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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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[
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,
2015]
We serendipitously discovered that many C. elegans wild isolates display a strong Mortal Germ Line (Mrt) phenotype (1) at high temperature, whereby upon chronic exposure to temperatures such as 25degC, they progressively become sterile after several generations. Here we will report on our systematic studies of this multigenerational onset of sterility in wild isolates of C. elegans. We assayed a reference panel of 97 genetically distinct isogenic C. elegans wild isolates (2) for the number of generations at 25degC after which full sterility occurred. Reproducible variation was found among these wild isolates showing that C. elegans displays natural variation in the Mrt phenotype, likely of genetic nature. The N2 strain is among the most resistant ones. In order to genetically map this natural variation in the multigenerational onset of sterility, we generated and investigated 120 recombinant inbred lines derived from parental isolate pairs with contrasted Mrt phenotypes. Previous studies (e.g. 3, 4) strongly suggest that the observed temperature-sensitive Mrt phenotype could be regulated through small RNAs and/or chromatin state modifications that are transmitted across generations. Therefore, we are investigating the evolution of small RNA contents over several generations in naturally Mrt and non-Mrt C. elegans isolates. The aim of this work is to test whether and how epigenetic inheritance systems are modulated by natural genetic variation. We are particularly intrigued by the evolutionary and ecological contexts that allow the evolution of conditional sterility defects.(1) Ahmed & Hodgkin. 2000. Nature. (2) Andersen et al. 2012 Nature Genetics (3) Ashe et al. 2012 Cell (4) Buckley et al. 2012 Nature..
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Braendle, Christian, Frezal, Lise, Demoinet, Emilie, Felix, Marie-Anne, Miska, Eric, Zhang, Gaotian
[
International Worm Meeting,
2017]
The mortal germ line phenotype was defined by Ahmed and Hodgkin as a multigenerational phenotype, whereby a selfing C. elegans line becomes sterile after several generations (1). The N2 reference strain does not display this phenotype, but Ahmed and Hodgkin could isolate mutants that are mortal. Some of these mutations are temperature-sensitive and affect small RNA and chromatin modification pathways, which may explain the multigenerational nature of the phenotype (e.g. 2,3). We discovered serendipitously that many C. elegans wild isolates display a strong mortal Germ Line (Mrt) phenotype: chronic exposure to high temperature, e.g. 25 deg C, progressively leads to sterility within several generations. Here we discuss this surprising finding. We first assayed a reference panel of 97 C. elegans wild isolates (4) for their Mrt phenotype, scoring the number of generations at 25 deg C after which full sterility occurred. Wild isolates showed strong, reproducible differences. We found no association between the severity of the Mrt phenotype and the climatic origin of C. elegans isolates. In the generations before full sterility, we observed a progressive brood size decrease, germ line differentiation defects, chromosomal aberrations at diakinesis and DNA damage. The mortal germline phenotype was however fully reversed by switching back from 25 deg C to 15 deg C before the last generation. Most surprisingly, germline immortality of some isolates was rescued by artificial infection with Nematocida microsporidia, natural intracellular C. elegans pathogens infecting intestinal cells, which suggests signaling from soma to germline. We suggest that the Mrt phenotype observed under laboratory conditions is likely not commonly displayed under natural conditions, yet provides an exciting model to test whether and how epigenetic inheritance systems are modulated by natural genetic variation. In order to pinpoint the molecular inheritance system underlying this mortal phenotype, we focused on determining the genetic basis of the quantitative variation among the C. elegans wild isolates, using both association mapping and laboratory crosses. The two approaches yielded three candidate regions, which we confirmed through introgression experiments. Thus, although the multigenerational nature of the Mrt phenotype may stem from inheritance of small RNAs and chromatin marks that are temperature-sensitive, phenotypic variation in the Mrt phenotype among C. elegans wild isolates is due to genetic variation. (1) Ahmed & Hodgkin. 2000. Nature; (2) Ashe et al. 2012 Cell; (3) Buckley et al. 2012 Nature; (4) Andersen et al. 2012 Nature Genetics.
