The proposed haem synthesis pathway in <i>Wolbachia</i>, showing structural intermediates.

<p>Enzymes are represented by red boxes, which contain the protein name in <i>Wolbachia</i> and the abbreviated enzyme name: ALAS, 5-aminolevulinate synthase; ALAD, 5-aminolevulinate dehydratase; PBGB, porphobilinogen deaminase; UROS, uroporphyrinogen III synthase; UROD, uroporphyrinogen III decarboxylase; CPO, coproporphyrinogen III oxidase; PPO, protoporphyrinogen IX oxidase; FC, ferrochelatase. Inhibitors of the pathway are represented by blue boxes, for which abbreviations used are as in the text.</p

Selected examples of phenotypes resulting from natural <i>Wolbachia</i> symbioses.

<p><i>Wolbachia</i> produces a large spectrum of phenotypes in their hosts ranging from parasitic to mutualistic traits existing as either facultative relationships or associations that have evolved to become obligate. Reproductive parasitism by <i>Wolbachia</i> is well recognised. For example, in the ladybird <i>Adalia bipunctata</i>, infection results in death of infected males during development to the benefit of female siblings (male killing) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003224#pntd.0003224-Hurst1" target="_blank">[72]</a>; in the woodlouse <i>Armadillidium vulgare</i>, infection causes development of infected genetic males into females (feminisation) <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003224#pntd.0003224-Bouchon1" target="_blank">[73]</a>; and in the mosquito <i>Culex pipiens</i>, <i>Wolbachia</i> strain <i>w</i>Pip produces cytoplasmic incompatibility (CI), in which crosses between infected males and uninfected females result in embryonic death. <i>Wolbachia</i> symbioses may also provide benefits to the host, such as increases in fecundity and longevity in <i>Drosophila melanogaster</i><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003224#pntd.0003224-Fry1" target="_blank">[74]</a>. In some species, mutualistic traits coexist with reproductive phenotypes, such as in <i>Culex pipiens</i>, where the CI-inducing strain <i>w</i>Pip also provides protection from mortality associated with <i>Plasmodium relictum</i><a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003224#pntd.0003224-Zl1" target="_blank">[13]</a>. In some host species, all individuals are infected and this association is often mutualistic, as in the bedbug <i>Cimex lectularius</i> in which <i>Wolbachia</i> supplies essential B vitamins <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003224#pntd.0003224-Hosokawa1" target="_blank">[10]</a>, or in the filarial parasite <i>Onchocerca ochengi</i>, where the presence of the bacteria is associated with the vertebrate host mounting an ineffective immune response <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003224#pntd.0003224-Hansen1" target="_blank">[75]</a>. However, in the parasitic wasp <i>Asobara tabida</i>, strain <i>wAtab3</i> is essential for oogenesis, making the relationship obligatory without any known benefits to the host <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003224#pntd.0003224-Kremer2" target="_blank">[43]</a>.</p

Diagrammatic view of the similarity of <i>Onchocerca</i> sigma-class GST gene models for <i>O. volvulus</i> GSTs 1a and 1b and the homologous <i>O. ochengi</i> sigma-class GST t09064.

<p>Gene models were aligned over the full-length sequence (total distance, 3,870 bp). Numbers associated with gene model exons (<i>I–VII shaded blocks</i>) and introns (<i>1–8 non-shaded blocks</i>) display the number of base-pairs within those sections over which the alignment is spaced. The three major differences between the genes (all insertions in <i>O. volvulus GST1a</i> intron 3) are highlighted in the diagram.</p

Alignment of partial gene sequences of glutathione <i>S</i>-transferases (GSTs) from <i>O. volvulus</i> (<i>OvGST1a</i>, <i>OvGST1b</i>) and <i>O. ochengi (OoGST1)</i> (A) and primer sets targeting <i>OvGST1a</i> (B).

<p>Primers are indicated by solid black arrows and dash arrows represent the binding regions of the loop forward (LFP) and loop back (LBP) primers respectively.</p

A comparison of LAMP and PCR methods to detect varying amounts of genomic DNA isolated from pools of black flies using different methods.

<p>A comparison of LAMP and PCR methods to detect varying amounts of genomic DNA isolated from pools of black flies using different methods.</p

Sensitivity of LAMP and PCR methods for the detection of <i>O. volvulus</i> using ten-fold serial dilutions of <i>O. volvulus</i> genomic DNA ranging from 0.001–1.0 ng.

<p>Detection of LAMP product using turbidity (<b>A</b>) or hydroxy napthol blue (<b>B</b>). PCR amplification of a ∼200 bp product using LAMP primers F3 and B3 was obtained when <i>O. volvulus</i> genomic DNA was used (<b>C</b>). Molecular weight marker (MW) is indicated.</p

Phylogenetic neighbour-joining tree showing the relationship of the sigma-class GSTs of <i>Onchocerca ochengi</i> to similar enzymes of nematodes, mammals and insects.

<p>Numbers shown alongside branches are bootstrap values of 1,000 replications. The key for protein sequence accession numbers and organisms displayed in the tree is as follows: <u>Nematodes</u>: Oo_GST_t09064, Oo_GST_t03844 and Oo_GST_t06414 glutathione transferase [<i>Onchocerca ochengi</i>]; Ov_GST_1b AAG44696.1 glutathione <i>S</i>-transferase Ia [<i>Onchocerca volvulus</i>]; Ov _GST_1a AAG44695.1 glutathione <i>S</i>-transferase Ia [<i>Onchocerca volvulus</i>]; Ll_GST XP_003139665.1 hypothetical protein LOAG_04080 [<i>Loa loa</i>]; Bm_GST_4 XP_001901855.1 glutathione <i>S</i>-transferase 4 [<i>Brugia malayi</i>]; As_GST_1 ERG83753.1 glutathione <i>S</i>-transferase 1 [<i>Ascaris suum</i>]; As_GST_4 ERG81431.1 glutathione s-transferase 4 [<i>Ascaris suum</i>]; Ce_GST-11 NP_508625.1 protein GST-11 [<i>Caenorhabditis elegans</i>]; Ce_GST-36 NP_509652.2 protein GST-36 [<i>Caenorhabditis elegans</i>]. <u>Mammals:</u> Hs_PGD NP_055300.1 hematopoietic prostaglandin D synthase [<i>Homo sapiens</i>]; Bt_PGD_x1 XP_002688181.1 PREDICTED: hematopoietic prostaglandin D synthase isoform X1 [<i>Bos taurus</i>]; Rt_PGD NP_113832.1 hematopoietic prostaglandin D synthase [<i>Rattus norvegicus</i>]; Mm_PGD NP_062328.3 hematopoietic prostaglandin D synthase [<i>Mus musculus</i>]. <u>Insects:</u> Md_GST_ NP_001273827.1 glutathione <i>S</i>-transferase [<i>Musca domestica</i>]; Dm_GST_s1 NP_725653.1 glutathione <i>S</i>-transferase S1, isoform A [<i>Drosophila melanogaster</i>]; Ph_GST XP_002426887.1 glutathione <i>S</i>-transferase, putative [<i>Pediculus humanus corporis</i>]; Tc_GST XP_970714.1 PREDICTED: glutathione <i>S</i>-transferase [<i>Tribolium castaneum</i>]. The GSTs from <i>O. volvulus</i> and their closest relative in <i>O. ochengi</i> are shown in bold.</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

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