Richaud, Aurelien et al. (2013) International Worm Meeting "Survey of C. elegans local polymorphism in an apple orchard by RAD-sequencing."

Stefanutti, Marine, Richaud, Aurelien, Felix, Marie-Anne

[International Worm Meeting, 2013]

Despite increased sampling of Caenorhabditis species in rotting vegetal substrates in the last years, we still lack ecological and evolutionary data to understand what drives Caenorhabditis distribution and diversity at a local scale. We thus sampled during 4 years a spatio-temporal set of rotting apples in an orchard in Orsay (France). We observed that C. elegans and C. briggsae were found every year and may occur in the same apples. However their temporal distributions differ: while C. briggsae is found mainly during summer, C. elegans is found in late fall. This temporal occurrence coincides with their temperature preference in the lab (Felix & Duveau, BMC Biol. 2012). In order to study the genetic structure of C. elegans at a local scale, we extracted genomic DNA from over 600 wild C. elegans "families" (progeny of a wild-caught worm) from the Orsay orchard. Preliminary results using phenotypically relevant polymorphisms indicate that genetic diversity can be found in the orchard, including within a given apple. For example, a polymorphism in drh-1, a gene involved in the defense against the Orsay virus (see abstract by Le Pen et al.) could be detected in the orchard. 87.5 % of sampled animals display the deleted allele. Both genotypes could sometimes be found in the same apple. Polymorphisms in plg-1 and tra-3 were also found, but not (so far) at the zeel-1 peel-1 incompatibility locus. In order to study the genetic structure more systematically, we initiated a RAD-sequencing approach, a method that detects Single Nucleotide Polymorphisms in an unbiased manner by partial Illumina sequencing next to restriction enzyme cutting sites. A first assay with the SbfI enzyme revealed an insufficient level of polymorphism. Therefore, we decided to shift to the 2b-RAD sequencing approach, an easier and less expensive variant method, that makes use of type IIb restriction endonucleases like BcgI (Wang et al. Nat. Meth. 2012). This approach will allow us to study different population genetic parameters such as genetic diversity, outcrossing rate, spatial distribution, migration, etc.

Richaud, Aurelien et al. (2017) International Worm Meeting "Investigating the tropism of vertically transmitted bunya-like viruses in Caenorhabditis."

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.

Richaud, Aurelien et al. (2015) International Worm Meeting "Survey of the genetic structure of a C. elegans wild population at a local scale by 2b-RAD-sequencing."

Richaud, Aurelien, Felix, Marie-Anne

[International Worm Meeting, 2015]

Historically, studies in ecology and evolutionary biology have focused mainly on non-model organisms and have neglected the nematode Caenorhabditis elegans. Despite increased sampling of Caenorhabditis species in rotting vegetal substrates in the last years, we still lack ecological and evolutionary data to understand what drives Caenorhabditis distribution and diversity at a local scale. We thus sampled during seven years (2008-2014) a spatio-temporal set of rotting apples in an orchard in Orsay (France). We observed that C. elegans and C. briggsae were found every year and may occur in the same apples. However their temporal distributions differ: while C. briggsae is found mainly during summer, C. elegans is found essentially in late fall. This temporal occurrence coincides with their temperature preference in the lab (Felix & Duveau, BMC Biol. 2012).To study the genetic structure of C. elegans at a local scale, we extracted genomic DNA from nearly 1000 wild C. elegans "families" (progeny of a wild-caught worm) from the Orsay orchard, sampled in a spatio-temporally structured manner, as well as from two other more natural sites, where the substrates are rotting stems, snails, slugs and isopods: Santeuil (2009-2014) and Plougasnou (2009). Preliminary results using phenotypically relevant polymorphisms indicate that genetic diversity can be found in the orchard, including within a given apple. For example, a polymorphism in drh-1, a gene involved in the defense against the Orsay virus, could be detected in the orchard. 87.5 % of sampled animals display the deleted allele (niDf250). Both genotypes were found every year except in 2009 and could sometimes be found in the same apple. Polymorphisms in plg-, tra-3 and at the zeel-1 peel-1 incompatibility locus were also found.In order to study the genetic structure more systematically, we initiated a 2b-RAD sequencing high-throughput approach, a method that allows to detect Single Nucleotide Polymorphisms in an unbiased manner by partial Illumina sequencing of the genome, next to type IIb restriction endonucleases sites like BcgI (Wang et al. Nat. Meth. 2012). With a multiplexing strategy combining 8 barcodes and 24 illumina indices, we were able to sequence 192 samples at the same time. We constructed and sequenced five libraries of 192 samples with an expected coverage of 50X.The genetic and genomic analyses of the sequencing data will allow us to study different population genetic parameters such as genetic diversity, outcrossing rate, spatial distribution, migration, etc.

Richaud, Aurelien et al. (2019) International Worm Meeting "Discovery of vertically transmitted RNA molecules encoding viral RNA-dependent RNA polymerase in Caenorhabditis nematodes."

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.

Blaxter, Mark et al. (2015) International Worm Meeting "The Caenorhabditis Genomes Project."

Koutsovoulos, Georgios, Chandrasekar, Sinduja, Stevens, Lewis, Blaxter, Mark

[International Worm Meeting, 2015]

The sequencing of the genome of Caenorhabditis elegans remains one of the milestones of modern biology, and this genome sequence is the essential backdrop to a vast body of work on this key model organism. "Nothing in biology makes sense except in the light of evolution" (Dobzhansky) and thus it is clear that complete understanding of C. elegans will only be achieved when it is placed in an evolutionary context. While several additional Caenorhabditis genomes have been published or made available, a recent surge in the number of available species in culture makes the determination of the genomes of all the species in the genus a timely and rewarding project.We have initiated the Caenorhabditis Genomes Project. From material supplied by collaborators we have so far generated raw Illumina short-insert data for sixteen species. Where possible we have also generated mixed stage stranded RNASeq data for annotation. The data are being made publicly available as early as possible (warts-and-all) through a dedicated genome website at htttp://caenorhabditis.bio.ed.ac.uk, and completed genomes and annotations will be deposited in WormBase as mature assemblies emerge. We welcome additional collaborators to the CGP, whether to assemble new genomes or to delve into the evolutionary history of favourite gene sets and systems.Species sequenced thus far in Edinburgh: Caenorhabditis afra, Caenorhabditis castelli, Caenorhabditis doughertyi, Caenorhabditis guadeloupensis, Caenorhabditis macrosperma, Caenorhabditis nouraguensis, Caenorhabditis plicata, Caenorhabditis virilis, Caenorhabditis wallacei, Caenorhabditis sp. 1, Caenorhabditis sp. 5, Caenorhabditis sp. 21, Caenorhabditis sp. 26, Caenorhabditis sp. 31, Caenorhabditis sp. 32, Caenorhabditis sp. 38, Caenorhabditis sp. 39, Caenorhabditis sp. 40, Caenorhabditis sp. 43.[Samples have been supplied by Aurelien Richaud, Marie-Anne Felix, Christian Braendle, Michael Alion, Piero Lamelza].