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[
International Worm Meeting,
2013]
Eukaryotic genome sizes range over 10,000-fold with a correlation between larger genomes and greater organismal size and complexity. In part, population genetic principles can explain this observation. Smaller organisms typically have larger population sizes which are more efficient at purging weakly deleterious mutations such as transposons and other insertions resulting in smaller genome sizes; larger organisms are the opposite. In contrast to the population genetic predictions, the reverse is observed for the species with known genome sizes within the Elegans group of the Caenorhabditis genus. Gonochoristic (male-female) species have larger genomes than hermaphroditic species despite the former predicted to have larger populations sizes than the latter. Why is this? Interestingly, Mendel's law of random chromosome assortment is violated in C. elegans males that are heterozygous for autosomal chromosomes of differing sizes whereby sons inherit the longer chromosome while the hermaphrodite daughters inherit the shorter chromosome, in a phenomenon which we call skew. Because a single hermaphrodite can start a new population, skew could explain how genomes of hermaphroditic species evolve to be smaller than the ancestral gonochoristic species. For this to be true, skew would be predicted to be a general property of the Caenorhabditis species. To this end, we are testing for the presence of skew in other Caenorhabditis species. We are also interested in understanding the mechanisms underlying skew and have initiated a forward genetic screen for genes that suppress skew.
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Yang, Fang-Jung, Le, Son Tho, Wang, John, Hsu, Jung-Chen, Lo, Yun-Hua, Chang, Tiffany
[
International Worm Meeting,
2015]
Most sexual species produce nearly equal sex ratios in their offspring. In nematodes, deviations from equal sex ratios occur in the self-fertile hermaphroditic species, which have evolved independently several times. Hermaphrodites produce predominantly hermaphrodite offspring, while sons are rarely observed, consistent with the high theoretical cost of male offspring in these species. In the genus Caenorhabditis, the three known hermaphroditic species also produce occasional males. When crossing occurs in C. elegans, the female-to-male offspring ratio is ~1:1, like most sexual species. In contrast, a previous study demonstrated that C. briggsae has a female-biased sex ratio (~2:1) possibly caused by competition between male X- versus O-bearing sperm.The female-biased sex ratios in C. briggsae may have evolved because of the probable advantages for fathers who produce more daughters, since a solitary hermaphrodite, but not son, can found a population. Alternatively, other conditions, such as local mate competition, may select for female-biased sex ratios. In the first model, only hermaphroditic species should have female-biased sex ratios, while in the second, female/males species could have female-biased sex ratios. To test these two possibilities, we conducted crossing experiments in the third hermaphroditic species, C. tropicalis, as well as 12 female/male species. We found that C. tropicalis produced an offspring sex ratio of ~1:1. For the female/male species, five exhibited ~1:1 ratios while seven others produced female-biased sex ratios. These results suggest that factors other than (or in addition to) hermaphroditism have selected for the evolution of female-biased sex ratios in this clade.
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[
International Worm Meeting,
2017]
Multiple pathways exist to repair DNA double strand breaks (DSBs), and most of the components of these pathways are conserved across species. Yet, which DSB repair pathway acts to repair a DSB depends on the cellular and genomic context and the type of break, and is highly regulated. While Homologous Recombination (HR) and polymerase Theta-Mediated End Joining (TMEJ) are the major DSB repair routes in the germline, the classical Non-Homologous End Joining (cNHEJ) pathway is the main DSB repair pathway in somatic tissues. To identify and characterise genetic factors involved in NHEJ and its regulation, we performed an unbiased forward genetic screen in nematodes carrying a transgenic NHEJ reporter. We isolated seven bona fide NHEJ mutants, three of these contained mutations in the well-known cNHEJ factors
cku-70 and
cku-80. The other four mutants carried mutations in genes of the THO ribonucleoprotein complex (
thoc-2,
thoc-5 and
thoc-7) and in
pnn-1. Both the THO complex and PNN play a role in RNA processing and are conserved in humans. We found that deficiency of PNN and the THO complex also leads to sensitivity to ionizing radiation in somatic tissues, but not in the germline, which is similar to the response of animals defective in
cku-70 and
cku-80, and points towards a role for the THO complex and PNN in cNHEJ. Transcriptome analysis by RNA sequencing identified a subset of transcripts to be differentially expressed and/or spliced in THO mutants but cNHEJ factors. To identify the mechanism by which the THO complex influences cNHEJ efficiency, a suppressor screen was performed in a
thoc-5 deficient background. We found that mutated
smg-1 rescues the NHEJ defect in THO complex mutants, but surprisingly this was independent of SMG-1's well-established role in nonsense-mediated decay. These findings classify the phosphatidylinositol 3-kinase-related kinase SMG-1 as a suppressor of NHEJ. To address the hypothesis that SMG-1 is hyperactive in THO complex deficient backgrounds we are currently performing phosphoproteomic experiments. Interestingly,
smg-1 mutants are hypersensitive to ionizing radiation-induced DSBs in the germline, similar to HR and TMEJ mutants. This hypersensitivity could suggest that SMG-1 stimulates repair of germline DSBs via HR or TMEJ. We postulate that SMG-1 regulates the repair of DNA double strand breaks by inhibiting NHEJ and promoting other DSB repair pathways.
