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Franzen da Silva, Aline, Valandro Soares, Marcell, Antunes Soares, Felix, Arantes, Leticia, Obetine, Fabiane, Lopes Machado, Marina, da Silveira, Tassia, Marafiga Cordeiro, Larissa
[
International Worm Meeting,
2021]
Huntington's disease (HD) is an autosomal dominant, progressive neurodegenerative disease. It occurs due to a mutation in the huntingtin gene with an abnormal CAG repeat, leading to a variable length N-terminal polyglutamine chain (poly-Q) which confers toxic functions to mutant Htt leading to neurodegeneration. Rutin is a flavonoid found in plants, buckwheat, some teas and also in apples. Although our previous studies have already indicated that rutin has protective effects in HD's models, more studies are needed to unravel its effects on protein homeostasis and the underlying mechanisms. In our study, we investigated the effects of chronic treatment with rutin in Caenorhabditis elegans model of HD focusing on ASH neurons and antioxidant defense. The synchronized L1 worms were placed on rutin-NGM plates and kept at 20°C. Rutin was added every 24 hours at concentrations of 15, 30, 60 and 120 muM. We assessed octanol response, neuronal polyQ aggregates and dye filling assay. In addition, we analyzed the downstream heat-shock protein-16.2 (HSP-16.2) and superoxide dismutase-3 (SOD-3). Overall, our data demonstrate that chronic rutin treatment maintains the function of ASH neurons in addition to decrease the degeneration of their sensory terminations. The mechanism proposed is antioxidant activity, through the overexpression of antioxidant enzymes and chaperones regulating proteostasis. Our findings provide new evidences about rutin playing a neuroprotective role in C elegans model. In addition to information for treatment strategies for neurodegenerative diseases and other diseases caused by age-related protein aggregation.
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[
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.
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[
Development,
2021]
Cell migration needs to be precisely regulated during development so that cells stop in the right position. A new paper in Development investigates the robustness of neuroblast migration in the <i>C. elegans</i> larva in the face of both genetic and environmental variation. To hear more about the story, we met the paper's four authors: Clement Dubois and Shivam Gupta, and their respective supervisors Andrew Mugler (currently Assistant Professor at the Department of Physics and Astronomy at the University of Pittsburgh, where his lab recently moved from Purdue University) and Marie-Anne Felix (Principal Investigator at Institut de Biologie de l'Ecole Normale Superieure in Paris and Research Director at CNRS).
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[
Parasitol Today,
1985]
The lymphatic filariases, Wuchereria bancrofti, Brugia malayi, and B. timori, infect nearly 100 million people throughout the tropics, but mainly in Africa and southeast Asia. Over 900 million people live in endemic, areas at risk to the infection. The filarial parasites reproduce slowly, whereas their mosquito vectors are quickly-reproducing opportunists. Thus, although vector control can reduce the risk of transmission, the parasite itself would seem a more vulnerable target for prolonged attack. In this article, Felix Partono discusses the clinical diagnosis of f lariasis and argues that the disease can be effectively controlled by attacking the parasites in infected communities, using diethyl-carbamazine (DEC) as the drug of choice.
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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.
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Meyer, Barbara J., Haag, Eric S., Schartner, Caitlin M., Ralston, Edward J., Korf, Ian, Thomas, Cristel G., Yin, Da, Schwarz, Erich M.
[
Evolutionary Biology of Caenorhabditis and Other Nematodes,
2014]
Sexual mode evolves rapidly in some eukaryotic lineages. This is expected to have pronounced consequences for population genetics, sexual differentiation and the nature and intensity of sexual selection, all of which may be reflected in the genome. C. elegans is a self-fertile species, derived recently from an obligately outcrossing male-female ancestor.This trait has evolved in at least two other species of the Elegans sub-genus, C. briggsae and C. sp. 11 (Kiontke et al. 2011). Previous studies indicate that selfing species have smaller genomes and several thousand fewer protein-coding genes than their outcrossing ancestors (Thomas et al. 2012). This reproducibility may be stimulated by an interaction between partial selfing and segregation distortion affecting large indels in male meiosis (Wang et al. 2010). However, the size, location, and gene content of specific deletions remain unknown for any natural system. To characterize the process of genome shrinkage, we have produced a genome assembly from the closest known outcrossing relative of C. briggsae, C. nigoni (formerly C.sp. 9; Felix et al. 2014). The C. nigoni genome is roughly 20 Mb (20%) larger than that of C. briggsae. By comparing C. nigoni contigs with the chromosome-level assembly for C. briggsae, we created an approximation of the C. nigoni physical map. Genome-wide sequence alignment showed the majority of the size reduction is located on the two arms of the five autosomes. Using C. sp.5 as an outgroup, we are able to identify gene family reductions, as well as specific genes recently lost in the C. briggsae lineage. Finally, we present detailed characterization of a family of rapidly evolving proteins that were independently lost in C. elegans and C. briggsae, the MSS (male-specific secreted) family, We have characterized their temporal and spatial expression, and find they are likely to be transferred to the female reproductive tract. We are now developing assays to reveal potential physiological responses to MSS proteins in females, including using calcium imaging to localize putative responder cells in female reproductive tract. References Felix, M.-A., C. Braendle, et al. (2014). "A streamlined system for species diagnosis in Caenorhabditis (Nematoda: Rhabditidae) with name designations for 15 distinct biological species." PLoS One 9:
e94723.Kiontke, K., M.-A. Felix, et al. (2011). "A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits." BMC Evol Biol 11: 339.Thomas, C. G., R. Li, et al. (2012). "Simplification and desexualization of gene expression in self-fertile nematodes." Curr Biol 22: 2167-2172.Wang, J., P. J. Chen, et al. (2010). "Chromosome size differences may affect meiosis and genome size." Science 329: 293.
