Evolutionary cost–benefit dynamics of <i>Wolbachia</i> in natural populations (below) and laboratory lines (above) of <i>Drosophila melanogaster</i>.

<p>While beneficial <i>Wolbachia</i> phenotypes such as virus protection (VP) enhance host fitness, increased <i>Wolbachia</i> densities are costly by decreasing lifespan (LS). The ancestral <i>Wolbachia</i> variant <i>w</i>MelCS is shown in blue, the laboratory-derived pathogen <i>w</i>MelPop that has been generated by irradiation (red flash) in red, and the recent worldwide infection variant <i>w</i>Mel in green.</p

SNP frequencies in <i>w</i>Cer of <i>R. cerasi</i> and <i>de novo</i> hosts.

<p>Frequencies were calculated for (<b>A</b>) <i>coxA</i>, (<b>B</b>) <i>ftsZ</i>, and (<b>C</b>) <i>gatB</i>. Column ‘bases’ gives the total number of sequenced bases. </p><p>^a SNP-frequency per kilobase;</p><p>^b SNP-frequency per kilobase minus calculated error base line (0.28/kb) of <i>Taq</i> polymerase;</p><p>^c two-tailed <i>P</i> values from χ² calculations with Yates Correction (2×2 contingency table) for ^a;</p><p>^d two-tailed <i>P</i> values from χ² calculations with Yates Correction (2×2 contingency table) for ^b.</p><p>Abbreviations: <i>nd</i> not determined.</p

Stop codons in <i>gatB</i>, <i>coxA</i>, and <i>ftsZ</i> of <i>w</i>Cer1 and <i>w</i>Cer2 <i>Wolbachia</i>.

<p>Lane three lists the position of the mutation corresponding to the size of the amplified MLST-gene fragment. Lines F37 to F42 represent <i>R. cerasi</i> individuals from different populations sampled in Sicily, Italy.</p

Cryptic co-infection with <i>w</i>Cer1 in <i>w</i>Cer2 carrying trans-infected lines.

<p>(<b>A</b>) Switch of strain prevalence from <i>w</i>Cer2 to <i>w</i>Cer1 in RC20. Asterisk represents time point of line establishment via microinjection in 200. Generations are indicated on x-axis. (<b>B</b>) Presence of <i>w</i>Cer1 in random samples of RC lines determined via strain-specific <i>wsp</i> PCR. First RC20 sample seems to carry <i>Wolbachia</i> at extremely low density below detection limit of <i>wsp</i> PCR whereas the other one gives a bright band with <i>w</i>Cer1-specific <i>wsp</i> primer set. DNA extracted from adult <i>Rhagoletis cerasi</i> served as positive controls (<i>w</i>Cer1 and <i>w</i>Cer1+2). (<b>C</b>) Random 36-bp fragment of the general <i>wsp</i> amplicon showing diagnostic <i>w</i>Cer1/<i>w</i>Cer2 sites. (<b>D</b>) Differentiation between <i>w</i>Cer1, <i>w</i>Cer2 and <i>w</i>Cer4 <i>Wolbachia</i> using VNTR-141 PCR. Abbreviations: CC <i>C. capitata</i>, M DNA size marker, T1-3 trans-infected RC line sample.</p

Ovary screen in trans-infected RC lines.

<p>Size of ovaries was compared on basis of number of mature eggs in one ovary. Bars represent ovary size per line determined for each ovary class: white is class I with no mature eggs; light grey class II (1–2 eggs); dark grey class III (3–9 eggs); and black is class IV with 10 or more eggs. Y-axis shows percent of ovaries per class; x-axis shows RC lines plus the <i>Wolbachia</i>-unifected <i>D. simulans</i> Nouméa TC control. Significances based on two-tailed <i>P</i> values from Fisher's exact test are indicated by asterisks.</p

Variable nucleotide positions in <i>gatB</i> (A) and amino acid positions in GATB (B) of <i>w</i>Cer2.