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[
International Worm Meeting,
2015]
We study the natural coevolution between Caenorhabditis briggsae and its two recently described RNA viruses called Santeuil and Le Blanc (1, 2). The main advantage of this system is to combine the access to wild host and virus populations with powerful molecular tools and experimental evolution designs. We characterized the incidence of the two C. briggsae viruses in France and found that they are found in sympatry. By monitoring the viral RNAs in wild-caught C. briggsae isolates using Fluorescent In Situ Hybridization, we demonstrated that the Le Blanc and Santeuil viruses could coexist in one host population, one animal and one intestinal cell. Molecular variation of the wild-caught viruses was assessed by sequencing their two RNA molecules. While both viruses' diversities are geographically structured, we detected balancing selection on the RNA-dependent RNA polymerase (RdRp) locus in one local Santeuil population. Despite the frequent incidence of coinfection in the wild, we found no evidence for genetic exchange (recombination or RNA reassortment) between the Santeuil and Le Blanc viruses. However, we found clear evidence for RNA reassortment between different Santeuil virus variants. Finally, we investigated natural variation in C. briggsae resistance to each virus. We tested a set of wild isolates -representative of C. briggsae worldwide diversity- for their sensitivity to the Santeuil and Le Blanc viruses. While temperate C. briggsae genotypes are generally susceptible to both viruses, the tested tropical C. briggsae genotypes are resistant to both viruses. Most interestingly, two Japanese C. briggsae genotypes show specific resistance to the Le Blanc virus. To understand the genetic basis of the general and virus-specific resistances of C. briggsae, we carried out a QTL-mapping approach using recombinant inbred lines between AF16 and HK104 (3) and identified a main QTL region on chromosome IV responsible for the variation in resistance to Santeuil virus infection.(1) Felix, Ashe, Piffaretti et al. 2011 PloS Biology. (2) Franz et al. 2012 Journal of Virology. (3) Ross et al. 2011 PLoS Genetics..
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[
International Worm Meeting,
2017]
The discovery of RNA viruses that naturally infect C. elegans and C. briggsae serves as an ideal model system to study antiviral immunity and host-pathogen co-evolution. The Orsay virus only infects C. elegans whereas Santeuil and Le Blanc viruses only infect C. briggsae. Intraspecifically, within both species we found a wide variation in viral sensitivity, as well as a positive correlation among wild isolates in sensitivity to both viruses in C. briggsae. An exception to this correlation is the C. briggsae strain HK104, which is specifically resistant to Le Blanc virus but sensitive to Santeuil virus. Taking advantage of this natural variation in the host, we use a genetic approach from the host side and use Recombinant Inbred Lines (RILs) to first map the recombinant genomic regions participating to the resistance/sensitivity in a general and/or specific manner. The RILs were phenotyped for the sensitivity to the relevant viruses using Fluorescent In Situ Hybridization (FISH). The genotype (SNP markers from pool sequencing) and phenotype (resistance/sensitivity from FISH) data were used to perform QTL analysis. Several Near Isogenic Lines (NILs) were created by introgressing the candidate regions. C. briggsae AF16 is resistant to both Santeuil and Le Blanc viruses while C. briggsae HK104 is specifically sensitive to the Santeuil virus. Using AF16xHK104 Advanced Intercrossed RILs (AIRILs) (Ross et al. 2011), two QTLs were detected on chromosomes III and IV for Santeuil virus sensitivity. The NILs in the AF16 background confirm both candidate regions. C. briggsae JU1498 is sensitive to both Santeuil and Le Blanc viruses. Using JU1498xHK104 RILs, a QTL on chromosome II was detected and is being introgressed. Once candidate polymorphisms associated with the virus sensitivity/resistance are identified, we will test them by RNAi knockdown, transformation rescue and/or CRISPR-mediated gene replacement.
