-
[
International Worm Meeting,
2011]
Many Caenorhabditis species proliferate in a variety of rotting vegetal substrates: fruits, flowers, plant stems. We will report on wide sampling of such substrates and possible carrier invertebrates, focusing on 1) French Guiana; 2) mainland France.
In French Guiana, we sampled mostly in wild tropical forest around the Nouragues CNRS Biological Station. Besides finding three new species, C. sp. 12, 17 and 18 (see abstract by Kiontke et al.), we determined the spatial distribution of six Caenorhabditis species at different scales and constituted a frozen isolate collection. Caenorhabditis species often co-occur in the same individual fruit or flower. Their spatial distribution over the location at one timepoint appears inhomogeneous.
In mainland France, rotting fruit and stem habitats were found to be shared by C. elegans and C. briggsae. Both species were isolated throughout most of France. In addition, C. remanei or C. sp. 13 were found each in one location. We sampled a systematic spatio-temporally structured set of rotting apples in an apple variety orchard in Orsay. We scored the prevalence of each species in different parts of the same apple, in different apples below a given tree, throughout the orchard and at 19 timepoints over three years. C. elegans and C. briggsae were abundantly found and may co-occur in the same apples. However, their temporal distribution did not coincide. C. briggsae was found alone in summer; both species co-occurred in early fall and C. elegans remained alone in late fall. This temporal sharing of the habitat coincides with their temperature preference in the lab. In relatively natural habitats of France (wood, heath), rotting stems of several plant species yielded Caenorhabditis. Both C. elegans and C. briggsae were found in the same plant species and sometimes in the same individual stem.
Most populations were analyzed on the day of sampling, which enabled us to determine the census and the stage of worms. Population sizes spanned a range of 1 to over 10,000 Caenorhabditis individuals in one fruit, flower or stem. Some populations of intermediate size contained all non-dauer stages expected from a proliferating population. Samples with high census always contained some L2d and dauers; some did not contain L3 and L4s at all, indicative of a population entering the dauer stage at the end of a proliferative stage.
-
[
International Worm Meeting,
2005]
The Caenorhabditis vulva is formed from a row of precursor cells in the ventral epidermis: P6.p normally adopts a central vulval fate (1), P(5-7).p a lateral vulval fate (2) and P(3,4,8).p a non-vulval fate (3). This spatial pattern ( 332123 ) is organized around the uterine anchor cell (AC). The AC emits a LIN-3/EGF signal that induces the 1 fate at high doses and the 2 fate at low doses. Activation of EGF-Ras signaling in P6.p has two consequences: it induces the 1 fate in P6.p and activates a lateral Notch pathway that induces the 2 and inhibits the 1 fate in P(5,7).p. In C. elegans, Pn.p fates are irreversibly determined by the time they divide: if the AC is ablated at this time, P6.p adopts a 1 fate and P(5,7).p a 2 fate. I found that in two close relatives of C. elegans in the same conditions, P6.p adopts a 2 fate (C. briggsae) and a 3 fate (C. remanei). More precisely, by ablating the AC at successive timepoints in the L3 stage, one uncovers a temporal series of P(5-7).p fate patterns (from 333 to 212), thus revealing relative activities of different vulva signaling pathways. In C. elegans N2, the transition is rapid (Wang & Sternberg, unpubl.), suggesting that P6.p, as soon as it is induced to a 1 fate, activates the 2 fate in P(5,7).p. In C. briggsae AF16 & HK104, P(5-7).p display an intermediate 222 pattern: P6.p adopts an inner 2 fate (vulCDDC), confirmed by L4-stage expression of
Cb-egl-17::GFP and
zmp-1::GFP. The 1 fate requires a late induction of P6.p progeny. In C. remanei PB4641 & PB228, the intermediate AC ablation results in a surprising 232 pattern: P6.p adopts a 3 fate yet induces the 2 fate in P(5,7).p, as they adopt a 3 fate upon simultaneous AC+P6.p ablations. This likely corresponds to changes in the relative weights of pathways downstream of Ras, with a high threshold for 1 fate induction and a low threshold for lateral signaling. In basal Caenorhabditis species and many other nematode genera, the intermediate 222 pattern is observed. When these changes are mapped onto the Caenorhabditis phylogeny (Kiontke et al.), the early specification of the 1 fate appears between the C. spp. DF5070 and PS1010 branches, and a further dramatic evolution occurs in the C. remanei + briggsae branch. Intra-specific variations are also detected (for C. elegans, cf Milloz et al.). Key changes in vulval patterning mechanism thus occurred in Caenorhabditis evolution, which are being further investigated using reporter genes for the different pathways.
