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

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

Overview of experimental design.

<p>Schematic showing how samples were processed for 16S rRNA amplicon sequencing.</p

Box and whisker plot showing DNA yield obtained by each pretreatment lysis method.

<p>Boxes extend from the lower quartiles to the upper quartiles with median values indicated by the line within each box. Whiskers represent maximum and minimum values, excluding any outliers (values indicated by circles which lie outside 1.5 times the interquartile range). Significant differences between methods are starred (* P <0.05; ** P ≤0.01; *** P ≤0.001).</p

Heat map showing most abundant operational taxonomic units (OTUs) with sample extracts arranged by hierarchical clustering.

<p>All OTUs that were present at 1% or higher in at least one sample are shown. Extracts are named according to the sample of origin followed by the pretreatment lysis method used and are arranged by Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering on the Bray-Curtis dissimilarity matrix. The coloured bar indicates which extracts have clustered most closely with all other extracts from the same sample (green) and those that have not (red). Reads have been assigned to OTUs based on 97% sequence similarity of the V3–V4 region. Note that in some cases this has resulted in multiple OTUs with the same taxonomic species identifier, which is most likely due to a high degree of intraspecies variability in this region of the gene, or incorrect base calling. <i>Lactobacillus</i> species that could not be identified to species level at the 97% cut-off have been assigned to genus subgroups: <i>L</i>. <i>gasseri</i> group (including <i>L</i>. <i>gasseri</i> and <i>L</i>. <i>johnsonii</i>), <i>L</i>. <i>acidophilus</i> group (including <i>L</i>. <i>acidophilus</i>, <i>L</i>. <i>helveticus</i>, <i>L</i>. <i>gallinarum</i>, <i>L</i>. <i>crispatus</i>, <i>L</i>. <i>jensenii</i> and <i>L</i>. <i>delbruekii</i>), <i>L</i>. <i>vaginalis</i> group (including <i>L</i>. <i>vaginalis</i> and <i>L</i>. <i>reuteri</i>) and <i>L</i>. <i>coleohominis</i> group (including <i>L</i>. <i>coleohominis</i> and <i>L</i>. <i>pontis</i>).</p

Principal coordinate analysis ordination of a Bray-Curtis dissimilarity matrix.

<p>Extracts are coloured by sample of origin. Extracts cluster closely with other extracts originating from the same sample and there is no observable effect of pretreatment lysis method. Extracts from samples that are dominated by <i>Lactobacillus iners</i> with variable proportions of <i>Gardnerella</i> have clustered on the left, extracts from samples that are dominated by <i>L</i>. <i>acidophilus</i> group have clustered on the bottom right and extracts from high diversity samples that contained a mixture of strict and facultative anaerobes cluster towards the top.</p

Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified bacterial gene fragments derived from field-collected individual female <i>Lu. longipalpis.</i>

<p>Lanes L1 to L10 corresponds to DGGE profiles of <i>Lu. longipalpis</i> collected from Lapinha cave-Minas Gerais; A1 to A5 correspond to DGGE profiles of <i>Lu. longipalpis</i> collected from Alagoas state/Brazil. Lanes labelled S correspond to standard DGGE markers prepared from a selection of bacterial 16S rRNA gene products to enable gel to gel comparison.</p

Denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified bacterial gene fragments derived from field-collected individual female <i>Lu. longipalpis.</i>

<p>Lanes A6 to A12 corresponds to DGGE profiles of <i>Lu. longipalpis</i> collected from Alagoas state/Brazil; C1 to C8 correspond to DGGE profiles of <i>Lu. cruzi</i> collected from Corumbá-Mato Grosso-Brazil. Lanes labelled S corresponds to DGGE markers prepared from a selection of bacterial 16S rRNA gene products to enable gel-to-gel comparison.</p

Abundance of bacterial phylotypes recovered from 16S rRNA gene clone libraries constructed from adult <i>Lutzomyia</i> sand flies.

<p>Between 26 and 28 clones were randomly selected each of the 4 pools of samples. The PCR products from GC clamped primers <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042531#pone.0042531-Muyzer1" target="_blank">[44]</a> were run on DGGE and clones were selected for sequencing on the basis of the gel profile. Gel profiles were grouped according to identical band distribution.</p

Bacterial phylotypes associated with <i>Lutzomyia</i> sand flies sampled from 4 field sites.

<p>Insects collected in field locations were suspended in 70% ethanol. DNA extracts from pooled samples of insects were subjected to PCR using primers specific for a 900 bp region of the 16S rRNA gene. The PCR products were cloned and colonies randomly selected. Purified plasmids from the clones were subjected to specific PCR, the products separated with DGGE and representative clones were selected on the basis of the band pattern of their DGGE profiles.</p