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[
International Worm Meeting,
2019]
Caenorhabditis elegans is one of the preeminent model organisms in modern biology, but only recently have we started to understand the details of its natural ecology and evolutionary history. Studying C. elegans alongside its closest relatives provides an important evolutionary context for the origins and constraints that have resulted in the particular instance analyzed in the laboratory. A focussed search for new Caenorhabditis species over the last decade has led to the discovery of over 50 species of Caenorhabditis. As part of an international collaboration, the Caenorhabditis Genomes Project (CGP), we have sequenced the genomes of all Caenorhabditis species currently in culture. We exploit these new genome sequences to perform the most comprehensive reconstruction of the Caenorhabditis phylogeny to date, providing an essential evolutionary framework for downstream analyses. We reveal extensive variation in genome size in the genus, ranging from the 48 Mb genome of C. drosophilae to the 160 Mb genome of C. vivipara. Investigating the origins of this variation, we find that protein-coding gene number is highly correlated with genome size, with C. drosophilae possessing just 13,000 genes due to extensive gene loss. Other genomic features, such as repeat (mobile element) proliferation also contribute to genome size changes. Among the non-coding portions of these genomes, we identified rapid and extensive intron loss across the genus, including in the group that includes C. elegans. We demonstrate the utility of these genome sequences for C. elegans research by analyzing the evolutionary history of key signaling pathways, revealing unexpected complexity. We have made these genomic resources, including genus-wide orthology sets, publicly available via the CGP website
(http://www.caenorhabditis.org) which includes a dedicated genome browser and a BLAST server. These new species and their associated genomic resources add to the arsenal of tools available to the C. elegans researcher to interrogate biology of this important nematode. The CGP is a collaboration between the following labs: Blaxter (Edinburgh), Felix (Paris), Ailion (Washington), Andersen (Northwestern), Braendle (Nice), Cutter (Toronto), Fitch (NYU), Fierst (Alabama), Kikuchi (Miyazaki), Rockman (NYU), Schwarz (Cornell), Wang (Academia Sinica).
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[
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].
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[
West Coast Worm Meeting,
2004]
The levamisole activated acetylcholine receptor in C.elegans is now known to constitute four major subunits- LEV-1, UNC-29, UNC-38 and UNC-63. Mutants for these genes are strongly resistant to the anthelminthic, levamisole. However, regulators of receptor subunit function have not all been fully characterised. Mutations in
lev-9 have been shown to confer partial resistance to both levamisole and nicotine, but do not affect levamisole binding in vitro (Lewis et al ., 1987). Thus, it has been suggested that
lev-9 is a possible modulator of levamisole receptor function. We have cloned
lev-9 and shown that it encodes a molecule homologous to the transmembrane glycoprotein, alpha sarcoglycan. In humans, the sarcoglycans are a major component of the dystrophin glycoprotein complex (DGC) and are associated with autosomal recessive forms of limb girdle muscular dystrophy. LEV-9 is mainly expressed in the muscles of the head, pharynx, body and vulva. Using fluorescently labeled a -bungarotoxin, we have shown that
lev-9 exhibits decreased surface receptor expression compared to wild type. Preliminary results using in vivo calcium imaging from vulval muscles in dissected worm preparations also suggest a difference between
lev-9 and wild type in the rate of calcium influx, in response to nicotine. Further experiments using fluorescent labeling to determine how LEV-9 affects the levamisole receptor surface expression and electron microscopic comparison of
lev-9 and wild types are in progress. P> P> Lewis JA, et al . (1987) J. Neurosci . 7(10), 3059-71
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Price, Jon, Willis, Alexandra, Stevens, Lewis, Miska, Eric, Fisher, Kinsey, Reinke, Aaron, Burton, Nick, Braukmann, Fabian, Baugh, L. Ryan
[
International Worm Meeting,
2021]
Despite reports of parental exposure to stress promoting adaptations in progeny in diverse organisms, there remains considerable debate over the ecological significance and evolutionary conservation of these multigenerational effects. Here, we investigate four independent examples of intergenerational adaptations to stress in C. elegans - bacterial infection, microsporidia infection, osmotic stress and starvation - across four different Caenorhabditis species. We found that all four intergenerational adaptations to stress are conserved in at least one other species, that the responses and evolutionary conservation patterns are stress specific, and that intergenerational adaptive effects have deleterious trade-offs in mismatched environments. By profiling the intergenerational and transgenerational effects of different stresses on gene expression across species, we identified 3,174 genes that exhibited intergenerational changes in expression in multiple species in response to stress. Furthermore we found that an inversion in the expression of certain stress response genes required for intergenerational adaptations, from increased expression in the offspring of stressed parents to decreased expression in the offspring of stressed parents, correlates with an inversion of an adaptive response to infection in C. elegans and C. kamaaina to a deleterious intergenerational effect in C. briggsae. By contrast, we did not observe any conserved transgenerational changes in gene expression in response to stress, suggesting that the intergenerational effects of stress on offspring gene expression are not maintained transgenerationally. Our results demonstrate that intergenerational responses to stress play a substantial, evolutionarily conserved, and largely reversible role in regulating animal physiology.