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Wang, David, Felix, Marie-Anne, Frezal, Lise, Richaud, Aurelien, Chen, Kevin, Jiang, Hongbing
[
International Worm Meeting,
2017]
Caenorhabditis elegans has recently became a model organism to study viral infection and antiviral immunity. So far, three Caenorhabditis natural viruses related to nodaviruses have been discovered, all infecting intestinal cells and transmitted horizontally: the Orsay virus infects C. elegans and the Le Blanc and Santeuil viruses infect C. briggsae (Felix and al., 2011). By a systematic sequencing approach, we discovered three related viral-like negative-sense RNA sequences in a C. brenneri strain from Colombia (JU1396) and two C. remanei strains from Japan and France (QG551 and JU2557). Using a BLAST approach against the Genbank protein reference sequences, we found that these reads mapped to the RNA-dependent RNA polymerase (RdRp) of bunyaviruses. We confirmed by RT-PCR the presence of these bunya-like fragments in the three strains, including after three rounds of bleaching, proving that they are vertically transmitted. By single-molecule FISH, we studied the host tropism of these bunya-like viruses and found that the RdRp RNA fragment is present in the germ line of male and female adults and also in some somatic tissues such as the pharynx. In the C. brenneri strain, the positive and negative strands appear to colocalize around the germ line nuclei. We screened for the presence of these fragments in other C. brenneri and C. remanei wild strains but did not find any other positive strains, even those sampled closed to the infected ones. We tested whether these strains were deficient in RNAi, which could explain the replication and persistence of virus-like RNA fragments. Indeed, we found by injecting
unc-22 dsRNA in the gonad of JU1396 and QG551 that they were unable to mount a RNAi response. Additionally, we are sequencing the small-RNAs from these two strains to see whether we can detect siRNA targeting the viral fragments and determine a specific small RNA size distribution and base composition pattern. Interestingly, we did not find so far any structural genes corresponding to bunyaviruses (or any other viruses) in these strains, but only that coding a RdRp gene, thus we still do not know whether viral particles may be active in these Caenorhabditis strains. To address this question, we plan to perform transmission electron microscopy on JU1396 and QG551 to see whether we are able to detect viral particles. We will also clone the fragment coding for one of the RdRp and inject it into a C. elegans strain insensitive to RNAi to test the hypothesis that this virus could be vertically transmitted through the germ line cytoplasm even lacking both glycoprotein and nucleoprotein genes.
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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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Felix, Marie-Anne, Frezal, Lise, Saber, Sayran, Baer, Charles, Saglio, Marie, Zhang, Gaotian, Noble, Luke
[
International Worm Meeting,
2021]
While heredity mostly relies on DNA sequence, other molecular and cellular features are heritable across generations. This non-DNA based memory could be of importance for adaptation of organisms to varying environments. Here we test whether and how non-genetic inheritance systems are modulated by natural genetic variation using two experimental paradigms: the mortal germline phenotype and memory of RNA interference. The mortal germline (Mrt) phenotype is a progressive onset of sterility over multiple generations. Temperature-sensitive Mrt mutations are known to affect small RNA pathways, histone methylation and thereby multigenerational inheritance. We previously showed that some C. elegans natural isolates display a temperature-sensitive Mrt phenotype. Using recombinants between two isolates, we had identified a globally rare causal polymorphism in the
set-24 gene. To identify polymorphisms explaining a greater part of natural variation in the Mrt phenotype, we performed a genome-wide association study using 95 isolates. A strong association on chromosome III was found, and replicated across two labs. Introgressions of chromosome III from the Mrt isolate JU775 in non-Mrt genetic backgrounds confirmed the association. These results show that a genetic variant underlying the multigenerational phenotype is found at intermediate frequency at the species scale. The sterility phenotype is likely suppressed in nature, by variation in temperature or potentially other interactions. We indeed find that naturally associated bacteria or infection by microsporidia strongly suppress the Mrt phenotype. Thus, the Mrt phenotype is likely revealed by unnaturally homogenous standard laboratory conditions, thereby providing an experimental handle on natural variation in transgenerational inheritance pathways. Importantly, we also reveal a positive, condition-dependent effect of an intestinal "pathogen", affecting the C. elegans germline. In addition to the Mrt phenotype, we turned to directly assay the memory of RNA interference, using a
pie-1::GFP silencing paradigm. C. elegans isolates also differ greatly in the number of generations of silencing memory of an RNAi trigger provided only in the first generation; some isolates do not show any memory. Moreover, genetic variation on chromosome III also underlies the short RNAi memory of JU775 compared to N2. Overall, we show that multigenerational memory is widely modulated by natural genetic variation in C. elegans.
