22 research outputs found

    Phylogenetic relationships of virus-like contigs from the dog whelk.

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    <p>Mid-point rooted maximum likelihood phylogenetic trees for each of the virus-like contigs associated with viRNAs in the dog whelk (<i>Nucella lapillus</i>). New virus-like contigs described here are marked in red, sequences marked ‘TSA’ are derived from public transcriptome assemblies of the species named, and the scale is given in amino acid substitutions per site. Panels are: (A) rhabdoviruses related to lyssaviruses, inferred using the protein sequence of the nucleoprotein (the only open reading frame available from this contig, which is likely an EVE); (B) orthomyxoviruses related to influenza and thogoto viruses, inferred using the protein sequence of PB1; (C) rhabdoviruses and chuviruses, inferred from the RNA polymerase. Support values and accession identifiers are presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.s002" target="_blank">S2 Fig</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.s019" target="_blank">S3 Data</a>, and alignments in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.s018" target="_blank">S2 Data</a>. Given the high level of divergence, alignments and inferred trees should be treated as tentative.</p

    Distribution of small RNA pathways across the Metazoa.

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    <p>Phylogeny of selected metazoan (animal) phyla (topology follows [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.ref180" target="_blank">180</a>]) with a table recording the reported range of modal lengths for miRNAs, piRNAs, and viRNAs detectable by bulk sequencing from wild-type organisms (miRNA modes taken from miRbase). Entries marked ‘No’ have been reported to be absent, and those marked ‘?’ are untested. Focal taxa in this study are marked in colour, and the target table entries are outlined. Vertebrate viRNAs are marked ‘(×)’ as mammalian virus-derived small RNAs are only detectable in tissues and experimental systems lacking viral suppressors of RNAi and/or an interferon response [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.ref031" target="_blank">31</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.ref035" target="_blank">35</a>]. Note that piRNAs are absent from some, but not all, nematodes [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.ref057" target="_blank">57</a>]. The column ‘dsRNA KD’ records whether dsRNA knockdown of gene expression using long dsRNA (i.e. a Dicer substrate) has been reported, as this may suggest the presence of an RNAi pathway capable of producing viRNAs from replicating viruses. The ‘Dcrs’ and ‘Agos’ columns record the inferred number of Dicers and (non-Piwi) Argonautes ancestrally present in each phylum, although the number of Dicers in Platyhelminthes is contentious as the putative second Dicer lacks the majority of expected Dicer domains. Broadly speaking, there are two competing hypotheses for the histories of Dicers and (non-Piwi) Argonautes in animals [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.ref047" target="_blank">47</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.ref050" target="_blank">50</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.ref181" target="_blank">181</a>]. The first (labelled H<sub>1</sub>), posits that an early duplication in Dicer and/or Argonaute (marked D<sup>+</sup> and A<sup>+</sup> in dark green on the phylogeny) gave rise to at least two very divergent homologues of each gene in the lineage leading to the Metazoa, followed by subsequent losses (D<sup>-</sup> and A<sup>-</sup> in dark red). The second (H<sub>2</sub>), suggests that divergent homologues are the result of more recent duplications (D<sup>+</sup> and A<sup>+</sup> in pale green), and where homologs have high divergence it is as a result of rapid evolution. Note that these hypotheses are independent for Argonautes and Dicers, and one may be ancient but the other recent. For Dicers, at least, the ‘ancient’ duplication is arguably better supported [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.ref047" target="_blank">47</a>], although it remains extremely difficult to determine orthology between the duplicates. In addition, Dicers and Argonautes have unambiguously diversified within some phyla (important examples marked A<sup>+</sup> and D<sup>+</sup> in grey)—as seen for the large nematode-specific WAGO clade of Argonautes (reviewed in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.ref141" target="_blank">141</a>]), and the multiple Argonautes in vertebrates.</p

    Small RNAs from RNA virus-like contigs.

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    <p>Panels to the left show the distribution of 20-30nt small RNAs along the length of the virus-like contig, and panels to the right show the size distribution small RNA reads coloured by the 5' base (U red, G yellow, C blue, A green). Read counts above the x-axis represent reads mapping to the positive sense (coding) sequence and counts below the x-axis represent reads mapping to the complementary sequence. For the dog whelk (A-D), only reads from the oxidised library are shown. Other dog whelk libraries display similar distributions and the small-RNA ‘hotspot’ pattern along the contig is highly repeatable (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.s006" target="_blank">S6 Fig</a>). Small RNAs from the two segments of the orthomyxovirus (A and B) show strong strand bias to the negative strand and no 5' base composition bias. Those from the first rhabdo-like virus (C) display little strand bias and no base composition bias, and those from the second rhabdo virus-like contig, which is a probable EVE (D), derive only from the negative strand and display a very strong 5' U bias. There were insufficient reads from the positive strand of this virus to detect a ping-pong signature. Small RNAs from the four dog whelk contigs all display 28nt peaks. Small RNAs from the bunya/phlebo-like virus identified in the brown alga (E) derive from both strands, and show a strong 5' U bias with a peak size of 21nt. The data required to plot the size distributions are provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.s014" target="_blank">S5 Table</a>.</p

    Small RNAs from TE-like contigs.