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[
International Worm Meeting,
2011]
Multiple neurological disorders are caused by defects in acetylcholine (ACh) signaling. However, molecular mechanisms that regulate and maintain proper post-synaptic ACh signaling are not fully understood. At the C. elegans neuromuscular junction, the appropriate balance of ACh and GABA signaling is required for coordinated muscle contraction and movement. Wild-type animals exposed to levamisole, a pharmacological agonist of levamisole-sensitive ACh receptors on the body-wall muscles, undergo time-dependent, hyper-contracted paralysis. Resistance and hypersensitivity to levamisole-induced paralysis can be used to identify genes that regulate GABA or ACh signaling. In a prior forward genetic screen, mutations in 13 different genes conferring resistance to 1 mM levamisole were identified (Lewis et al., 1980). However, mutants with weak resistance or hypersensitivity, as well as sterile and lethal mutants, were not isolated. To identify additional factors required for ACh/GABA signaling we performed a genome-wide RNAi screen for gene knockdowns that cause hypersensitivity or resistance to 0.4 mM levamisole. We developed a liquid levamisole swim assay performed in 24 well plates and screened each well at two time points in order to identify both hypersensitive and resistant mutants. This assay did not require picking or prodding animals and enabled us to screen 17,469 clones, representing 90% of the genome, in duplicate. Primary hits were retested in quadruplicate and validated hits tested positive in at least 4/6 trials. We identified 193 genes with altered levamisole response (25 resistant; 168 hypersensitive). Among our hits were several of the genes isolated in the original Lewis et al. screen, suggesting that our screen identified bona fide regulators of synaptic signaling. Of the 193 genes at least 53% have evidence for expression in muscle and 87% have human homologs. Many of the genes we identified have an annotated sterile or lethal phenotype and would not have been isolated in the Lewis et al. screen. The set of genes identified in our genome-wide levamisole screen does not show significant overlap with sets of genes identified in previous aldicarb resistant and hypersensitive screens (Sieburth et al., 2005; Vashlishan et al., 2008) indicating that these screens reveal distinct synaptic regulators. We are currently using genetic, cell biological, and optogenetic approaches to determine how these 193 genes influence synapse formation, maintenance, and/or signaling.
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Na, Huimin, Andersen, Erik C., Baugh, L. Ryan, Dekker, Job, Walhout, Marian, Stevens, Lewis, Moya, Nicolas D., Tanny, Robyn E., Chitrakar, Rojin
[
International Worm Meeting,
2021]
Decades of research have led to the development of comprehensive genome resources that have been essential to study the Caenorhabditis elegans species. In parallel, the emergence of Caenorhabditis briggsae as a model system has been useful to make interspecies comparisons. Despite the importance of C. briggsae as a model, its genome resources have not been developed to the same extent as C. elegans. The current genome of C. briggsae reference strain AF16 contains thousands of unresolved gaps and numerous mis-assemblies. Because of these issues, C. briggsae gene models remain incomplete and have numerous structural errors in protein-coding genes. We sought to exploit the latest sequencing technologies and computational tools to provide the highest quality C. briggsae genome resources to date. First, we generated high-quality genome assemblies for two strains of C. briggsae: QX1410 (a "tropical" strain isolated in Saint Lucia that is closely related to AF16) and VX34 (a divergent strain isolated in China). These genome assemblies incorporate high coverage Oxford Nanopore PromethION long reads and chromosome conformation capture (Hi-C) data. Second, we genotyped 99 recombinant inbred lines generated from reciprocal crosses between QX1410 and VX34. Using these data, we produced a high-quality recombination map that validated the placement of scaffolds after genome assembly. Third, we sequenced the transcriptomes of each strain to high coverage using Pacific Biosciences SMRT and Illumina platforms. We developed a computational pipeline that leverages long and short RNA reads to generate a genome annotation for each strain. These new genome annotations have improved accuracy and completeness relative to the AF16 genome. Fourth, our research group currently maintains over 1,600 C. briggsae wild strains, comprising the largest collection worldwide. We sequenced the genomes of this entire collection to high coverage using the Illumina platform. We mapped the sequences of all wild strains to the QX1410 genome to call single nucleotide variants across the entire population. These high-quality genome resources will facilitate new avenues of research, including quantitative and population genetic studies of C. briggsae, and enable informative comparisons with C. elegans.