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[
International Worm Meeting,
2013]
Species involved in host-pathogen relationships exert selective pressures on each other. This co-evolution situation results in an arms race between host and pathogen, which may lead to specialisation of their interactions.
We recently found three related horizontally-transmitted RNA viruses that naturally infect C. elegans or C. briggsae, called Orsay, Santeuil and Le Blanc viruses (Felix et al. 2011, Franz et al. 2012). Here we study their specificity for C. elegans vs. C. briggsae, and at the intraspecific level in C. briggsae.
We first used viral filtrates to infect a set of C. elegans and C. briggsae isolates, and measured by RT-PCR the virus ability to replicate. We find that the Orsay virus can infect C. elegans but not C. briggsae, whereas Santeuil and Le Blanc viruses infect C. briggsae, but not C. elegans. Thus, each virus shows specificity toward one of these two Caenorhabditis species.
Given that C. briggsae can be infected by two viruses, we then measured viral replication after infection of C. briggsae isolates by either Santeuil or Le Blanc viruses, using RT-qPCR. We observed 1) wide variation among C. briggsae isolates; 2) correlation between the sensitivities to each virus; 3) an exception to the correlation. Schematically, C. briggsae isolates can be separated into two groups: sensitive isolates, in which the viruses replicate efficiently; and resistant ones, in which the viruses either disappear or are barely maintained. Strikingly, all sensitive strains belong to the temperate C. briggsae clade, raising the possibility that sensitivity is derived within this clade. The exception to the correlation in sensitivity is HK104, a temperate-clade isolate from Japan. HK104 is sensitive to the Santeuil virus, but resistant to Le Blanc. This result opens the possibility to study specificity of host-pathogen interactions through genetic analysis.
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[
International Worm Meeting,
2013]
We recently found three viruses, Orsay, Santeuil and Le Blanc, which naturally infect Caenorhabditis nematodes (1,2). These ss(+)RNA viruses cause intestinal cell symptoms and are horizontally transmitted. Whereas C. elegans can so far only be infected by the Orsay virus, European C. briggsae genotypes are susceptible to both Santeuil and Le Blanc viruses, and both viruses have been found in the same locations. This vulnerability of C. briggsae to two viruses enables studies of in vivo viral competition and of the mechanisms driving their short-term evolution, as well as the impact of their competition on worm fitness.
RNA viruses may evolve rapidly through both high mutation rates and recombination events. The impact of recombination widely varies from one viral species to another but in all cases, for recombination to occur, different virus types have to infect the same host cell. The first step is thus to assess whether different virus species can co-infect the same worm population, the same animal and the same cell.
By using quantitative RT-PCR, we demonstrate that the Le Blanc and Santeuil viruses can coexist in a worm population, even when originally introduced at widely different concentrations. The two viruses are jointly maintained over 10 worm generations. We presently investigate the co-infection at the whole organism and single cell levels by tracking the viral RNAs in co-infected worms using Fluorescent In Situ Hybridization.
1- Felix, Ashe, Piffaretti et al. 2011 PloS biology.
2- Franz et al. 2012 Journal of virology.