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[
International Worm Meeting,
2009]
In C. briggsae, patterns of genetic diversity among strains from across the globe correlate perfectly with the geographic origin of the natural isolates, corresponding to clades of worms from temperate regions, the tropical circles of latitude, and near the equator (Cutter et al. 2006; Dolgin et al. 2007). Ecologically, these geographic regions differ dramatically in temperature regime, begging the question of whether heritable phenotypic differences might also conform to the geographic partitioning of variation in a potentially adaptive manner. An association between the temperature at which a particular isolate is optimally fecund and the temperature of the isolate''s clade of origin could indicate local adaptation and provide insight into C. briggsae ecology and evolution. To address this issue, we tested the thermal tolerance, as quantified by self-fecundity, of 10 wild-isolate strains originating from the three latitudinal regions when the strains were subjected to extreme high and low temperatures. Our results demonstrate a decline to zero progeny production at 32 deg C that was exhibited by worms from all three regions, indicating an upper fertile limit between 30 deg C and 32 deg C for C. briggsae as a species. However, at 30 deg C we observed a significant 4-fold difference in lifetime fecundity for strains from the Tropic circles of latitude clade compared to those of both the temperate and equatorial clades, suggesting a tolerance of the tropical isolates to higher temperatures. Ongoing work explores fecundity at low temperatures (12 deg C - 16 deg C) to test for heritable differences among strains at cooler temperatures. Cutter, A.D., M.A. Felix, A. Barriere & D. Charlesworth. 2006. Patterns of nucleotide polymorphism distinguish temperate and tropical wild isolates of Caenorhabditis briggsae. Genetics. 173: 2021-2031. Dolgin, E.S., M.A. Felix & A.D. Cutter. 2008. Hakuna nematoda: genetic and phenotypic diversity in African isolates of Caenorhabditis elegans and C. briggsae. Heredity. 100: 304-315.
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Koutsovoulos, G., Besnard, F., Vargas-Velazquez, A., Dubois, C., Felix, M-A., Blaxter, M.
[
International Worm Meeting,
2017]
Oscheius tipulae is a common free-living nematode in the same clade as the parasitic taxa Heterorhabditis and strongylids, and closer to the model species Caenorhabditis elegans than the outgroup Pristionchus pacificus. This hermaphroditic species is thus informative for comparative genetics, developmental and evolutionary studies. However, the genetic toolbox for non-model organisms such as O. tipulae is still underdeveloped. In model species with fully assembled and annotated genomes, mapping-by-sequencing has become a standard method to map and identify phenotype-causing mutations. Candidate variants are pinpointed using a cross to a divergent mapping strain and sequencing of a pool of mutant segregants (e.g. ref. 1). Chemical mutagenesis (EMS) performed about 15-20 years ago by Marie-Anne Felix's lab on the O. tipulae strain CEW1 generated several vulval development and other morphological mutants, including dumpy, roller and uncoordinated phenotypes (refs 2-6). Using the mapping-by-sequencing approach, a draft genome sequence for O. tipulae CEW1 and crosses with the divergent wild isolate JU170, we have identified for the first time relevant candidate genes for these phenotypes in O. tipulae. Our success suggests that a draft assembly with multiple scaffolds per chromosome is sufficient to perform mapping-by-sequencing. Other recent genetics tools such as CRISPR/Cas9 system have been developed and widely used on Caenorhabditis species to perform targeted mutagenesis, and we propose to confirm candidate genes by developing CRISPR/Cas9 system on O. tipulae. We argue here that the mapping-by-sequencing approach and genomic analysis tools can be easily used in non-model organisms. This brings non-model species firmly into genomics-enabled science, and provides tools to investigate the biology of non-model species and improve our understanding of evolution and developmental mechanisms. (1) Doitsidou et al. PLOS One 2011; (2) Felix et al. Nematology, 2000; (3) Dichtel et al. Genetics 2001; (4) Louvet-Vallee et al. Genetics 2003; (5) Dichtel-Danjoy et al. Dev Biol 2004; (6) Felix, Oscheius tipulae, WormBook, 2006.
