Wild M et al. (1983) International C. elegans Meeting "C elegans actin genes."

Begley M, Wild M, Hirsh DI, Krause MW, Landel CP

[International C. elegans Meeting, 1983]

Fitch, D. et al. (2013) International Worm Meeting "RhabditinaDB: online database for wild worms."

Kiontke, K., Fitch, D.

[International Worm Meeting, 2013]

New species of rhabditid nematodes are being discovered at an increasing rate. In Caenorhabditis alone, more species have been discovered in the last decade than in the previous 100 years since C. elegans was described. Although this discovery provides more candidate species for comparative biology and genomics, it poses a challenge for taxonomists and for keeping track of comparative data. In response to this challenge, we have created RhabditinaDB, a curated, online, open-access database to provide information on all known rhabditid nematodes, including data on taxonomy, phylogeny, distribution, living and non-living resources such as strains and type specimens, images (including DIC stacks, electron micrographs, and camera lucida drawings), species descriptions and relevant literature, phenotypic character data and molecular sequences. Additionally, we have implemented tools for querying the data, such as keyword searching, BLAST, PhenoBLAST (modified from Gunsalus et al., 2004) and a species-comparison page. Currently, the database shows information for all known species in genus Caenorhabditis, but will be expanded in the coming years to show data for all rhabditid species. Some problems in rhabditid systematics have arisen from the difficulty of obtaining taxonomic materials, lack of good keys or ways to compare new species to those already described. Specifically, there has been a high rate of synonymous descriptions and the accumulation of newly discovered but undocumented species. Our database should mitigate these problems by providing as much data as possible via one resource, along with a recent taxonomy (Sudhaus 2011), based primarily on our molecular phylogeny (Kiontke et al., 2007, 2012). Not only will RhabditinaDB facilitate taxonomy, but it will also provide data on rhabditid biodiversity. This in turn should facilitate phylogenetically informed taxon choices for comparative studies and genomics. As RhabditinaDB is a work in progress, we welcome suggestions for improvements, and data submissions. RhabditinaDB can be accessed at: http://wormtails.bio.nyu.edu/Databases . Thanks to M. Katari, J. Lorenzana, NYU Bobst Library, W. Sudhaus. References: Gunsalus et al. 2004, Nucl. Acids Res. 32:D406-D410; Sudhaus 2011, J. Nematode Morphol. Syst. 14(2):113-178; Kiontke et al. 2007, Curr. Biology 17(22):1925-1937.

M Ailion et al. (1999) International C. elegans Meeting "27 in the life of wild and mutant worms"

JH Thomas, M Ailion

[International C. elegans Meeting, 1999]

Dauer formation in C. elegans is strongly induced at 27deg, a temperature just below the highest temperature that permits growth and reproduction. At this temperature, dauer formation is much more strongly induced by pheromone than at 25deg and even in the absence of exogenous pheromone, N2 makes some dauers at 27deg. This basal level of dauer formation is probably not dependent on endogenous pheromone since daf-22 mutant animals also form dauers at a similar frequency at 27deg. All twenty-one natural isolates of C. elegans that we've tested have virtually the same thermal limit and all induce dauer formation at 27deg. Dauers formed at high temperatures have normal fertility if recovered at lower temperatures, suggesting that the strong induction of dauer formation at stressful temperatures was selected for as a protection against sterility or death. In contrast to the low wild-type level of dauer formation at 27deg, many mutants are strongly Daf-c at 27deg but are not Daf-c at 25deg. Mutants with this phenotype include unc-64 , unc-31 , unc-3 , daf-3 and the dyf mutants. We have performed screens for Daf-c mutants at 27deg and isolated 100 mutants. We isolated twenty-one alleles of known Daf-c genes (many weak alleles), fifteen alleles of dyf or Daf-d genes, five alleles of unc-31 or unc-3 and 13 alleles of new Daf-c genes. Many other mutants have only been partially characterized. Of the new Daf-c mutations, two are alleles of pdk-1 and one is an allele of aex-6 . The Daf-c phenotype of many of the mutants is fully suppressed by mutations in daf-16 , suggesting that these genes act in the daf-2/age-1 (insulin-receptor /PI3 kinase) pathway. These include unc-64 and unc-31 , which encode neurosecretory proteins that may be involved in insulin secretion, and pdk-1 which encodes a PI3-dependent protein kinase that acts downstream of age-1 . The aex-6(sa699) , sa573 and sa691 mutations also appear to affect this pathway and may encode novel components of the insulin pathway. In addition to their Daf-c phenotype, the aex-6(sa699) and sa691 mutants also have defects in movement and defecation. We have mapped sa691 to a narrow region of the X chromosome between lin-32 and unc-2 and are attempting to clone it.

