTSV transovarially into their F1 progeny and H. flava might be one of major vectors of SFTSV in Korea.

A surveillance of chigger mites was performed to monitor the incidence of scrub typhus vectors at 4 environmental collection points of 6 locations from September to November 2014 in Korea. During the survey period, 420 chigger mites were collected and the dominant species was Leptotrombidium scutellare (42.6%). The first appearance of chigger mite was at 37th week (9.3.-9.10.) and the collected numbers of chigger mites was the highest at 43rd week (10.17.-10.23.). In Goryeong-gun, 299 chigger mites were collected, whereas 5 chigger mites were collected In Yesan-gun. The high environmental collecting rates were recorded at rice field (56%) and waterway (20%). The annually collected numbers (2012-2014) of chigger mites were compared with the average temperatures in August. This result suggests that the average temperature in August might be related with the annual incidence of scrub typhus vectors in Korea. However, the relationship between climate factors and the density of chigger mites needs to be studied by long-term periodical surveillance.

Trombiculid mites are known to be the vector of scrub typhus by transmitting rickettsial pathogen, Orientia tsutsugamushi, to human. In this study, we tried to establish a monitoring system for trombiculid mites using chigger mite collecting traps instead of the conventional rodent-capture method. For selection of collecting points, 10 environmental points were chosen from three regions (Taean, Jinan and Chungju) and two field collections were performed in spring (March-May) and autumn (October-November) seasons from 2013 to 2014. Among 10 environmental points, waterway (37.9%), grass field (28.0%), rice field and field near mountain side (11.4%) and reservoir/wet field (7.3%) showed high collecting rates and they should be included for the representative collecting points for surveillance using chigger mite collecting trap. In order to test the possibility that the dried chigger mites from collecting trap can be used for detection of O. tsutsugamushi, we pooled 30, 10 and 5 chigger mites separately and performed the nested PCR. The infection of O. tsutsugamushi was successfully detected from 5 chigger mites pooling sample. This study shows that chigger mite collecting trap could be an alternative method for monitoring system of scrub typhus vectors.

We developed the smart mosquito counter device (height 1080 mm x width 560 mm x diameter 320 mm, 220V 60Hz 30 W), which can attract the blood sucking female mosquitoes by emitting CO2 gas (300ml/min), can count the number of the captured mosquitoes by an infra-red beam area sensor, can identify the some mosquito species by analyzing mosquito image patterns using RGB (Red, Green, and Blue), and can send the captured mosquitoes’ number and pictures through the CDMA module at the real time. We operated the smart mosquito device and we could know when any mosquito peak time was in a day as well as what the mosquito species are and the captured mosquito numbers are. We found that the accuracy of the smart device was about over 95% compared the real mosquito data including numbers and images and transmitted data by CDMA. Because we can know the mosquito occurrence peak times, locations, and mosquito species by this device, we can efficiently control mosquitoes at the right time, and we can alert the public for any mosquito-born diseases based on the mosquito occurrence data. We can improve the quality of mosquito vector control with the functions of this device and we will develop the smart device to identify more mosquito species.

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.

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 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.

We are currently developing the smart mosquito counter device (height 1080mm × width 560mm × diameter 320mm, 220V 60Hz 30W), which can attract the blood sucking female mosquitoes by emitting CO2 gas (300ml/min), can count the number of the captured mosquitoes by an infra-red beam area sensor, and can send the captured mosquitoes’ number through the CDMA module at real time. We operated the 8 – 16 devices with mosquito sensor networks and a server at the Youngdeungpo-gu areas in Seoul city of south Korea for three years (2011-2013) and we could efficiently control mosquitoes at the high mosquito density area based on the mosquito sensor’s data. We found that the accuracy of the device was about over 93% compared the real mosquito data and transmitted data by CDMA. We also found the water reservoir areas to control floods in Seoul metro city had relatively higher mosquito density than other normal areas, because those flat water areas were preferred by mosquitoes. Because our pest control office knew the mosquito occurrence peak times and areas from mosquito data transmitted by the mosquito sensor devices, we efficiently controlled mosquito larva and adults at the right time. We could accomplish very systematic mosquito control policy and we had the high credibility with the results. Based on the mosquito occurrence data, we selectively and scientifically controlled mosquitoes. Therefore, we reduced pesticide usage and saved annually pesticide expenses up to 2/3. Conclusively, if there is no mosquito, we don’t have to control mosquitoes.

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.

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.