A Major Locus Controls a Genital Shape Difference Involved in Reproductive Isolation Between Drosophila yakuba and Drosophila santomea

International audienceRapid evolution of genitalia shape, a widespread phenomenon in animals with internal fertilization, offers the opportunity to dissect the genetic architecture of morphological evolution linked to sexual selection and speciation. Most quantitative trait loci (QTL) mapping studies of genitalia divergence have focused on Drosophila melanogaster and its three most closely related species, D. simulans, D. mauritiana, and D. sechellia, and have suggested that the genetic basis of genitalia evolution involves many loci. We report the first genetic study of male genitalia evolution between D. yakuba and D. santomea, two species of the D. melanogaster species subgroup. We focus on male ventral branches, which harm females during interspecific copulation. Using landmark-based geometric morphometrics, we characterized shape variation in parental species, F1 hybrids, and backcross progeny and show that the main axis of shape variation within the backcross population matches the interspecific variation between parental species. For genotyping, we developed a new molecular method to perform multiplexed shotgun genotyping (MSG), which allowed us to prepare genomic DNA libraries from 365 backcross individuals in a few days using little DNA. We detected only three QTL, one of which spans 2.7 Mb and exhibits a highly significant effect on shape variation that can be linked to the harmfulness of the ventral branches. We conclude that the genetic architecture of genitalia morphology divergence may not always be as complex as suggested by previous studies

Evolution of Susceptibility to Ingested Double-Stranded RNAs in Caenorhabditis Nematodes

International audienceBACKGROUND: The nematode Caenorhabditis elegans is able to take up external double-stranded RNAs (dsRNAs) and mount an RNA interference response, leading to the inactivation of specific gene expression. The uptake of ingested dsRNAs into intestinal cells has been shown to require the SID-2 transmembrane protein in C. elegans. By contrast, C. briggsae was shown to be naturally insensitive to ingested dsRNAs, yet could be rendered sensitive by transgenesis with the C. elegans sid-2 gene. Here we aimed to elucidate the evolution of the susceptibility to external RNAi in the Caenorhabditis genus. PRINCIPAL FINDINGS: We study the sensitivity of many new species of Caenorhabditis to ingested dsRNAs matching a conserved actin gene sequence from the nematode Oscheius tipulae. We find ample variation in the Caenorhabditis genus in the ability to mount an RNAi response. We map this sensitivity onto a phylogenetic tree, and show that sensitivity or insensitivity have evolved convergently several times. We uncover several evolutionary losses in sensitivity, which may have occurred through distinct mechanisms. We could render C. remanei and C. briggsae sensitive to ingested dsRNAs by transgenesis of the Cel-sid-2 gene. We thus provide tools for RNA interference studies in these species. We also show that transgenesis by injection is possible in many Caenorhabditis species. CONCLUSIONS: The ability of animals to take up dsRNAs or to respond to them by gene inactivation is under rapid evolution in the Caenorhabditis genus. This study provides a framework and tools to use RNA interference and transgenesis in various Caenorhabditis species for further comparative and evolutionary studies

Natural and Experimental Infection of Caenorhabditis Nematodes by Novel Viruses Related to Nodaviruses

Novel viruses have been discovered in wild Caenorahbditis nematode isolates and can now be used to explore host antiviral pathways, nematode ecology, and host-pathogen co-evolution

Outline of the RNAi test.

<p>(<b>A</b>) Experimental design of the RNAi test. a) L4 larvae were transferred on day 1 onto two RNAi plates seeded with bacteria producing <i>Oti-actin</i> dsRNAs. b) On day 2 (24 hrs later), the animals were isolated onto a new RNAi plate seeded with the same bacteria. c) On day 3 (after 16 hrs), the adults were removed. d) On day 4, the progeny was scored for the number of wild-type larvae, deformed larvae and embryos (these embryos must be highly delayed, arrested or dead). Experiments were performed at 23°C. (<b>B</b>) Morphological phenotype in the actin RNAi experiments. a) Deformed L1 larva of the <i>C. elegans</i> N2 strain on HT115 bacteria expressing <i>Oti-actin</i> dsRNAs (visualized here using Nomarski microscopy but visible under a dissecting microscope). b) Control L1 larva of the <i>C. elegans</i> N2 strain grown on control HT115 bacteria expressing <i>Cbr-lin-12</i> dsRNAs.</p

Complementing <i>C. briggsae</i> and <i>C. remanei</i> with <i>Cel-sid-2</i>.

<p>(<b>A</b>) Sensitivity to ingested <i>Oti-actin</i> RNAi of two <i>C. briggsae</i> integrated transgenic lines, JU1018 and JU1076, transformed with <i>Cel-sid-2</i> genomic DNA and a <i>myo-2::DsRed2</i> marker. Both lines are rendered sensitive to external actin dsRNA application, compared to the reference strain AF16. JU1018 has a lower brood size than the other lines. Statistical comparisons were made to the results of control experiments with <i>Cel-rol-6</i> or <i>Cbr-lin-12</i> dsRNA using a Mann-Whitney-Wilcoxon rank sum test on the number of normal larval progeny of each parent. Note that this test is very conservative and has low power. The significance of the difference is depicted as follows: (<b>NS</b>) non-significant, (<b>*</b>) 0.01<<i>p</i><0.05, (<b>**</b>) 0.001<<i>p</i><0.01, (<b>***</b>) <i>p</i><0.001. Error bars indicate the standard error of the mean over individuals (n = 6–12, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029811#pone.0029811.s001" target="_blank">Table S1</a> for details). (<b>B</b>) Sensitivity to <i>Cre-unc-22</i> RNAi by feeding of a <i>C. remanei</i> transgenic line, JU1184, transformed with <i>Cel-sid-2</i> genomic DNA and a <i>myo-2::DsRed2</i> marker. The transgenic strain <i>C. remanei</i> JU1184 displayed the characteristic twitching phenotype when <i>Cre-unc-22</i> dsRNAs were administered by feeding, whereas no twitcher was seen in the reference strain <i>C. remanei</i> PB4641. χ² test: <i>p</i> = 10⁻⁷⁵.</p

Transgenesis of <i>Caenorhabditis</i> species.