Hill, Robin L. (2011) International Worm Meeting "Characterizing wild nematode isolates in an undergraduate research lab."

Hill, Robin L.

[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.

Rose, Jacqueline et al. (2021) International Worm Meeting "Collection of Wild Isolates as a Remote Hands-on Research Experience"

Long, Nathan, Rose, Jacqueline

[International Worm Meeting, 2021]

In response to the COVID-19 pandemic, teaching was moved to remote learning design including experiential lab courses. With classes offered remotely, many students opted to stay home and continue their classes online. However, as lab courses provide students some of the only hands-on laboratory experience for many students prior to graduation, it was our aim to create a hands-on lab experience that was transportable and therefore accessible to all enrollees. The major challenge in creating a remote C. elegans lab experience was that our university would not permit the shipment of E. coli, in any form, to students. This created a challenge for shipping worms to students, as well as for providing a food source once worms arrived. To circumvent this obstacle, we designed the class around the idea of students each collecting wild-type worms locally. As well, a vegan broth was utilized as a substitute food source. The lesson plan consisted of multiple assignments beginning with a literature review to develop an appreciation for the contributions and potential power of using wild isolates in research. Following this, students independently developed their own collection protocol. Materials that would allow for the extraction of worms from soil using the Baermann funnel method were then shipped or provided for pickup. Finally, following collection students determined if the worms were Caenorhabditis by screening animals for self-replication and by physical observation of the pharyngeal structure. As part of the final assignment, students completed the wild isolate submission form from the Caenorhabditis elegans Natural Diversity Resource (www.elegansvariation.org). Students were encouraged to be innovative in their method design and implementation and to utilize notebooks to record unique steps and deviations from initial method proposals. Upon completion, students were required to return a sample of their worm collection to the university for further identification. By the conclusion of the term, half of the students had successfully collected worms. In the future, this lesson plan can be expanded to include additional identification procedures such as crossing wild-isolates with the N2 strain and/or DNA sequencing.

Shingai R et al. (2000) Japanese Worm Meeting "Durations of Locomotion of Wild Type and GABAergic Mutants of C.elegans"

Hoshi K, Shingai R

[Japanese Worm Meeting, 2000]

We studied durations of locomotion of wild-type nematodes (N2) and the GABAergic mutants unc25 and unc47 which moved on the surface of agarose plates. We identified forward movement, backward movement, resting and turns by watching images on video and computer displays. Forward movement lasted longer and rests were briefer without, than with bacteria. Frequency distributions except for backward movement fitted a sum of two exponential functions. The durations of resting in these mutants were much longer than in the N2 strain, especially in the absence of bacteria. The turn frequency of unc47 nematodes in the absence of bacteria had a higher short component than that of the wild type N2 and unc25 nematodes. These results show that the quantitative measurement reveals behavioral differences between the mutants and those induced by different external environments.

Vergara, Ismael A. et al. (2011) International Worm Meeting "Loss-of-Function genomic variations in wild Caenorhabditis elegans."

Wang, Jun, Tarailo-Graovac, Maja, Chen, Nansheng, Vergara, Ismael A.

[International Worm Meeting, 2011]

As revealed by an explosive number of genome sequencing projects, humans carry an enormous amount of genomic variations (GVs). Strikingly, many of these GVs found in apparently healthy individuals are loss-of-function mutations previously associated with genetic diseases, including many types of cancer and mental retardations. How these GVs function in apparently normal and disease humans is not well understood, due to two major challenges. First, the large size of human genes, the large intergenic regions, and the large quantity of GVs residing within each gene together make it challenging to correlate GVs and affected genes. More importantly, it is difficult if not impossible to interrogate the impact of most functionally important GVs on the fitness of their carrier, hence impeding further understanding of their contribution to the pathogenesis of disease conditions in humans. Using Roche/454 (4X coverage) and Illumina sequence reads (72X coverage) in a complementary manner, we have identified various types of GVs with base pair resolution in the wild C. elegans strain CB4856, which was first isolated in an island in Hawaii. Using N2 strain as reference, we have identified 313,219 putative SNPs, 21,332 small indels, 533 large deletions and 208 large insertions, some of which are larger than 10,000 bp. Also, we have detected hundreds of insertions and deletions co-occurring at the same breakpoint. Inspection of the breakpoints has revealed patterns of local duplications as well as events of non-allelic homologous recombination. Among these GVs, we have identified candidate loss-of-function mutations in 141 different essential genes in C. elegans, nine of which are orthologs of human disease genes (OMIM). The biological impact of these loss-of-function GVs on the fitness of CB4856 is being analyzed.

