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[
International C. elegans Meeting,
1999]
In C. elegans , the 3 fates of the Pn.p vulva precursor cells are specified around the same time by a graded induction by the anchor cell and lateral signaling among the precursors. This mechanism is not conserved in other nematodes. Considerable variations are found within the Rhabditidae (family including C. elegans ), Panagrolaimidae and Cephalobidae. One other vulva patterning mechanism relies on 2 successive nested inductions by the anchor cell: the first induction specifies vulval vs. non-vulval fates, the second specifies inner vs. outer vulval fates in the Pn.p daughters. We have started a genetic analysis in such a species, Oscheius/Dolichorhabditis CEW1. One advantage of this species is its simple vulva lineage. We have isolated about 60 vulva mutants in an F2 Egl screen. We found several Hyperinduced mutants affecting the first induction only, in which P4.p and P8.p adopt an outer vulval fate (2deg), but no Multivulva mutants with an alternation of 1deg and 2deg fates. Many mutants affect the number of vulval cell divisions, irrespective of fate induction. Some have an additional third round of division for P5.p and P7.p; some have a fourth round in the P6.p lineage (unknown phenotype in C. elegans ). In other mutants conversely, P(4-8).p, or P4.p and P8.p only, divide too few times. Laser ablation studies indicate that the number of divisions of P(5-7).p depends on a gonadal signal, but can be uncoupled from fate specification. Some of these mutants also have fate induction defects, but we did not find Vulvaless mutants defective in fate induction only. Other frequent phenotypic categories are: i. P(4-8).p, the competence group, behave like non-competent Pn.ps (
lin-39 or
bar-1 phenotype in C. elegans ). ii. The competence group is enlarged to P3.p. iii. The vulva is miscentered on P7.p. This spectrum of vulva mutations is very different from that of C. elegans . It reflects the 2-step mechanism of vulval induction, and also suggests that the relation between vulval cell cycle and fate differs from that of C. elegans . We are currently analysing the role of the Ras pathway in Oscheius (see abstract by Delattre and Felix).
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[
International Worm Meeting,
2019]
The QR neuroblast migrates anteriorly in the first stage larva of Caenorhabditis elegans, while undergoing three rounds of division. The two daughter cells of QR.pa, QR.paa and QR.pap (henceafter called QR.pax), stop their migration in an anterior body position and acquire a neuronal fate. Mentink et al. (Dev Cell 2014) found that QR.pax migration stops upon expression of the Wnt receptor MIG-1, which surprisingly is not induced by positional clues but by a position-independent timing mechanism. Given this temporal regulation, we wondered 1) how precise QR.pax positioning was when confronted to stochastic noise, environmental variation and body size variation and 2) whether and how QR.pax final position varies among C. elegans wild isolates. To assess the robustness of QR.pax position to stochastic noise, we compared the variance in its position to that of other neurons migrating during embryogenesis. The variance of QR.pax final position is similar to the variance of ALM, CAN and HSN positions, while the BDU neuron, which only undergoes a short-range embryonic migration, displays a smaller variance. We further manipulated the environment. At higher temperatures, the mean position of QR.pax is posteriorly shifted. Sustained starvation after hatching increases its variance. However, the position of the QR.pax cell bodies in the observed range of migration does not impair axon formation. Given the temporal mechanism of
mig-1 regulation, we then manipulated embryo and L1 size using mutants and tetraploid animals. As expected from the temporal mechanism, smaller mutants display more anterior QR.pax cells, while longer mutants and tetraploids display posteriorly shifted QR.pax cells. We then measured QR.pax final position in a set of C. elegans wild isolates. We could detect highly significant variation among isolates, albeit in a relative tight window. We wondered whether this variation could be accounted for in part by variation in embryo size. Indeed, we found a significant correlation between embryo size and QR.pax position in wild isolates, which explains part of the natural variation in QR.pax final position. Thus, we demonstrated that 1) the mechanism of QR.pax positioning makes it sensitive to variation in body size; 2) natural variation in positioning of these neurons can be accounted for in part by variation in body size.