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Wang, David, Jiang, Hongbing, Wu, Guang, Franz, Carl, Renshaw, Hilary, Chen, Kevin
[
International Worm Meeting,
2015]
Model organisms have played a critical role in our understanding of innate immunity. The recent discovery of Orsay virus, the 1st virus capable of infecting C. elegans, and the discoveries of Santeuil and Le Blanc viruses which infect C. briggsae, provide a unique opportunity to define virus host interactions in these model hosts. In order to identify candidate antiviral genes, we have performed a time course transcriptional profiling with RNA-seq. In C. elegans, we identified 151 genes that were differentially expressed upon Orsay virus infection. In this set, only 36 have annotation; 22 genes contain domains involved in ubiquitin-mediated proteolysis. By further defining the transcriptional response of the orthologous genes in C. briggsae to Santeuil and Le Blanc virus infection, we identified 39 conserved genes induced in both hosts by the three viruses. Strikingly, 17 of the 39 conserved response genes are paralogs of a single gene family that is exemplified by C17H1.3. This gene family has a human ortholog, but no known function has been associated to these orthologous genes. The conserved induction of these genes in response to infection by multiple viruses strongly suggests they may play a role in antiviral defense. Efforts to define such function by targeted gene deletion and overexpression are underway. .
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Franz, Carl J., Frezal, Lise, Jiang, Yanfang, Wang, David, Felix, Marie-Anne, Renshaw, Hilary
[
International Worm Meeting,
2013]
Orsay, Santeuil and Le Blanc viruses were recently discovered, enabling for the first time the study of virus-host interactions using a natural pathogen in the well-established model organism Caenorhabditis elegans and its relative Caenorhabditis briggsae. All three viruses share less than 50% amino acid identity and are most closely related to nodaviruses, which are positive sense RNA viruses with bipartite genomes. Comparison of their complete genomes demonstrated unique coding and noncoding features absent in known nodaviruses. Le Blanc virus, similar to Santeuil virus, was capable of infecting wild C. briggsae isolates but not the AF16 C. briggsae laboratory reference strain nor any tested C. elegans strains. We characterized the tissue tropism of infection in Caenorhabditis nematodes by all three viruses. Using immunofluorescence assays targeting viral proteins, as well as in situ hybridization, we demonstrated that viral proteins and RNAs localized primarily to intestinal cells in larval stage Caenorhabditis nematodes. The viral proteins could be detected in one to six of the 20 intestinal cells present in Caenorhabditis nematodes. In Orsay virus-infected C. elegans, viral proteins could be detected as early as six hours post infection. Furthermore, the RNA-dependent RNA polymerase and capsid proteins of Orsay virus exhibited different subcellular localization patterns from each other. Collectively, these observations broaden our understanding of viral infection in Caenorhabditis nematodes.
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[
International C. elegans Meeting,
2001]
Serotonin (5HT) is a neurotransmitter which often functions as a modulator of tissue excitability and behavioral states. In C. elegans , one behavioral effect it produces is the stimulation of egg-laying. 1 One mechanism by which it appears to do this is by shifting the worm's vulval muscles from a quiescent or inactive mode to a more active one during which eggs are laid in a cluster. Through a combination of genetic and pharmacologic approaches, we have identified mutants in several genes to be 5HT-resistant for this behavior:
egl-19 (L-type voltage gated Ca 2+ channel alpha-1 subunit),
tpa-1 (PKC homolog),
acy-1 (adenylate cyclase), &
gpa-14 (novel G-protein). 2,3 While implicated in 5HT-signalling, these molecules may also constitute effectors from other neurotransmitter systems known to interact with 5HT in egg-laying, including neuropeptide signalling: Neuropeptides derived from the
flp-1 gene, for example, are known to potentiate 5HT-response. 4,5 To gain more insight into these issues, we have been utilizing Cameleon, a genetically encodable and ratiometric Ca 2+ -sensor 6,7 in both intact & cut-worm preparations to study how vulval muscle physiology changes in the presence or absence of 5HT & and how it responds to agents like forskolin or FLP peptides in N2s and in various mutant backgrounds. Preliminary results indicate that, in the absence of 5HT, vulval muscles in N2 worms typically show sporadic but often clustered Ca 2+ transients. Upon application of exogenous 5HT (5 mg/ml), this pattern gives way to a more rhythmic train of small transient events (~0.5 Hz). This data, along with initial mutant characterization, will be presented at the June meeting. 1 Horvitz HR et al , Science 216 :1012-4 2 Waggoner LE et al , Neuron 21 :203-214 3 Shyn S & Schafer W, 2000 WCWM abstract 228 4 Schinkmann K & Li C, J Comp Neurol 316 :251-260 5 Waggoner LE et al , Genetics 154 :1181-1192 6 Miyawaki A et al , Nature 388 :882-887 7 Kerr R et al , Neuron 26 :583-594