<p>Transgenes that provide transgenesis markers are in bold.</p>#<p>GFP marker is not expressed. “nd”: not determined.</p

Comparison of sensitivity to ingested <i>Oti-actin</i> dsRNAs of <i>C. elegans</i> N2 to two examplary species.

<p>(<b>A</b>) <i>C.</i> sp. 18 JU1857 is insensitive to RNAi by feeding, whereas <i>C. elegans</i> N2 shows a response. <i>C.</i> sp. 18 JU1857 and <i>C. elegans</i> N2 progeny were scored using the experimental design described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029811#pone-0029811-g001" target="_blank">Figure 1</a>, using <i>Oti-actin</i> and <i>Cbr-lin-12</i> (negative control). (<b>B</b>) <i>C.</i> sp. 15 QG122 is highly sensitive to ingested <i>Oti-actin</i> dsRNAs. Here, feeding of <i>Oti-actin</i> dsRNA resulted in complete sterility of the mothers, whereas in the same experiment, treated N2 mothers produced arrested embryos and larvae. Statistical comparisons were made to the results of control experiments with <i>Cbr-lin-12</i> dsRNA, which has no effect on either strain, using a Mann-Whitney-Wilcoxon rank sum test on the number of normal larval progeny of each parent. The significance of the difference is depicted as follows: (<b>NS</b>) non-significant, (<b>*</b>) 0.01<<i>p</i><0.05, (<b>**</b>) 0.001<<i>p</i><0.01, (<b>***</b>) <i>p</i><0.001. Error bars indicate the standard error of the mean over individuals (n = 6–12, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029811#pone.0029811.s001" target="_blank">Table S1</a> for details).</p

Evolution of RNAi sensitivity in the <i>Caenorhabditis</i> genus.

<p>The RNAi results are displayed on the phylogeny from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029811#pone.0029811-Kiontke1" target="_blank">[17]</a>, for the different dsRNA administration methods and for different genes. The red stars indicate a loss of sensitivity to ingested <i>Oti-actin</i> dsRNAs in a parsimonious evolutionary scenario (see text). Note that the absence of an external <i>Oti-actin</i> RNAi response may correspond to an inability to respond either to all ingested dsRNAs (as in <i>C. briggsae</i> and likely in <i>C. drosophilae</i>), or only to specific dsRNAs (as in <i>C. elegans</i> JU1580 and <i>C. angaria</i>). In blue: results from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029811#pone.0029811-Flix2" target="_blank">[13]</a>. In green: result`s from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029811#pone.0029811-Winston2" target="_blank">[5]</a>. The dsRNAs for the <i>ama-1</i> gene were specific to the tested species. We did not find differences among different tested isolates of <i>C. briggsae</i> and <i>C.</i> sp. 7 (see Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029811#s2" target="_blank">Methods</a> for the identity of other tested strains), nor did Winston et al. for different isolates of <i>C. brenneri </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029811#pone.0029811-Winston2" target="_blank">[5]</a>. <i>C. brenneri</i> can respond to <i>ama-1</i> dsRNAs if they are injected into the gonad, indicating that this species is defective in the systemic spread of the signal from body cavity to gonad. I: <i>Elegans</i> supergroup of <i>Caenorhabditis</i> species; II: <i>Drosophilae</i> supergroup as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029811#pone.0029811-Kiontke1" target="_blank">[17]</a>.</p

RNAi sensitivity of <i>C.</i> sp. 11, <i>C. drosophilae</i> and <i>C. angaria</i>: feeding versus injection.

<p>(<b>A</b>) <i>C.</i> sp. 11 JU1373 was found to be insensitive to <i>Oti-actin</i> RNAi using the feeding protocol (a), but sensitive to <i>Oti-actin</i> dsRNAs introduced by injection (b). (<b>B</b>) <i>C. drosophilae</i> DF5077 was found to be insensitive to RNAi by feeding using both <i>Oti-actin</i> (a) and <i>Cdr-ama-1</i> (b), but sensitive to <i>Oti-actin</i> dsRNAs introduced by injection (c). <i>C. drosophilae</i> has a smaller brood size than <i>C. elegans</i> in all experiments. (<b>C</b>) <i>C. angaria</i> RGD1 was found to be insensitive to RNAi by feeding using <i>Oti-actin</i> (a) but not <i>Can-ama-1</i> (b). (c) Compared to control animals injected with <i>Cel-unc-22</i> dsRNAs, <i>C. angaria</i> RGD1 animals injected with <i>actin</i> dsRNAs showed a significantly reduced number of larvae in their progeny. Injections of <i>actin</i> dsRNAs into <i>C. angaria</i> RGD1 animals were performed in two replicate experiments (shown in blue and red). The proportion of injected P0 that became fully sterile is reported below. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029811#s3" target="_blank">Results</a> for <i>C. elegans</i> are shown as positive control for the efficiency of the dsRNA treatment.</p