Autofluorescence microscopy for paired-matched morphological and molecular identification of individual chigger mites (Acari: Trombiculidae), the vectors of scrub typhus

PLoS One. 2018 Mar 1;13(3):e0193163. doi: 10.1371/journal.pone.0193163. eCollection 2018.

Abstract

Background: Conventional gold standard characterization of chigger mites involves chemical preparation procedures (i.e. specimen clearing) for visualization of morphological features, which however contributes to destruction of the arthropod host DNA and any endosymbiont or pathogen DNA harbored within the specimen.

Methodology/principal findings: In this study, a novel work flow based on autofluorescence microscopy was developed to enable identification of trombiculid mites to the species level on the basis of morphological traits without any special preparation, while preserving the mite DNA for subsequent genotyping. A panel of 16 specifically selected fluorescence microscopy images of mite features from available identification keys served for complete chigger morphological identification to the species level, and was paired with corresponding genotype data. We evaluated and validated this method for paired chigger morphological and genotypic ID using the mitochondrial cytochrome c oxidase subunit I gene (coi) in 113 chigger specimens representing 12 species and 7 genera (Leptotrombidium, Ascoschoengastia, Gahrliepia, Walchia, Blankaartia, Schoengastia and Schoutedenichia) from the Lao People's Democratic Republic (Lao PDR) to the species level (complete characterization), and 153 chiggers from 5 genera (Leptotrombidium, Ascoschoengastia, Helenicula, Schoengastiella and Walchia) from Thailand, Cambodia and Lao PDR to the genus level. A phylogenetic tree constructed from 77 coi gene sequences (approximately 640 bp length, n = 52 new coi sequences and n = 25 downloaded from GenBank), demonstrated clear grouping of assigned morphotypes at the genus levels, although evidence of both genetic polymorphism and morphological plasticity was found.

Conclusions/significance: With this new methodology, we provided the largest collection of characterized coi gene sequences for trombiculid mites to date, and almost doubled the number of available characterized coi gene sequences with a single study. The ability to provide paired phenotypic-genotypic data is of central importance for future characterization of mites and dissecting the molecular epidemiology of mites transmitting diseases like scrub typhus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • DNA / genetics
  • Genotype
  • Microscopy, Fluorescence / methods*
  • Optical Imaging / methods*
  • Phylogeny*
  • Trombiculidae / anatomy & histology
  • Trombiculidae / classification
  • Trombiculidae / genetics*
  • Trombiculidae / ultrastructure*

Substances

  • DNA

Grants and funding

This study was supported in part by a scholarship for doctoral degree students from the National Research Council of Thailand granted to Rawadee Kumlert for the FY2017; the LKS Strategic Award (LG34) “The Natural Immune Response in Pediatric Scrub Typhus in Chiang Rai” (DHP is PI); and a supportive research grant from the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand to Rawadee Kumlert for writing her PhD thesis. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.