Blanc, Caroline et al. (2021) International Worm Meeting "Meiosis modifications at the origin of asexuality in Mesorhabditis pseudogamous nematodes"

Saclier, Nathanaelle, Galtier, Nicolas, Rey, Carine, Delattre, Marie, Blanc, Caroline

[International Worm Meeting, 2021]

Sexuality is widespread and asexuality is a derived character. Despite the recognized costs associated with sex, asexuals remain rare, which constitutes one of the most intriguing paradox in evolutionary biology. To understand the adaptative value of sex, there is clearly a need for further exploration of the asexual world, and in particular of the transition to asexuality. We recently characterized the nematode genus Mesorhabditis, in which regular sexual species are found, as well as species featuring progressive loss of males and sexuality. These pseudo-sexual species are composed of 90% females and only 10% males. Males and sperm are needed for most eggs to develop by gynogenesis (the sperm is needed to activate the oocytes but its DNA is not used, i.e pseudogamy). In the few cases where the sperm DNA is incorporated, the eggs develop as males, because only the Y-bearing sperm of males are competent to fertilize (Grosmaire & al. Science 2019; Launay & al. BMC Evol Bio 2020). In this intriguing system, females are thus produced asexually whereas males are produced sexually. Mesorhabditis nematodes represent an ideal system to study the evolutionary consequences of transition to asexuality in closely related species and to explore the molecular origins of asexuality. There are many ways to lose sex. Here we asked which modifications to meiosis led to the production of diploid oocytes in asexuals. Using cytological descriptions, we found that meiosis of asexual females is similar to sexual species up to anaphase I. Chromosome pairing and crossing-over occur and bivalent forming chiasmata are present in diakinesis. Next, the first meiotic spindle forms and bivalent chromosomes initiate their segregation. However, chromosome segregation eventually stops and all univalents realign at metaphase of meiosis II. Meiosis II then proceeds normally with the segregation of sister chromatids and formation of the single polar body. Hence, meiosis in these asexuals is characterized by the maintenance of recombination and the assortment of non-sister chromatids in the diploid oocyte. In parallel, we are comparing the level of recombination in the sexual and asexual species of this genus. For that, we have sequenced the genome of a dozen strain for one sexual and one asexual species, to measure linkage desequilibrium. The theoretical consequence of such modified meiosis is genome-wide homozygosity, except maintenance of heterozygosity on chromosome centers. This will be contrasted with the actual measure of heterozygosity.

Frezal, Lise et al. (2015) International Worm Meeting "Caenorhabditis briggsae and its two natural viruses, coevolution in a 'menage a trois'."

BRESARD, Gautier, Felix, Marie-Anne, Frezal, Lise

[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..

BRESARD, Gautier et al. (2013) International Worm Meeting "Specificity of interaction between Caenorhabditis and their natural viruses."

Felix, Marie-Anne, BRESARD, Gautier

[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.

Alkan , Cigdem et al. (2017) International Worm Meeting "Interactions between Caenorhabditis nematodes and viruses infecting them naturally."

Ruaud, Albane, Alkan , Cigdem, BRESARD, Gautier, Felix, Marie-Anne

[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.

Frezal, Lise et al. (2013) International Worm Meeting "Microevolution and coexistence of Santeuil and Le Blanc viruses in Caenorhabditis briggsae. ."

Frezal, Lise, Felix, Marie-Anne

[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.

Chen, Kevin et al. (2015) International Worm Meeting "Exploring novel antiviral immunity in C. elegans."

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. .

Franz, Carl J. et al. (2013) International Worm Meeting "Tissue Tropism and Infection Characterization of Novel Viruses in Caenorhabditis Nematodes."

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.

Belicard, Tony et al. (2011) International Worm Meeting "Evolution in viral sensitivity to the Orsay virus in natural C. elegans isolates."

Belicard, Tony, Felix, Marie-Anne

[International Worm Meeting, 2011]

We isolated the first natural viruses infecting Caenorhabditis nematodes: the Orsay virus in C. elegans isolate JU1580 and the Santeuil virus in C. briggsae JU1264 (Felix & al., 2011). We more recently found a third virus in C. briggsae JU1498 (Le Blanc virus). These viruses cause disorders in intestinal cells of their host and are horizontally transmitted. Their genomes are composed of two single-stranded positive RNA segments carrying 3 ORFs. One of them, the ORF d, has no homology with any known ORF (Felix & al., 2011). We aim to identify its role during infection. We thus cloned it and are currently expressing it in a JU1580 background in order to know whether it affects the anti-viral response of the worm. In order to evaluate natural variation in sensitivity to these viruses, we scored the susceptibility of natural isolates and standard laboratory strains of C. elegans and C. briggsae. The results reveal i) a species specificity of infection by each virus and ii) intraspecific variation in sensitivity within both species for their respective viruses. First, we found a species specificity of each virus for a specific Caenorhabditis host species. Indeed, the Santeuil and Le Blanc viruses do not infect JU1580, while the Orsay virus does not infect JU1264 and JU1498 Second, we evaluated the geographic and genetic distribution of Orsay virus susceptibility in a worldwide set of 25 C. elegans isolates representing wild genetic diversity. We measured the viral load by RT-qPCR. Preliminary results suggest that only a subset of isolates from the Old world are sensitive to the virus and none of the "New World". This diversity seems to be partially linked with their ability to perform a small RNA response that acts in anti-viral defense (Felix & al., 2011; poster by Nuez & Felix). We plan to determine the genetic architecture and identify the molecular basis for this intraspecific variation in Orsay virus susceptibility. One approach is to cross closely related sensitive and resistant strains to obtain Recombinant Inbred Lines. We will test the susceptibility to the virus in these lines in order to find loci involved in the last evolutionary event causing resistance/sensitivity to the virus. By identifying these loci, we will be able to describe the last step in the "arms race" between C. elegans and its natural virus.

Caroline Bowen et al. (2006) European Worm Meeting "RNA Degradation in the Nematode Worm C.elegans"

Caroline Bowen, Alison Woollard

[European Worm Meeting, 2006]

Caroline Bowen and Alison Woollard RNA turnover is necessary to maintain basal levels of proteins, through mRNA regulation, and to remove aberrant mRNA transcripts. Here we examine processes of RNA degradation in C.elegans. This approach is based on a functional investigation of the C.elegans components of the RNA degradation pathways, identified from homology to other organisms.. The 5-3 RNA degradation pathway in C.elegans has been previously identified (Newbury and Woollard 2004). Here we investigate the 3-5 pathway in C.elegans, starting with the identification of its core and accessory components. 3-5 exoribonucleic acid decay, first identified in S.cerevisae, is brought about by a complex of exoribonucleases, helicases and associated factors, known as the exosome, (Mitchell et al 1997). The exosome was hailed as a proteosome for RNA, due to the degradation properties of the heteromeric complex.. We have used RNAi and an available mutant to show that disruption of the exosome complex, through the targeted destruction of individual components, results in worms with severe developmental problems including slowed growth rates. A full length GFP tagged exosome component, has shown the component to be largely confined to the nucleus of all cells, including the germline. Phenotypes associated with depletion of the exosome components include slow growth and developmental problems. These are attributed to effects on rRNA processing. When exosome components are silenced, there is a failure of the degradation of specific cleaved rRNA precursors, which in turn effects ribosome function and subsequently protein synthesis. We will present our findings on the nature of these rRNA processing defects, which will help to clarify the molecular basis of the phenotypes we observe.

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.