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 new WHO bottle bioassay method to assess the susceptibility of mosquito vectors to public health insecticides: results from a WHO-coordinated multi-centre study.

BACKGROUND: The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both. METHODS: A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration-response curves for each insecticide-species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively. RESULTS: Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species-insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were < 10% for most mosquito species-insecticide combinations. CONCLUSION: Our findings, based on the largest susceptibility dataset ever produced on mosquitoes, showed that the new WHO bottle bioassay is adequate for evaluating mosquito susceptibility to new and promising public health insecticides currently deployed for vector control. The datasets presented in this study have been used recently by the WHO to establish 17 new insecticide discriminating concentrations (DCs) for either Aedes spp. or Anopheles spp. The bottle bioassay and DCs can now be widely used to monitor baseline insecticide susceptibility of wild populations of vectors of malaria and Aedes-borne diseases worldwide

Geometric morphometrics of the scutum for differentiation of trombiculid mites within the genus Walchia (Acariformes: Prostigmata: Trombiculidae), a probable vector of scrub typhus

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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

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

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

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