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Genome/transcriptome analysis of the chigger mite Leptotrombidium pallidum, a major vector for scrub typhus, with a special focus on genes more abundantly expressed in larval stage

https://doi.org/10.1016/j.aspen.2020.07.011Get rights and content

Highlights

  • The genome size of L. pallidum was determined to be 193 Mb and 15,842 genes were predicted.

  • A total of 10,885 protein-coding genes were constitutively expressed in all four stages.

  • A total of 4,078 genes were differentially expressed in all four stages.

  • Whole genome amplification is a reliable option for genome sequencing of a single tiny individual.

Abstract

Leptotrombidium pallidum is the major vector mite for Orientia tsutsugamushi, the causative agent of scrub typhus, in Asian countries, including Korea. Despite its medical importance, L. pallidum has little genetic information available to date. To analyze the L. pallidum genome, we extracted genomic DNA (gDNA) from a single female of a 7-generation inbred L. pallidum colony and amplified the gDNA by whole genome amplification (WGA). The resulting amplified gDNA was used to construct paired-end and mate-pair libraries that were sequenced using Illumina platforms (HiSeq2000 and MiSeq). More than 45 Gb of sequence reads from both paired-end and mate-pair libraries of the WGA gDNA were trimmed and then de novo assembled using CLC Assembly Cell v.4.0 for contig assembly and SSPACE for scaffolding. The assembly generated approximately 6,545 scaffolds with an N50 value of 92,945 and total size of ~ 193 Mb. For gene predictions, the PASA and GeneWise models were used, and ab initio gene predictions were performed independently, resulting in the prediction of 15,842 genes. RNA-Seq expression profiles revealed constitutive expression of 11,572 unique protein-coding genes in larva, 12,364 in protonymph, 12,872 in male adult, and 12,617 in female adult stages. Of the 15,842 predicted genes, 10,885 were commonly expressed through all L. pallidum stages. Genes selectively over-transcribed in the larval stage, which is when host parasitization and disease transmission occur, were further annotated, and their putative roles were discussed.

Introduction

Scrub typhus (tsutsugamushi disease) is an acute, febrile disease caused by the obligate intracellular bacterium Orientia tsutsugamushi. Typical symptoms of scrub typhus include fever, rash, muscle pain, and pneumonitis (Kawamura and Tanaka, 1988). If not treated with appropriate antibiotics, mortality rates by scrub typhus range from 1 ~ 40% depending on area and O. tsutsugamushi strain (Tamura, 1995). One million cases of scrub typhus are identified annually, and more than one billion people are at risk of this disease (Lerdthusnee et al., 2002). This disease is distributed extensively from the eastern parts of Russia to northern Australia in the south and Afghanistan in the west (Kelly et al., 2009), but some cases have also been reported in Africa (Ghorbani et al., 1997, Osuga et al., 1991), South America (Balcells et al., 2011) and the Middle East (Izzard et al., 2010).

Scrub typhus is vectored by the larval stage (chigger) of some Trombiculid mites belonging to the genus Leptotrombidium, which is a genus of very small mites (0.2–0.4 mm) (Eamsobhana et al., 2009). Only the larval stage of Trombiculid mites is parasitic, but mites can maintain O. tsutsugamushi by transovarial transmission as well as transstadial transmission (Roberts et al., 1977). Ground-dwelling rodents are the main host of Trombiculid mites, whereas humans are minor incidental hosts. Trombiculid mites are not blood-feeding, but the larvae pierce the skin of host animals with their mouth parts, inject saliva with digestive enzymes, and then ingest the digested tissues. Among Trombiculid mites, Leptotrombidium pallidum is widely distributed as the primary vector of scrub typhus in the Korean Peninsula (Chang, 1995, Kim et al., 2008, Seong et al., 2001) and Japan (Kawamura and Tanaka, 1988, Takahashi et al., 2004). In addition to scrub typhus, Leptotrombidium mites are known to harbor other pathogens, including hantavirus (Houck et al., 2001) and Bartonella bacteria (Kabeya et al., 2010). Despite their medical importance, little molecular information on Leptotrombidium mites is available to date. In addition, the molecular and genetic bases of their vector competence are unknown, and there are no available methods for effective control.

Recently, the genome sizes of two major Leptotrombidium mites (L. pallidum and L. scutellare) in Korea were estimated by quantitative PCR (qPCR) to be approximately 191 and 262 Mb, respectively, which is within a similar range to those of other Acariform mites (Kim et al., 2014). This relatively small genome size is a great advantage in whole-genome analysis. Whole-genome sequencing of Leptotrombidium mites would provide fundamental genetic information to inform scrub typhus prevention and assess vector competence. Furthermore, discovering new target sites for novel acaricides and repellents and eventually designing efficient measures to prevent scrub typhus would be feasible once genomic information is available. In addition to the completed genomes of Homo sapiens as the host and O. tsutsugamushi as the pathogen, genomic information about Leptotrombidium mite vectors would enhance understanding of mite vector-host-pathogen interactions.

In this study, we performed whole-genome sequencing of L. pallidum, the vector mite of scrub typhus. For this, genomic DNA (gDNA) extracted from a single inbred female was amplified by whole genome amplification (WGA). The resulting amplified gDNA was sequenced, and the trimmed sequences were de novo assembled. A total of 15,842 putative genes was predicted from the assembled sequences. Genes specifically expressed in the parasitic larval stage were identified, and their putative roles were discussed. L. pallidum genome represents an immediate resource for molecular and genetic studies of Leptotrombidium mite vectors.

Section snippets

L. pallidum rearing

Engorged L. pallidum larvae (chiggers) were collected from wild black-striped mice, which were captured using Sherman traps in April 2014 from Cheolwon and Chungju, Korea. Black-striped mice were captured and handled according to an animal use protocol that was reviewed and approved by the Korea Center for Disease Control & Prevention-Institutional Animal Care and Use Committee (KCDC-046-13-2A). The collected L. pallidum population was maintained as described previously (Shin et al., 2014). In

Genome sequencing and assembly

The Illumina HiSeq sequencing system was used to sequence four genomic libraries of L. pallidum with insert sizes ranging from 540 bp to 8 kb. This generated a total of 47.9 Gb of paired-end reads, ranging in size from 100 to 300 bp. Quality filtering and correction of paired-end reads for very small and/or bad-quality sequences yielded 26.9 Gb of high-quality sequences (~60-fold coverage), which were subsequently assembled into a 193.7 Mb genomic sequence scaffold with an N50 length of

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We are grateful to the Division of Medical Entomology at the Korea National Institute of Health for technical support for L. pallidum collection and colony maintenance. This work was supported by a grant (2013E5500800) from the Korea Centers for Disease Control and Prevention. Authors are grateful to Dr. Jaeyoung Choi at Smart Farm Research Center, Korea Institute of Science and Technology for the technical support.

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