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Tanny, Robyn, Fisher, Kinsey, Stevens, Lewis, Evans, Kathryn, Antoshechkin, Igor, Chen, Jingxian, Powell, Maya, Baugh, Ryan, Andersen, Erik, Chitrakar, Rojin, Webster, Amy
[
International Worm Meeting,
2021]
The genetic basis of natural variation in starvation resistance is not well understood though it is a fundamental, biomedically important trait. We developed a population selection and sequencing approach (MIP-seq) to measure starvation resistance for a large number of wild C. elegans strains in a single culture. We identified three quantitative trait loci (QTL) affecting starvation resistance. These QTL overlap with hyper-divergent regions and contain multiple members of several large gene families involved in environmental interactions. In particular, we identified 16 members of the insulin/EGF receptor-like domain (irld) family with variants within starvation resistance QTL. We generated and assayed loss-of-function mutations for four irld family members, all of which increased starvation resistance. We show that the transcription factor
daf-16/FoxO, a critical effector of insulin/insulin-like growth factor signaling (IIS) known to promote starvation resistance, is required for increased resistance of
irld-39;
irld-52 mutants, that these mutants affect DAF-16 target gene expression, and that the IIS receptor
daf-2/InsR is epistatic to these irld genes. We propose that IRLD proteins bind insulin-like peptides (ILPs) to modify signaling in the sensory nervous system thereby affecting organismal physiology. This work demonstrates the efficacy of using population sequencing to investigate natural variation of a complex trait, and it identifies irld genes that regulate IIS and starvation resistance. Furthermore, it shows that variation in a rapidly evolving large gene family modifies activity of a deeply conserved signaling pathway to affect a fitness-proximal trait.
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[
West Coast Worm Meeting,
2002]
Mutants with defects in sensory cilium structure are unable to respond to sensory stimuli and also exhibit complex and unexpected changes in development and behavior. Lewis and Hodgkin initially reported that cilium-defective mutants such as
che-2 and
che-3 are smaller than wild-type animals (1977). We have previously demonstrated rescue of the
che-2 body size defect by expressing the
che-2 cDNA in amphid sensory neurons. These observations lead us to the hypothesis that an animal regulates its body size by sensing an environmental cue such as food. We have also demonstrated that cilium-defective mutants exhibit distinctive tracking patterns on food, although the mutants are not uncoordinated. Computerized analysis of locomotion revealed that a wild-type animal has two distinct locomotory states on food, dwelling and roaming, defined based on turning rate and speed. The roaming state is significantly decreased in cilium-structure mutants.
