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

<div><p>Background</p><p>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.</p><p>Methodology/Principal findings</p><p>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 (<i>coi</i>) in 113 chigger specimens representing 12 species and 7 genera (<i>Leptotrombidium</i>, <i>Ascoschoengastia</i>, <i>Gahrliepia</i>, <i>Walchia</i>, <i>Blankaartia</i>, <i>Schoengastia</i> and <i>Schoutedenichia</i>) from the Lao People’s Democratic Republic (Lao PDR) to the species level (complete characterization), and 153 chiggers from 5 genera (<i>Leptotrombidium</i>, <i>Ascoschoengastia</i>, <i>Helenicula</i>, <i>Schoengastiella</i> and <i>Walchia)</i> from Thailand, Cambodia and Lao PDR to the genus level.</p><p>A phylogenetic tree constructed from 77 <i>coi</i> gene sequences (approximately 640 bp length, n = 52 new <i>coi</i> 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.</p><p>Conclusions/Significance</p><p>With this new methodology, we provided the largest collection of characterized <i>coi</i> gene sequences for trombiculid mites to date, and almost doubled the number of available characterized <i>coi</i> 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.</p></div

A RAPID IN VITRO METHOD FOR THE EVALUTION OF CANDIDATE REPELLENTS AGAINST LEPTOTROMBIDIUM CHIGGERS

Scrub typhus is an acute febrile zoonotic disease resulting from infection with the Gram-negative intracellular bacteria Orientia (formerly Rickettsia) tsutsugamushi (Hyachi) (Seong et al. 2001). The disease is endemic in much of south and central Asia, with approximately one million cases each year and more than a billion people at risk worldwide (Rosenberg 1997). Scrub typhus is transmitted by several species of larval trombiculid mites which are commonly known as chiggers (Tanskul et al. 1998). Repellents provide an effective method of protecting individuals from arthropods (Gupta and Rutledge 1994). In this study 6 essential oils were tested to evaluate their repellent activity against the chigger, Leptotrombidium imphalum Vercammen-Grandjean and Langston. A rapid and economic in vitro procedure which requires only 5 min and a small number of chiggers was used to determine the median effective doses. The results showed that clove oil was significantly more effective than others with ED50 of 53.2 µg followed by vetiver oil

A RAPID IN VITRO METHOD FOR THE EVALUATION OF CANDIDATE REPELLENTS AGAINST LEPTOTROMBIDIUM CHIGGERS

Scrub typhus is an acute febrile zoonotic disease resulting from infection with the gram-negative intracellular bacteria Orientia (formerly Rickettsia) tsutsugamushi (Hyachi) (Seong et al. 2001). The disease is endemic in much of south and central Asia, with approximately one million cases each year and more than a billion people at risk worldwide (Rosenberg 1997). Scrub typhus is transmitted by several species of larval trombiculid mites which are commonly known as chiggers (Tanskul et al. 1998). Repellents provide an effective method of protecting individuals from arthropods (Gupta and Rutledge 1994). In this study 6 essential oils were tested to evaluate their repellent activity against the chigger, Leptotrombidium imphalum Vercammen-Grandjean and Langston. A rapid and economic in vitro procedure which requires only 5 min and a small number of chiggers was used to determine the median effective doses. The results showed that clove oil was significantly more effective than others with ED50 of 53.2 µg followed by vetiver oil

A RAPID IN VITRO METHOD FOR THE EVALUTION OF CANDIDATE REPELLENTS AGAINST LEPTOTROMBIDIUM CHIGGERS

Scrub typhus is an acute febrile zoonotic disease resulting from infection with the Gram-negative intracellular bacteria Orientia (formerly Rickettsia) tsutsugamushi (Hyachi) (Seong et al. 2001). The disease is endemic in much of south and central Asia, with approximately one million cases each year and more than a billion people at risk worldwide (Rosenberg 1997). Scrub typhus is transmitted by several species of larval trombiculid mites which are commonly known as chiggers (Tanskul et al. 1998). Repellents provide an effective method of protecting individuals from arthropods (Gupta and Rutledge 1994). In this study 6 essential oils were tested to evaluate their repellent activity against the chigger, Leptotrombidium imphalum Vercammen-Grandjean and Langston. A rapid and economic in vitro procedure which requires only 5 min and a small number of chiggers was used to determine the median effective doses. The results showed that clove oil was significantly more effective than others with ED50 of 53.2 µg followed by vetiver oil

Fluorescence microscopy for trombiculid mite identification.

<p>(A) UV light imaging (no filter) with distinct yellow-orange autofluorescence of the trombiculid mite dorsal scutum. (B) Characteristics of setae or claw structures are more delineated using multilayer bright-field imaging with a FITC filter where multiple composite images are combined into one; <i>Walchia ewingi lupella</i> leg III (scale bar 35 μm). (C) Autofluorescence (AF) imaging with a FITC filter provides clear scutum images of high resolution, ideal for measurements. Note the prominently fluorescing double eyes; <i>Blankaartia acuscutellaris</i> (scale bar 35 μm). (D) Comparison of AF and bright-field (BF) images with FITC filter of the same specimen by switching light-mode; morphological scutum details and setae insertions are rendered more precisely by AF alone, while in panel (E) setae, legs and gnathosome details are sharper when AF is combined with BF illumination, example <i>Helenicula</i> sp. (scale bar 10 μm). (F) The usually difficult-to-see setae on coxa III are clearly visible using AF-BF microscopy with FITC filter (scale bar 10 μm).</p

Comparison of autofluorescence (top panels) and bright-field (bottom panels) microscopy of the chigger mite scutum.

<p>Fluorescence microscopy enables enhanced visualization of morphological outline, shape and details such as setae insertion points of the scuta. Images represent <i>Ascoschoengastia</i> sp. (A, F), <i>Walchia</i> sp. (B, G), <i>Schoengastiella</i> sp. (C, H) <i>Leptotrombidium</i> sp. (D, I), and <i>Helenicula</i> sp. (E, J).</p

Summary of rodents and mites collected from the Lao study, with subsequent morphotyping and genotyping.

<p>Summary of rodents and mites collected from the Lao study, with subsequent morphotyping and genotyping.</p

Schematic overview of images required for morphotyping (template panel).

<p>A minimum set of 16 defined images are required to retrospectively confirm and differentiate chigger mites to the species level; images; 1 Scutum shape; 2 Scutum details; 3 Scutum eye; 4 Dorsal body setae; 5 Chelicerae; 6 Galeal setae; 7 Dorsal palpi; 8–10 Legs I-III; 11 Ventral body; 12 Ventral body setae; 13 Ventral palpi; 14–16 Coxa I-III. <u><i>Note</i></u>: <i>the schematic drawing was prepared by co-author Kittipong Chaisiri</i>.</p

Mite characteristics requiring autofluorescence (AF) or bright-field (BF) based imaging.

<p>Mite characteristics requiring autofluorescence (AF) or bright-field (BF) based imaging.</p