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[
International Worm Meeting,
2019]
C. elegans is associated in nature with a species-rich, distinct microbiota, which was characterized only recently [1]. Our understanding of C. elegans microbiota function is thus still in its infancy. Here, we identify natural C. elegans microbiota isolates of the Pseudomonas fluorescens subgroup that increase C. elegans resistance to pathogen infection. We show that different Pseudomonas isolates provide paramount protection from infection with the natural C. elegans pathogen Bacillus thuringiensis through distinct mechanisms [2] . The P. lurida isolates MYb11 and MYb12 (members of the P. fluorescens subgroup) protect C. elegans against B. thuringiensis infection by directly inhibiting growth of the pathogen both in vitro and in vivo. Using genomic and biochemical approaches, we demonstrate that MYb11 and MYb12 produce massetolide E, a cyclic lipopeptide biosurfactant of the viscosin group, which is active against pathogenic B. thuringiensis. In contrast to MYb11 and MYb12, P. fluorescens MYb115-mediated protection involves increased resistance without inhibition of pathogen growth and most likely depends on indirect, host-mediated mechanisms. We are currently investigating the molecular basis of P. fluorescens MYb115-mediated protection using a multi-omics approach to identify C. elegans candidate genes involved in microbiota-mediated protection. Moreover, we are further exploring the antagonistic interactions between C. elegans microbiota and pathogens. This work provides new insight into the functional significance of the C. elegans natural microbiota and expands our knowledge of immune-protective mechanisms. 1. Zhang, F., Berg, M., Dierking, K., Felix, M.A., Shapira, M., Samuel, B.S., and Schulenburg, H. (2017). Caenorhabditis elegans as a model for microbiome research. Front. Microbiol. 8:485. 2. Kissoyan, K.A.B., Drechsler, M., Stange, E.-L., Zimmermann, J., Kaleta, C., Bode, H.B., and Dierking, K. (2019). Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group. Curr. Biol. 29.
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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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[
International Worm Meeting,
2019]
From ecological sampling, we find that wild Caenorhabditis nematodes are commonly associated with a diverse array of microbes, including bacteria, viruses, fungi, and microsporidia. In Bangalore, India, a wild C. briggsae strain (JU3205) was found with an unknown microbe adhering to the intestinal epithelial cells in the lumen of the gut. Phenotypically, this microbe appears to grow perpendicular along the internal sides of the intestinal lumen, giving it a bristle-like appearance. This microbe appears to be pathogenic, as the infected wild worm strain grows slower and has intestinal cells that are severely reduced in size. We see a near 100% penetrance with this microbe in the wild C. briggsae strain, and the microbe can easily be transferred to the wild-type N2 C. elegans through growth on the same plate. In order to identify this intestinal-adhering microbe, we extracted a section of the C. briggsae intestine and verified that the adhering bacteria were still present and intact. Then, we conducted PCR using universal 16S bacterial primers and identified a new species of bacteria in the Enterobacteriaceae family, equally close in sequence to the Enterobacter and Escherichia genera. To verify this identification, we created a unique fluorescence in situ hybridization (FISH) probe to this Enterobacteriaceae species and found it bound to bacilli in the lumen of the intestine, while a control wild C. elegans strain with a different adhering bacteria showed no signal. Furthermore, we used a series of cleaning protocols to remove other contaminating microbes from strain JU3205 and verified via FISH that the Enterobacteriaceae species is still present while other bacteria species appeared to be absent (using a universal bacteria FISH probe). Altogether, we have discovered and identified a new species of Enterobacteriaceae bacteria that can bind to the apical side of intestinal epithelia cells in C. briggsae and C. elegans. Our future plan with this bacteria is to discover host factors necessary for bacterial binding to intestinal cells. As proof of principle for a forward genetic screen, we used an intestinal GFP C. elegans strain to validate an easy visual phenotype for bacterial adherence to the intestine. Given the pathogenic phenotypes in the animals and their near 100% penetrance, we believe it can be established as a model system to study natural host/bacteria interactions in the intestine.
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[
International Worm Meeting,
2017]
Partnerships between animals and microbes are ancient and coevolved relationships are common and born out of mutual benefit. Proper establishment and maintenance of these relationships relies on strong lines of host-microbial communication. Our goal is to develop a comprehensive understanding of the host-microbial signaling pathways that regulate these processes. To do this, we employ the genetically tractable, high-throughput amenable and microbially 'tuned' nematode C. elegans as it harbors a simple community of microbes in the wild (its 'microbiome). Through collaborative meta-analyses, we defined its core microbiome that is largely distinct from that of corresponding substrates- Enterobacteriaceae, Pseudomonadaceae, Xanthomonadaceae, Sphingomonadaceae, three Bacteroidetes families and several less numerous families. To comprehensively examine host selection of its microbiome, we defined a community of 68 bacterial isolates with >98% identify to meta-analyses from our collection [>550 microbes] and fed this model microbiome to wild C. elegans strains (42) for comparison. Using a high-throughput gut colonization method that we developed, we observed host strain specific colonization levels over a 30 fold range with N2 at an extreme. As in the wild, sequencing of animals over time demonstrated strong host role in selecting its microbiome distinct from the lawns. This process appears to be deterministic, as specific wild C. elegans strains are highly adapted to manage levels their native microbes- i.e., C. elegans JU1218 (an apple worm) is resistant to the deleterious effects of its native Bacteroidetes (Chryseobacterium sp. JUb44) and maintained lower levels of colonization. To dissect the genetics of these adaptations, we generated and screened an RNAi sub-library of 364 genes (based on functional SNPs between JU1218 and N2) for clones that improved the overall health and/or reduced colonization levels of the lab strain of C. elegans (N2) when challenged with the Chryseobacterium. The majority of the RNAi clones further enhanced JU1218 resistance, suggesting partial loss of functions in the wild strain, but also identified 26 promising candidates in signaling pathways (e.g., a GPCR and several transcription factors) influence Chryseobacterium impact on N2. Thus, through these and additional studies, we hope to leverage C. elegans as a robust natural genetic system to catalogue the pathways that are important managing microbiome form and function.