<p><b>A</b>) Position 1 in the presented 404 bp fragment corresponds to position 981 of the full <i>gatB</i> locus of <i>w</i>Ri infecting <i>Drosophila simulans</i> Riverside (GenBank accession number CP001391). Aa position 1 in (<b>B</b>) corresponds to aa position 148 of the full GATB protein of <i>w</i>Ri (protein ID:ACN94961.1). Frequency of SNP indicates which SNPs are singletons or occur recurrently in what host system. Nonsense mutations leading to a stop codon are indicated by asterisks. Abbreviations: aa amino acid.(</p

Effect of <i>Wolbachia</i> strains on viral titers.

<p>(A–B) Relative viral titer in (A) DCV- and (B) FHV-infected flies. Relative titers are normalised by the mean titer of <i>Wolbachia</i>-free controls (uninfected). Error bars are standard errors. Symbols above the bars give the significance relative to the <i>Wolbachia</i>-free controls based on a Dunnett's test (*: <i>P</i><0.05; **: <i>P</i><0.01; ***: <i>P</i><0.001).</p

<i>Wolbachia</i> strains used in this study.

<p>The <i>Wolbachia</i> strains were traninfected into <i>D. simulans</i> STCP either by</p>a<p>by microinjection or</p>b<p>introgression. The transinfection was done.</p>c<p>during this study, or previously by</p>d<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004369#ppat.1004369-Veneti1" target="_blank">[83]</a>,</p>e<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004369#ppat.1004369-Poinsot1" target="_blank">[82]</a>or</p>f<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004369#ppat.1004369-Zabalou1" target="_blank">[23]</a>. The <i>Wolbachia</i> strains were obtained from and/or described by</p>g<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004369#ppat.1004369-Mateos1" target="_blank">[97]</a> (San Diego Drosophila Species Stock Center),</p>h<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004369#ppat.1004369-Sheeley1" target="_blank">[98]</a>,</p>i<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004369#ppat.1004369-Dyer1" target="_blank">[21]</a>,</p>j<p><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004369#ppat.1004369-Chrostek2" target="_blank">[45]</a> and</p>k<p>Wolfgang Miller (unpublished). The fly line names either refer to</p>l<p>the transinfected <i>D. simulans</i> stock or</p>m<p>the original host.</p><p><i>Wolbachia</i> strains used in this study.</p

Correlation between protection, viral titers and <i>Wolbachia</i> density.

<p>Dots indicate mean value of the traits for each <i>Wolbachia</i> strain. Error bars are standard errors. Solid lines show predicted values from linear regressions for illustrative purposes. <i>r_g</i> is the genetic correlation between traits. (A) Correlation of survival between DCV- and FHV-infected flies (negative natural log of hazard ratios). (B) Correlation between DCV and FHV titers. (C–D) Correlation between viral titer and survival following (C) DCV infection or (D) FHV infection. Viral titers were estimated as viral RNA concentrations relative to the <i>Drosophila</i> gene <i>EF1α100E</i>. (E–F) Relationship between <i>Wolbachia</i> density and survival in (E) DCV- and (F) FHV-infected flies. <i>Wolbachia</i> density was estimated as the ratio between copy numbers ofthe Wolbachia gene <i>atpD</i> and the <i>Drosophila</i> gene <i>Actin 5C</i>.</p

Phylogeny of <i>Wolbachia</i> strains and respective level of protection and within-host density.

<p>(A) The phylogeny is based on the sequence of the MLST genes <i>16S rRNA</i>, <i>aspC</i>, <i>atpD</i>, <i>ftsZ</i>, <i>sucB</i>, <i>groEL</i>, <i>coxA</i> and <i>fbpA</i>. Branch labels represent posterior support values. Nodes with less than 50% support were collapsed. The scale bar indicates time in coalescent units. (B–C) Flies were either infected with (B) DCV or (C) FHV. Survival is expressed as the negative natural log of the hazard ratio compared to <i>Wolbachia-</i>free flies, as estimated from a Cox's mixed-effect model. Error bars are standard errors. Symbols above the bars give the significance relative to the <i>Wolbachia</i>-free controls (*: <i>P</i><0.05; **: <i>P</i><0.01; ***: <i>P</i><0.001). (D) <i>Wolbachia</i> density is expressed as the ratio of <i>Wolbachia</i> genomic DNA to <i>Drosophila</i> genomic DNA, as estimated by quantitative PCR. Different letters indicate significant differences based on a Tukey's honest significance test on ln-transformed data.</p