Date Published: March 1, 2018
Publisher: Public Library of Science
Author(s): Rawadee Kumlert, Kittipong Chaisiri, Tippawan Anantatat, Alexandr A. Stekolnikov, Serge Morand, Anchana Prasartvit, Benjamin L. Makepeace, Sungsit Sungvornyothin, Daniel H. Paris, Sebastian D. Fugmann.
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.
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.
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.
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 Asia-Pacific 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). Unfortunately, detailed classification schemes are highly limited for the soil-dwelling and free-living nymphs and adults [6, 7].
This study has demonstrated that a combination of AF and BF microscopy imaging can provide sufficient resolution and information to characterize trombiculid mites to the species level, while preserving the mite genomic DNA for subsequent genotyping of the same individual chigger.