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

Benjamin L Makepeace,Sebastian D Fugmann,Kittipong Chaisiri,Rawadee Kumlert,Serge Morand,Anchana Prasartvit,Sungsit Sungvornyothin,Alexandr A Stekolnikov,Daniel H Paris,Tippawan Anantatat

doi:10.1371/journal.pone.0193163

Abstract

BackgroundConventional 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 findingsIn 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/SignificanceWith 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.

Highlights

The larval stage of trombiculid mites is commonly termed “chiggers” [1, 2]
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
Aiming to improve mite characterization without any special preparation, we devised a novel approach based on a combination of fluorescence and brightfield microscopy, taking advantage of the autofluorescent properties of the exoskeletons of arthropods. We examined if these autofluorescent properties could be exploited for mite identification following the morphological criteria of previously defined keys for trombiculid mites

Summary

Introduction

The larval stage of trombiculid mites (family Trombiculidae, subclass Acari) is commonly termed “chiggers” [1, 2]. These larvae are small (approximately 0.2–0.3 mm long) and are recognized vectors of Orientia tsutsugamushi, the causative agent of scrub typhus in the AsiaPacific region. Their bites can result in intense irritation and dermatitis, known as trombidiosis or trombiculiasis in humans and animals, but the frequency and occurrence of this has not been well characterized [3–5]. Taxonomic classification of trombiculid mites is based on exact morphological and morphometric criteria of external characteristics, which have been developed predominantly for the larval stages (usually found during feeding on rodents or other small, terrestrial vertebrates). Conventional gold standard characterization of chigger mites involves chemical preparation procedures (i.e. specimen clearing) for visualization of morphological features, which contributes to destruction of the arthropod host DNA and any endosymbiont or pathogen DNA harbored within the specimen

Methods

Results

Conclusion