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Frezal, Lise, Kaur, Taniya, Wang, David, Tahan, Stephen, Richaud, Aurelien, Felix, Marie-Anne, Zhao, Guoyan
[
International Worm Meeting,
2019]
Caenorhabditis elegans has recently become a model organism to study viral infection and antiviral immunity. So far, three Caenorhabditis natural viruses related to nodaviruses have been discovered, all infecting intestinal cells and transmitted horizontally: the Orsay virus infects C. elegans and the Le Blanc and Santeuil viruses infect C. briggsae (Felix and al., 2011). By a systematic sequencing approach, we discovered in three gonochoristic Caenorhabditis species (C. remanei, C. brenneri and C. zanzibari) multiple RNAs fragments coding for putative RNA-dependent RNA polymerases sharing similarity to different RNA viruses including bunyaviruses, narnaviruses and sobemoviruses. The presence of these RNAs has been confirmed by RT-PCR and their persistence in progeny after bleach treatment indicates a vertical transmission. By single molecule FISH we detected several of these RNAs in the cytoplasm of the male and female germline of their host, but also in some somatic tissues such as the pharynx. We tested whether these strains were deficient in exogenous RNAi, which could explain the replication and persistence of virus-like RNAs. Indeed, we found by injecting dsRNA targeting endogenous genes into the gonad of two of these strains (JU1396 and QG551) that they were unable to mount a RNAi response. Specific patterns of small RNAs complementary to the different viral-like RNAs were observed, suggesting that the different RNAs have a different biology within their host. Interestingly, we did not find so far any structural genes coding for capsid or accessory proteins in these strains, but only that coding a RdRP. While vertical transmission of viruses in the family of Narnaviridae, which are known as capsidless viruses, has been described in fungi, we hypothesize that these RNA molecules propagate in the germline as nematode capsidless viruses.
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[
International Worm Meeting,
2019]
Germline proliferation and maintenance are essential processes in development and are required to ensure the proper transmission of genetic and epigenetic information to the offspring. In the C. elegans germline, Argonaute-mediated small RNA pathways direct genome surveillance to ensure proper inheritance and maintain fertility. Two of these are the piRNA pathway, which guides the repression of parasitic genomic elements, and the germline nuclear RNAi pathway, which mediates environmentally-induced transgenerational gene silencing. Defects in these pathways lead to sterility, with mutant individuals exhibiting a mortal germline (Mrt) phenotype, in which a progressive decline in fertility accumulates across generations, ultimately resulting in complete sterility. This phenotype is enhanced by environmental stress conditions such as high temperatures. Interestingly, the heat-dependent Mrt phenotype typical of small RNA pathway mutants can be found in some C. elegans wild isolates. Recent work (Frezal, et al., 2018) has identified the
set-24 gene as the major causal locus of the strong Mrt phenotype observed in specific wild strains. Set-24 encodes a germline-specific protein with an N-terminal SET domain. Set-24 mutant animals reach sterility in less than 20 generations when chronically exposed to restrictive (25 deg C) temperatures. Preliminary results indicate that SET-24 is dispensable for piRNA activity and global H3K9me3 deposition, but it is required for the transgenerational maintenance of silencing via the nuclear RNAi pathway, as inferred from the progressive re-expression of a germline-specific GFP transgene after GFP RNAi. Ongoing work is aimed at elucidating the contribution of SET-24 to the nuclear RNAi pathway by identifying SET-24 interactors, antagonists and targets. This will broaden our understanding of how epigenetic memory is established and transmitted at the molecular level.
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[
International Worm Meeting,
2021]
C. elegans is a popular model organism that has proved very useful for studying the cell biology of intracellular infections. However, its use as a model for the study of host-virus interactions has been limited by the fact that only one natural viral pathogen of C. elegans has been identified to date (Felix and Wang, 2019; Franz et al., 2014). The goal of this project is to identify novel natural nematode viruses capable of infecting C. elegans by mobilizing ordinary citizens to collect wild nematodes. Studying the interactions of different types of viruses with their host's cells can provide new insights into cell biology and host-pathogen interactions. To date, only four viruses naturally infecting Caenorhabditis nematodes have been identified, and of those only one (Orsay virus) infects C. elegans (Felix et al., 2011; Frezal et al., 2019). In the past, identification of intracellular pathogens in wild-caught nematodes has relied on detection by microscopy of morphological changes caused by the infection (Felix et al., 2011; Troemel et al., 2008). This approach is relatively low throughput and requires an expert screener. Our approach instead uses a fluorescent reporter-based method, taking advantage of a set of genes which are expressed at low levels in basal conditions but highly upregulated during infection by intracellular pathogens (Bakowski et al., 2014; Reddy et al., 2017, 2019). Co-culturing infected nematodes together with C. elegans expressing these intracellular infection reporters produces fluorescence which is easily detected on a fluorescence dissecting microscope. By using this method on a large sampling of wild-caught nematodes, we hope to identify novel nematode viruses which can be transmitted to C. elegans. In the pilot phase of this project, we established protocols for wild nematode collection which require minimal supplies and can be performed at home by people with no particular science background after viewing a series of short training videos. We have successfully cultured wild nematodes from these samples in the lab, and have established systems for sample intake, expansion and frozen stocking of the strains, performing co-culture experiments, and sharing experimental results with the original collectors. In the fall of 2021, we hope to expand this project by partnering with educators at a variety of levels on a larger scale who would be interested in incorporating nematode hunting into their science curriculum.