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Stevens, Lewis, Schwarz, Erich M., Yin, Da, Chandrasekar, Sinduja, Schartner, Caitlin M., Haag, Eric S., Meyer, Barbara J., Ralston, Edward J., Koutsovoulos, Georgios, Anderson, Erika C., Blaxter, Mark
[
International Worm Meeting,
2019]
Hermaphroditism has independently evolved at least three times within the Caenorhabditis genus, and six times in Pristionchus. This has often coincided with substantial losses of protein-coding genes, which are often implicated in male reproduction. However, the hermaphrodite C. tropicalis challenges this pattern. Although C. tropicalis has a substantially reduced genome (83 Mb in size, versus ~130 Mb in several male-female Caenorhabditis species), its closest male-female relative (C. wallacei) has an almost equally small genome (85 Mb). One explanation might be that genome shrinkage in C. tropicalis arose independently of hermaphroditism; this would fit the recent discovery of male-female Caenorhabditis with remarkably compact genomes, such as C. sulstoni with 65 Mb. An alternative explanation might be that C. wallacei reverted to male-female sexuality after hermaphroditism had already shrunk the genome of its shared tropicalis/wallacei ancestor. To begin testing these hypotheses, we used PacBio, Illumina, and Hi-C sequencing to produce third-generation genome assemblies for C. tropicalis and C. wallacei, each having six complete chromosomal scaffolds. Both assemblies are 98.6%-98.7% complete as scored by BUSCO, which matches the score for C. elegans (98.6%). In hermaphroditic C. briggsae versus its male-female sister species C. nigoni, ~7,000 genes lost in C. briggsae disproportionately include small genes with male-biased gene expression, such as the male secreted short (mss) gene family; the mss family encodes sperm surface glycoproteins, found only in outcrossing species, that are required for sperm competitiveness in mating. In contrast, C. tropicalis has only ~1,400 fewer protein-coding genes than C. wallacei (19,722 versus 21,017), 20% the disparity of C. briggsae vs. C. nigoni. Two clustered multigene families with male-biased expression conserved widely in male-female species (mss and a CAP-domain family that includes CRE28795) are absent not only in C. tropicalis but also in C. wallacei. More generally, gene families with conserved XO- or XX-biased expression have consistently fewer members in C. wallacei than in male-female species C. nigoni, C. remanei, or C. brenneri, and the diminished gene numbers of C. wallacei approach or equal those seen in hermaphrodites (for XO-biased and XX-biased gene families, respectively). These data suggest that C. wallacei might indeed be an atypical male-female Caenorhabditis species that underwent a temporary period of hermaphroditism, and jettisoned male reproductive genes during that period.
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Dilks, Clayton, Stinson, Loraina, Stevens, Lewis, Zhang, Gaotian, Roberto, Nicole, Crombie, Tim, Evans, Kathryn, Buchanan, Claire, Lee, Daehan, Cook, Daniel, Wang, Ye, Lu, Dan, Andersen, Erik, Zdraljevic, Stefan, Tanny, Robyn
[
International Worm Meeting,
2021]
Caenorhabditis elegans isolated from the Hawaiian Islands are known to harbor a high degree of genetic diversity relative to non-Hawaiian isolates. It was recently suggested that Hawaiian C. elegans can be partitioned into at least four genetically distinct groups. An analysis of geospatial environmental data further suggested that the genetic groups might associate with environmental parameters such as elevation and temperature, although the sample size for that study was small (n = 43 isolates). To better characterize the niche and genetic diversity of Hawaiian C. elegans and further define the associations of genetic groups with environmental parameters, we sampled different substrates and niches across the Hawaiian Islands six times over a three-year period. In total, we isolated 7,107 nematodes from 2,400 of 4,506 substrate samples (53% success rate). Among the nematodes we isolated, we identified five Caenorhabditis species, including 499 C. elegans, 377 C. briggsae, and 55 C. tropicalis isolates. We measured several environmental parameters at each sampling site and combined them with environmental parameters from geospatial databases to reveal that C. elegans is typically found in cooler and relatively drier climates at higher elevation than the other two selfing Caenorhabditis species. We isolated C. elegans most frequently from montane-alpine mesic forest habitat dominated by plant species native to the Hawaiian Islands. When possible, we cryopreserved C. elegans isolates and sequenced their genomes. To date, including Hawaiian isolates from collaborators, we have sequenced the genomes of 505 Hawaiian C. elegans isolates. With these data, we grouped the isolates into 163 isotypes (strains belonging to a single isotype have >0.9997 genome-wide concordance). We found that some of the isotypes were collected from the same locations over the three-year sampling period, and most of the collections of the same isotype were found within 500 meters of each other. Principal component analysis (PCA) of genetic variation revealed that the 163 isotypes fall into seven genetically distinct groups, three more than previously found on the islands with a smaller sample. Taken together, our findings begin to outline the spatiotemporal patterns of C. elegans genetic diversity on the Hawaiian Islands and raise new questions about evolutionary forces driving the genetic structure we have uncovered. For example, are these groups isolated by ecological or geographic distances, or perhaps both, and to what extent do reproductive incompatibilities contribute to the structure we have observed?