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.

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.

Repellent and acaricidal activities of eucalyptus oil, permethrin, and DEET against Leptotrombidium pallidum larvae, which are a vector transmitting tsutsugamushi disease, were evaluated under laboratory conditions using a filter paper impregnated method. The LD₅₀ values of eucalyptus oil and DEET were 0.025 and 0.018 ㎎/㎠, respectively while that of permethrin was higher than 0.2 ㎎/㎠. In the repellency test of these materials at 6.14 ㎎/㎠, eucalyptus oil gave complete repellency, and the larvae crossed the treated zone killed. But permethrin showed 60% repellency at 9.20 ㎎/㎠ and the mites croosed the zone were not killed. The percent repellency of DEET at 0.53 ㎎/㎠ was 8.3 and 2.8 times higher than that of permethrin and eucalyptus oil, respectively. The acaricidal activities of emulsifiable concentrates-pump sprayers containing the eucalyptus oil as an active ingredient were assayed. The emulsifiable concentrates containing 1% and 3% eucalyptus oil showed weak mortality at 1 hour after treatment, while all ones containing more than 6% oil produced 100% activity against L. pallidum larvae. The mortality also increased as exposure time to the concentrates increase. These results suggest that the potential of eucalyptus oil highly expected to be used as a control or repellent agent against L. pallidum larvae may be very high.