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    <p>The threecolumns show (left to right): the distribution of 20-30nt small RNAs along the length of a TE-like contig; the size distribution of small RNA reads (U red, G yellow, C blue, A green); and the sequence ‘logo’ of unique sequences for the dominant sequence length. Read counts above the x-axis represent reads mapping to the positive sense (coding) sequence, and counts below the x-axis represent reads mapping to the complementary sequence. For the sequence logos, the upper and lower plots show positive and negative sense reads respectively, and the y-axis of each measures relative information content in bits. Where available, reads from the oxidised library are shown (A-F), but other libraries display similar distributions (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.s008" target="_blank">S8 Fig</a>). These examples from sponge (A), sea anemone (B), starfish (C), earthworm (D), dog whelk (E-F) and brown alga (G) were chosen to best illustrate the presence of the ‘ping pong’ signature, but other examples are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.s008" target="_blank">S8 Fig</a>. Note that the size distribution of TE-derived small RNAs varies substantially among species, and that the dog whelk (E and F) displays at least two distinct patterns, one (F) reminiscent of that seen for some RNA virus contigs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.g003" target="_blank">Fig 3C</a>). The data required to plot these figures is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007533#pgen.1007533.s014" target="_blank">S5 Table</a>.</p

    Isolation of KV and growth in flies.

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    <p>(A) Density gradient and virus titre: Kallithea virus (purple) was effectively separated from DAV (green) at 1.18 g/mL (dotted line) in fractions 15 and 16 of an iodixanol gradient. (B) Transmission electron micrograph of KV-positive fractions showed KV to be a rod-shaped enveloped particle, as has been described previously for other nudiviruses [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007050#ppat.1007050.ref055" target="_blank">55</a>]. We did not observe unenveloped KV particles, bacteria, or RNA viruses in the isolate. (C) Relative viral titres normalised by the number of fly genomic copies and virus levels at time zero in each sex. Each point represents a vial of 10 flies. Viral titres peaked at 10 days post-infection, and were generally higher in females (red) than males (blue) late in infection. (D) We were able to infect adult flies orally by applying the viral isolate to <i>Drosophila</i> medium, although relative copy number of the virus was very low and infection was inefficient, with only 2 of 16 vials (each of 10 flies) having increased titre after one week, indicating an infectious rate lower bound of ~1% at 5x10<sup>3</sup> ID50.</p

    Trait means, genetic variance (V<sub>G</sub>), total phenotypic variance (V<sub>P</sub>), heritability (H<sup>2</sup>), and coefficient of genetic variation (CV<sub>G</sub>) in titre and mortality following KV infection in the DGRP.

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    <p>Trait means, genetic variance (V<sub>G</sub>), total phenotypic variance (V<sub>P</sub>), heritability (H<sup>2</sup>), and coefficient of genetic variation (CV<sub>G</sub>) in titre and mortality following KV infection in the DGRP.</p

    KV causes male-biased mortality, increased lethargy, and decreased fecundity.

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    <p>(A) Injection of KV virus into <i>OreR</i> flies led to sex-specific mortality. Infected females (red dotted line) experienced a small but significant increase in mortality, but males (blue dotted line) experienced a significantly larger rate of mortality after day 10. Flies injected with control gradient solution were unaffected (solid lines). Each point is the mean and standard error for the proportion of flies alive in each vial (10 vials of 10 flies). (B) Although females remained alive for longer, they were more lethargic. We assessed daily movement of flies injected with either chloroform-inactivated KV (green) or active KV (purple). KV-infected flies moved less from days 3–7 post-infection. (C) Females also displayed altered egg laying behaviour. Thirty pairs of flies were injected with inactive chloroform treated KV (green) or active KV (purple). KV-infected flies laid a slightly, but not significantly, higher number of eggs during early infection (1 and 2 DPI) but laid significantly fewer eggs in late infection (7 and 8 DPI). This reduction in egg laying is due to a shutdown of oogenesis before vitellogenesis (D, E), and ovaries from KV-infected flies house a lower proportion of ovarioles that include late-stage and mature egg chambers (F) and a higher proportion which contain apoptotic nurse cells (G). Ovaries were analysed 10 DPI, and error bars (F,G) show the standard error.</p

    Confirmation of antiviral genes identified in GWAS.

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    <p>KV titre was measured in flies expressing a foldback hairpin targeting 18 genes identified in the GWAS, using GAL4 lines that knock each down in either the whole fly or specifically in the gut. (A) The data were used to estimate random effects associated with each gene knock down, plotted with 95% highest posterior density intervals. (B) Knock-down of the most confident association in the GWAS, <i>Cip4</i>, caused reduced <i>Cip4</i> RNA levels and (C) increased viral titre. (D) The associated variant (3L_4363810_SNP), was polymorphic (G/A), representing a nonsynonymous polymorphism in some splice variants, and survival following KV infection was significantly increased in fly lines with the “A” genotype, especially in females. Each point in comparison of survival in the two genotypes is a line mean. (*MCMCp < 0.05).</p

    Genome-wide association of polymorphism in the DGRP with KV-induced titre and mortality.

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    <p>Manhattan plots showing the p-value for the effect of each polymorphism on viral titre (purple) and mortality (green). The top SNPs for each phenotype are shown in expanded inset panels, including surrounding genes. For clarity “CG” is omitted from gene identifiers. Horizontal lines show significance thresholds obtained through randomisation (p<sub>rand</sub> = 0.05 in blue; p<sub>rand</sub> = 0.01 in red).</p

    Genetic variation in resistance to KV.

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    <p>(A) We measured LT50 in both sexes, and titre in females, following KV injection in the DGRP. For titre, each bar represents the mean (and standard error) titre relative to fly genome copy-number, as assessed by qPCR for 5 vials of 10 flies for each of 125 DGRP lines. For LT50, each bar represents the mean time until half the flies (in a vial of 10) were dead, for three vials per line, per sex. (B, C) We used a multi-response linear mixed model to calculate genetic correlation between the traits. Shown are the raw data (left), and the estimated line effects (right) after accounting for any injection date and qPCR plate effects, and for the estimated variance among lines. Each point is a DGRP line measured for both phenotypes. We find a strong positive correlation between male and female LT50 values (B). We also observe a weak positive correlation between titre and LT50 (C).</p
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