Additional file 1 of Molecular insights versus morphological traits: rethinking identification of the closely related Angiostrongylus cantonensis and Angiostrongylus malaysiensis

Additional file 1: Figure S1. Illustration of the ITS2 PCR–RFLP band patterns for A. cantonensis, A. malaysiensis, and their hybrid for

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

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

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

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

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

Morand_dryad

ID: Identification number of rodent individuals species: species name of rodents sex: F (Female), M (Male), unknown (missing information) age: adult, sub adult, young (based on size and development of genital organs) accuracy: accuracy of trapping point using GPS, 1 = inferior to 10 m, 2 = inferior to 100 m habitat: main habitat of the trapping= lowland, upland (non flooded lands), forest, settlement habitat2: description of habita site: locality in Thailand, Laos and Cambodian longitude: GPS coordinates latitude: GPS coordinates elevation: in meter slope: in % Distance to forest: in meter Distance to agriculture steep: in meter Distance to agriculture flat: in meter Distance to built-up: in meter Distance to water: in meter SHDI: Shannon’s Diversity Index Patch density: density of patches of the different land cover types Edge density: density of the contours of all patches Total_area: in square meter Water: in % of total area Agriculture steep: in % of total area Agriculture flat: in % of total area Agricultre irrigated: in % of total area Built-up areasForest: in % of total are