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[
International Worm Meeting,
2011]
Small Public Liberal Arts Colleges pose multiple challenges, particularly in teaching laboratory courses. Resources are limited and the majority of students have no lab experience. In addition, faculty struggle to maintain a full-time active research program due to heavy teaching loads. A significant percentage of students expressing an interest in the sciences also express a desire for graduate or professional studies. As many of these programs desire "hands-on" scientific experience, providing research opportunities could be vital to their future success. To address these issues, a full semester research-based project was developed around isolating and identifying wild nematode isolates. An overview of nematode phylogeny and population genetics studies is coupled with students working in small groups to develop a research project, isolate nematodes and characterize the isolates using morphological and molecular analysis. Results are presented in a format similar to graduate lab meetings. The molecular techniques utilized during the pilot project included species-specific PCR (
glp-1) (1) as well as sequencing of the 18S ribosomal RNA gene (2). I adapted universal rice primers (URP) as a fingerprint assay (3). Morphological analysis included buccal (lips, buccal tube, pharynx) and tail (shape, papillae and spicule structure) morphology as well as mating system (gonochoristic vs. hermaphroditic). Strains from the CGC have been used for comparison (both morphological and molecular) to wild isolates. Mating crosses with C.elegans and C.briggsae males have been performed with hermaphroditic species. Males from gonochoristic strains can be employed for gonochoristic isolates. Hermaphroditic isolates were obtained from leaf litter in wooded settings, open grasslands and under a rotting pumpkin. Species-specific PCR as well as mating crosses reject identification as C.elegans or C.briggsae. Sequencing of the 18S RNA gene is ongoing. Gonorchorisitc isolates were obtained from both household and grass cutting compost. Morphological analysis based on tail morphology suggests the genus Rhabditis, subgenus Rhabditella and Cephalopoides. Student feedback was uniformly positive. Isolates were easily obtained and the self-directed nature of the projects deepened their understanding of lab methods and research design. The range of analysis methods allows the course to be adapted to resource availability, therefore providing a method of research experience while enhancing knowledge of the model species. (1) Barriere, A., and Felix M.A. (2005). Curr. Biol. 15,1176-184. (2) Floyd, R. et al. (2002). Mol Ecol. 11, 839-850. (3) Kang, H. W. et al. (2002). Mol. Cells. 2, 281-287.
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Koneru, S., Barkoulas, M., Essmann, C., Felix, M.A, Osman, G.A, Fasseas, M.K.
[
International Worm Meeting,
2017]
In its natural habitat, C. elegans encounters a plethora of other organisms, including some that are pathogenic. Over the last years, C. elegans has been widely used as a model to study mechanisms of innate immunity to various pathogens including bacterial and fungal species. Here we present a new class of natural eukaryotic pathogens of C. elegans, the oomycetes, which are morphologically similar, yet evolutionary distinct to fungi. Oomycetes are known to cause disease in both plants and animals, including humans, but until now they have been relatively understudied. We have sampled and established culture conditions for two oomycete species belonging to the Myzocytiopsis and Haptoglossa genera, which are both obligate pathogens of C. elegans. We present here the life cycle and infection strategies of these new pathogens that allow them to colonise and eventually kill the nematode host. We have performed RNAseq experiments to address pathogen-induced changes of host gene expression. We show that the transcriptional response to oomycete infection consists of upregulation of already known antimicrobial peptides and other putative immunity genes, as well as members of a previously uncharacterised family of chitinase-like (chil) genes. Using reporter constructs and smFISH, we demonstrate the chil genes are induced at the epidermis. We also demonstrate that chil induction is an oomycete-specific response and cannot be mounted against other pathogens or upon environmental stresses. Intriguingly, we present evidence that infection is not required for chil gene induction since a non-infectious pathogen extract is also capable of triggering the same transcriptional response. Using functional genetics and atomic force microscopy, we demonstrate that the chil genes play a role to antagonise the oomycete infection by modifying the biochemical properties of the cuticle. Our work introduces some new natural pathogens of C. elegans and paves the way for the discovery of the molecular components of oomycete recognition and transcellular signalling involved in the innate immunity response.
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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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[
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.