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[
International Worm Meeting,
2003]
In order to study how a cell lineage varies during evolution, we use a microevolutionary approach with the nematode vulva as a model system. The vulva is formed by precursors in the ventral epithelium, called the Pn.p cells, and each Pn.p cell has a specific fate. The same vulva lineage characters that diverge between closely related species of Caenorhabditis are also polymorphic between strains (Delattre & Felix, 2001). The frequency of P3.p division varies within the Caenorhabditis genus (between species) and within C. elegans (between strains). In order to understand the processes that are responsible for variation in P3.p division frequency, our aims are to define the number of genes involved and to understand the molecular and cellular mechanisms that underlie the vulva lineage variations: 1)We performed a genetic analysis of P3.p division frequency between divergent strains of C. elegans. Recombinant inbred lines between the N2 and CB4857 strains and between the N2 and CB4856 strains (the latter set a kind gift of M. Hammarlund & E. Jorgensen) were analyzed for P3.p division frequency. Non-parental (intermediate or transgressive) levels for the lineage character were observed, showing that the P3.p division polymorphism between two strains is due to variations at several loci. 2)Using Single Nucleotide Polymorphisms in relation to the phenotype of the N2xCB4857 lines, we could not detect a major locus that could be at the origin of the difference; we could rule out candidate loci such as
lin-39.3)P3.p is competent to form vulval tissue after P(4-8).p ablation in C. elegans, but not in C. briggsae (Delattre & Felix, 2001). The frequency of P3.p division is not fully correlated with its competence. We are analyzing more strains and species in order to draw phylogenetic hypotheses on the polarity of changes in P3.p division and competence.4) The frequency of P3.p division may be regulated by the (non)-fusion of the cell in the L2 stage.Therefore, we are studying the frequency and timing of P3.p fusion using the AJM-1::GFP marker, and will compare it to the frequency of its division. The fusion process could have consequences on the observed level of P3.p competence. The understanding of the relationships between the processes of division, fusion and competence could help understand the evolution of the size of the competence group from the intraspecific to the supraspecific level.
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[
International Worm Meeting,
2007]
A wide variety of bacterial and fungal pathogens have been shown to cause lethal intestinal infections in C. elegans. However, relatively little is known about the relevant pathogens that C. elegans encounters in the wild, and no bacterial pathogens have yet been shown to invade C. elegans intestinal cells. Antoine Barriere and Marie-Anne Felix recently identified a strain of C. elegans that harbors intracellular bacteria during a sampling of French compost pits (1) and they generously sent us this strain for characterization. The intracellular bacteria in this strain are only found in the intestine, and they can be transmitted from one C. elegans animal to another by co-incubating infected and uninfected animals on the same plate. The infection can be cured in the next generation by bleaching, or by removing adults from the plate immediately after egg-laying, indicating that it is not transmitted vertically. We have found that the infection can be transmitted by taking a streak of bacteria from a plate of infected worms, transferring it to a new plate, and then adding recipient worms immediately. However, if the streak of bacteria is incubated for 24 hours or more before adding recipient worms, worms will not become infected, indicating that the bacteria lose infectivity outside of their hosts. Through 16S rDNA cloning and sequencing we have identified the infecting intracellular bacteria as a species in the Ochrobactrum genus. Ochrobactrum are Gram negative bacteria commonly found in the soil, and are generally considered harmless, as they are only rarely associated with infection in humans. However, the closest relative of Ochrobactrum is Brucella, which is an intracellular pathogen of mammals that can infect and kill humans. Brucella is considered a potential bioterrorism weapon and is a BSL3 (biosafety level 3) organism. Perhaps because of the difficulties in working with Brucella, little is known about how it infects its host, and its genome contains few classical virulence factors. Thus, the C.elegans/Ochrobactrum model may provide a safer alternative for understanding how this clade of intracellular bacteria infect and kill their hosts.
1) Barriere and Felix (05) Current Biology
Subsequent experiments have indicated that the intracellular pathogen is not the bacterium Ochrobactrum, but instead corresponds to a new species of microsporidia, which are eukaryotic pathogens.