Samuel, Buck S. et al. (2013) International Worm Meeting "Influence of the Microbiome on C. elegans Growth in the Wild."

Samuel, Buck S., Ruvkun, Gary, Rowedder, Holli, Felix, Marie-Anne, Braendle, Christian

[International Worm Meeting, 2013]

Like us, C. elegans lives in a microbial world. In its natural habitats of rotting fruits and vegetation, these nematodes proliferate as they dine on an array of microbes. Interactions with microbes span a spectrum from constant confrontation (pathogens) to relative indifference (food) to perhaps even mutual benefit (symbionts). This study identifies these natural microbes and addresses whether microbiome composition influences proliferation of C. elegans in the wild. To examine this question, we sequenced bacterial 16S (SSU) rDNA amplicons from habitats with wild C. elegans populations collected in France and Spain. Our results show that C. elegans encounters a broad array of bacteria in the wild-especially the divisions (phyla) of Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria. An abundance-weighted comparisons of phylogenetic differences (UniFrac) showed distinct clustering by habitat type, as rotting apples clustered separately from other habitats sequenced. Further, rotting apples clustered by large presence of proliferating or small non-proliferating (dauer) populations of worms. C. elegans appear to proliferate in apples with 'simpler' microbiomes (lower diversity, fewer species and Proteobacteria-rich). Specific alpha-proteobacteria were particularly enriched in apples with proliferating worms, while a number of genera were consistently found in apples with non-proliferating worms (e.g., Pseudomonas, several Bacteroidetes, etc.). Population size also correlated with apple rottenness, suggesting bacterial load is key to growth as well. Similarly, Proteobacteria content does affect C. elegans (N2) growth rate in the lab, as worms grew faster on mixtures (and single isolates) with 80% Proteobacteria versus those with 40% Proteobacteria. Together, these studies define the microbial diet of C. elegans and implicate the natural microbiome as a key determinant of C. elegans' growth in the wild.

Goldstein P (1981) International C. elegans Meeting "synaptonemal complex analysis of wild-type and him mutants of C elegans."

Goldstein P

[International C. elegans Meeting, 1981]

Josselin Milloz et al. (2007) International Worm Meeting "Evolution of vulva development among wild C. elegans isolates."

Marie-Anne FELIX, Isabelle Nuez, Josselin Milloz

[International Worm Meeting, 2007]

The C. elegans vulva arises from a row of 6 competent cells (P3.p to P8.p). Under normal conditions, three cells adopt a vulval fate: the anchor cell in the somatic gonad sends a LIN-3/EGF signal that strongly activates a MPK-1/MAPK signaling pathway in the most proximal cell, P6.p, and less strongly in P5.p and P7.p. In response, P6.p adopts a 1 vulval fate and releases Delta-like proteins towards P5.p & P7.p. LIN-12/Notch signaling in these cells leads them to adopt a 2 vulval fate and to repress MAPK signaling. A third pathway -BAR-1/<font face=symbol>b</font>-catenin- cooperates with the MAPK signaling to induce vulval fates. We have explored the evolution of this signaling network within C. elegans. As vulva development is invariant within and between all wild isolates, we sensitized the system to unravel potential cryptic genetic differences. To this aim, we introgressed 13 mutations that affect the activity of the three pathways into 6 wild C. elegans isogenic backgrounds (CB4856, JU258, PB303, PB306, PS2025 and AB1). The effect of each mutation varies significantly between different genetic backgrounds. For example, the mean number of induced cells ranges from 0.6 in N2 to 2.2 in AB1 (n&#62;50) with let-23(sy1rf). Interestingly, the defects caused by a bar-1(ga80lf)/<font face=symbol>b</font>-catenin are much stronger in JU258 (1.8 induced cells) than in N2 (2.5 induced cells, n=100). As a consequence, genetic screens performed in other wild strains than N2 would give different alleles, and even different genes, e. g. more strong alleles of the Wnt pathway with JU258 or less strong alleles of the Ras pathway with AB1. To understand the mechanistic basis of some differences, we focused on N2 vs. AB1. All the mutations affecting the Ras pathway induce more cells in AB1, as if Ras pathway activity was higher in AB1 than in N2. Indeed, the downstream activity of the Ras pathway is twice higher in AB1 than in N2 (measured by quantification of egl-17::CFP, a downstream reporter, at the lethargic L2). In conclusion, the vulva signaling network has evolved quantitatively within C. elegans, in the absence of change in the final pattern. We now aim at unraveling the genetic architecture of the differences using recombinant inbred lines (see contribution by F. Duveau et al.). We simultaneously perform an in silico approach (see contribution by E. Hoyos et al.) to get a better understanding of the system and its evolution.