-
Eric Miska, Josephine Piffaretti, Guang Wu, David Wang, Alyson Ashe, Guoyan Zhao, Mabel Sanroman, Leonard Goldstein, Tony Belicard, Isabelle Nuez, Yanfang Jiang, Marie-Anne FELIX
[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2010]
We found natural viral infections in wild isolates of both C. elegans and C. briggsae. The infections visibly affect the intestinal cells. Infected worm lysates passed through 0.2 um filters could be used to infect uninfected worms, which could be further passaged for many generations. Two highly divergent but related RNA viruses in the family Nodaviridae, tentatively named Orsay nodavirus and Santeuil nodavirus, were detected and sequenced. The viruses were subject to processing by the RNAi machinery as evidenced by the detection of virally derived small RNAs that mapped to the entire viral genome. These data demonstrate that nodaviruses are natural parasites of nematodes in the wild. We detect intra-specific variation in sensitivity to the virus among different C. elegans or C. briggsae isolates. Further study of the interactions between these viruses and nematodes is likely to provide insight into the natural ecology of nematodes and may reveal novel innate immune mechanisms that respond to viral infection.
-
[
European Worm Meeting,
2002]
We compare vulval patterning mechanismsbetween Caenorhabditis elegans and Oscheius sp. 1 CEW1. In Caenorhabditis elegans, the pattern of Pn.p vulval precursor cell fates is set by several cellinteractions: an inductive signal from the anchor cell and a lateral signalbetween Pn.p cells. In Oscheius sp. 1 CEW1, the same fate pattern is specified by two successive anchorcell inductions, first on P(5-7).p, and then on P6.p daughters. In order to study vulval development in Oscheius, weperformed genetic screens for egg-laying mutants and found the threefollowing vulva development mutant categories. 1) We obtained numerousdov mutants, which show an abnormal number of divisionof vulval precursor cells (Dichtel etal., Genetics 2001). Similar mutants are scarce (and sterile) in C. elegans. 2) We also obtained many cov mutants (competence and/or centering of the vulva). 3) Unlike in C. elegans, we foundfew iov mutants (abnormal induction of vulval precursor cell fates), and they show different phenotypescompared to the C. elegans induction mutants. Thevulval-defective screens in C. elegans and Oscheius thus show strikingdifferences in the mutant phenotypes that were reached, which points todifferent evolutionary potentials. A corresponding greater variability inPn.p division number of the 3 cells was observed in natural populations of Oscheiusspecies (Delattre and Flix, Curr. Biol. 2001). Thus, these celldivisions are less robust against genetic variations in Oscheius. In C. elegans, vulval induction occurs through the RAS/MAPKinase pathway. In order to study the role of this pathway in Oscheius, we used U0126, an inhibitor of MEK (a downstream effector of RAS). U0126blocks the induction in C. elegans. In Oscheiussp. 1, both inductions are affected, suggesting that MEK could play a rolein both steps of vulval induction. Using anchor cell ablation experiments in dov and cov mutants that show a partial penetrance, we observed that the anchor cellinfluences the competence and the divisions of the Pn.p cells, in additionto inducing the vulval fates. We will present the epistatic relationshipsof the iov mutations with the cov/dov mutations, and the possiblerole of MEK in these different anchor cell roles in Oscheius.
