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

Overview of PCR-based and DNA sequencing results.

*<p>positivity criteria in analogy to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002163#pntd.0002163-Bustin1" target="_blank">[19]</a>. GenBank accession numbers:</p>1<p>BankIt1587796 Seq2 KC283067.</p>2<p>BankIt1587796 Seq3 KC283068.</p>3<p>BankIt1587796 Seq1 KC283066.</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

Reports of apparent mixed infections in Asia that included rickettsioses.

<p>Reports of apparent mixed infections in Asia that included rickettsioses.</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

Phylogenetic tree of all currently available <i>coi</i> gene sequences of morphotyped trombiculid mites (n = 52 new; n = 25 from GenBank).

<p>This study provided 52 new <i>coi</i> gene sequences (marked by *, approx. 640 bp length) from Lao PDR (n = 47), Thailand (n = 4), Cambodia (n = 1), and included all available <i>coi</i> sequences from NCBI (n = 25). The phylogenetic tree constructed from these <i>coi</i> gene sequences demonstrated distinct grouping of assigned morphotypes at the genus levels. Although evidence of both genetic and morphological plasticity was found and sample sizes for each species were small, there was preliminary evidence of sub-structuring of chigger populations below the species level. Different branch colors indicate morphological classification of Trombiculidae [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193163#pone.0193163.ref025" target="_blank">25</a>]; blue = <i>Walchia</i>, purple = <i>Neotrombicula</i>, green = <i>Ascoschoengastia</i>, yellow = <i>Schoutedenichia</i>, orange = <i>Schoengastia</i>, pink = <i>Blankaartia</i>, red = <i>Leptotrombidium</i>. Black branch represents sequences of house dust mites (out group). This indicated that DNA extracted from specimens used for autofluorescence analysis was of sufficient quality for downstream PCR amplification.</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

Polygala stipulacea

<p>Dot plots demonstrates ICAM-1 levels in inoculated mouse sera. (Mock inoculation; n = 2 per time point, <i>O. tsutsugamushi</i> inoculation; n = 6 per time point). Bars indicate median and error bars represent interquartile range. The asterisks indicate significant differences (<i>P</i><0.05).</p

Serum levels of circulating soluble VCAM-1.

<p>Dot plots demonstratesVCAM-1 levels in inoculated mouse sera. (Mock inoculation; n = 2 per time point, <i>O. tsutsugamushi</i> inoculation; n = 6 per time point). Bars indicate median and error bars represent interquartile range. The asterisks indicate significant differences (<i>P</i><0.05).</p