-
[
European Worm Meeting,
2008]
Most C. elegans biology is studied under one standard laboratory. condition in one reference genetic background (N2). However, its ecological. and evolutionary framework matters to understand the evolutionary origin of. the mechanisms studied in the laboratory.. C. elegans mostly proliferates in rotting fruits (Barriere and Felix,. Genetics 2007). The examination of C. elegans and C. briggsae individuals. taken directly from rotting fruits samples shows that:. 1. fungi in addition to bacteria can be found in the intestine;. 2. constipation is a recurrent problem;. 3. individuals of some populations are infected by various pathogens,. some of which are unidentified and produce abnormal phenotypes,. such as intestinal cell fusion.. A library of bacterial, fungal and slime mold strains was constructed. from individual rotting fruits from which C. elegans and C. briggsae were. also isolated. The microbes are identified by sequencing a fragment of. 16S/18S rDNA. This library includes putative food and pathogens.. Caenorhabditis species were isolated from wild and domesticated. rotting fruits collected worldwide. We will report on the isolation,. culture and preliminary phylogenetic position of several putative new. Caenorhabditis species of the Elegans group (so far including C. elegans,. C. briggsae, C. remanei and C. brenneri). These new species include:. - C. sp. 5 JU727 (China), a close relative of C. briggsae with a. gonochoristic (male/female) mode of reproduction. - C. sp. 7 JU1199, gonochoristic, a putative outgroup to the four. described species (which is much easier to culture than the next outgroup,. C. japonica). - C. sp. 9 JU1325 and C. sp. 10 JU1333, two gonochoristic species. from India - C. sp. 11, a hermaphroditic species from La Reunion (an. island next to Madagascar).. I am happy to distribute strains. See
http://www2.ijm.jussieu.fr/worms for. worm strains. I thank V. Robert, M. Hermann, J. Milloz and many others for. collecting samples, I. Nuez for technical help and E. Troemel and J.. Hodgkin for characterization of two associated microbes (see abstract by. Hodgkin et al.).. Lab priority: If you wish a single talk per lab, I would put F. Duveau''s. abstract as a priority.
-
[
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.
-
[
International Worm Meeting,
2009]
Gene functions change throughout evolutionary history and understanding how this occurs is a key goal of molecular evolutionary biology. To date researchers have systematically looked at changes in gene function by comparing loss of function phenotypes in orthologs from different model organisms, such as S. cerevisiae and C. elegans. However, since there has been such a large evolutionary divergence between these organisms their body plans are so different and many phenotypes are impossible to compare. It is much more practical to compare phenotypes from organisms with similar body plans and ecological niches. Here we use Caenorhabditis nematodes to systematically look at changes in gene function through evolution because loss of function phenotypes map easily between species. In order to address this problem we are building an RNAi library for C. briggsae to look for changes in gene phenotype from those that were observed in C. elegans. Genome scale RNAi has been successfully used in C. elegans and thus we are going to use it in C. briggsae. In order to do this we take advantage of the
sid-2 transgenic C. briggsae line (gratefully contributed by Marie-Anne Felix''s group) which has been shown to uptake RNAi by feeding. We will present preliminary screening data from the C. briggsae RNAi library and showcase the changes in gene function we have found so far.
-
[
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.
-
[
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.
-
[
International Worm Meeting,
2013]
Symbiosis is the living together of unlike organisms. These interactions vary across a cost-benefit spectrum from those imposing a high cost on the host (parasitism) to those benefitting the host (mutualism). Indeed, some of these associations are critical for normal host development and survival. Yet the role of many commensal (non-host-harming) strains remains unknown. I have begun to examine Caenorhabditis elegans and C. briggsae for gut-associated microbes. As established genetic model systems, these worms have numerous molecular-genetic tools available. Worms maintained in the laboratory lose their associated microbiota due to frequent bleaching. Thus, I am working with Caenorhabditis sampled from their natural environment, leveraging historical Felix lab collections.
First, I will characterize associated gut bacteria by 16S rDNA sequencing to assess microbial diversity. Second, I will isolate strains on appropriate culture media, then re-inoculate germ-free host worms with individual bacterial strains and monitor key features of host fitness under various environmental stresses to determine their effect on survival and fecundity. Third, I will focus on those bacterial strains showing the largest differences between infection and control strains, interrogating them under a wide variety of conditions, probing specificity and details of recognition, acquisition & maintenance using molecular genetic methods.
Host-gut bacterial associations may be particularly complex, with hosts striving to maintain their bacterial populations at optimal levels. In this case, the host has the difficult task of balancing inevitable exposure to bacteria consumed intentionally or inadvertently as a food source (necessary and likely beneficial to the host), against the likelihood of ingested bacteria profiting from the intestinal environment and becoming pathogenic at high loads. This study seeks begin documenting the role of associated gut microbiota in Caenorhabditis, establishing them as a genetic model system to study symbiosis.