Cook, Daniel et al. (2017) International Worm Meeting "The C. elegans natural diversity resource."

Andersen, Erik, Roberts, Joshua, Zdraljevic, Stefan, Cook, Daniel

[International Worm Meeting, 2017]

Caenorhabditis elegans is used as a model in over 1,100 labs around the world and has enabled biological discoveries in many diverse fields. These advances have been fueled by an extensive genetic toolkit, including strains, reagents, and databases. However, the majority of C. elegans research focuses on the laboratory-domesticated N2 strain, neglecting potential insights gleaned from natural populations. Studies of C. elegans natural variation can identify the genetic factors underlying biomedically relevant traits and genome evolution. To address the need for resources to study natural variation, we developed the Caenorhabditis elegans Natural Diversity Resource (CeNDR) - available at www.elegansvariation.org. The web-based CeNDR platform includes three areas: (1) a central repository for the deposition, organization, and dissemination of wild C. elegans strains, including detailed information for each strain (e.g. collection date, GPS location, substrate, and elevation); (2) a data portal for dissemination of whole-genome sequence data in BAM or CRAM formats and variant data in VCF format for each of the 383 wild isolates, including a powerful interactive genome browser that can be used to interrogate genetic variation across the population for genes or regions of interest; (3) a genome-wide association mapping portal to enable mappings of quantitative traits measured using wild C. elegans strains, including a comprehensive report of significance, variation, measures of selection, etc. We believe that CeNDR will become an indispensable tool within the C. elegans genetic toolkit to enable researchers to examine natural populations and identify interesting new biological phenomena.

Yang, Heeseung et al. (2021) International Worm Meeting "Genome-wide association study for nictation behavior of the nematode C. elegans"

Cook, Daniel, Lee, Daehan, Paik, Young-Ki, Lee, Junho, Andersen, Erik, Kim, Heekyeong, Yang, Heeseung

[International Worm Meeting, 2021]

Dispersal is crucial for many organisms. There are various strategies for efficient dispersal, and phoresy is one example. Animals can disperse efficiently by attaching to another organism, like hitchhiking. Caenorhabditis elegans dauers show a stage-specific standing behavior, called nictation, that can facilitate phoretic interactions of dauers. Standing and waving their body, dauers can interact with other organisms such as isopods. It helps dauers to escape from harsh conditions and move into a better environment for re-growing and reproduction. As C. elegans are found most frequently as dauer larvae in the wild, nictation is thought to play an important role in their life cycle. However, the genetic basis and the underlying regulatory mechanism of nictation are not well understood. Here, we try to figure out the genetic factors that regulate nictation and make nictation diversity between C. elegans wild isolates. Wild isolates from the worldwide region showed diverse nictation fractions in the same experimental conditions. A genome-wide association mapping of the nictation of 137 wild isolates identified a quantitative trait locus (QTL) for nictation. Using near-isogenic lines, we identified a QTL of 90 kb interval, and also found that this QTL affects other dauer-related phenotypes. Now we are testing and confirming candidate genes using CRISPR mutants and RNAi experiments. Elucidating nictation regulatory mechanisms will provide new insights into the genetic basis of phoretic behavior.

Zamanian, M et al. (2017) International Worm Meeting "Evidence of small RNA involvement in nematode benzimidazole resistance."

Lee, Junho, Zdraljevic, Stefan, Andersen, Erik, Rodriguez, Briana, Lee, Daehan, Tanny, Robyn, Cook, Daniel, Brady, Shannon, Zamanian, M

[International Worm Meeting, 2017]

Parasitic nematodes impose a debilitating health and economic burden across much of the world. The morbidity and mortality inflicted by these pathogens are partly curtailed by mass drug administration programs that depend on the continued efficacy of a limited portfolio of anthelmintic drugs. Benzimidazoles are WHO-designated "Essential Medicines" and an indispensable component of this limited arsenal. Nematode resistance to benzimidazole chemotherapy threatens parasite control efforts in both human and veterinary medicine. Despite this threat, the genetic landscape of potential resistance mechanisms to thiscritical drug class remains largely unexplored. There is an urgent and recognized need to identify molecular mechanisms and genetic markers that cause benzimidazole resistance. In order to identify conserved nematode drug responses, we exploit natural variation in two model roundworms, Caenorhabditis elegans and Caenorhabditis briggsae, to discover quantitative trait loci (QTL) that control benzimidazole sensitivity. Surprisingly, we found that resistance to benzimidazoles mapped to syntenic piRNA-enriched regions of the genome with few protein-coding genes in both Caenorhabditis species. We used near isogenic lines (NILs) to narrow the major-effect benzimidazole QTL in C. elegans to a smaller region of the genome and demonstrate that the benzimidazole-resistance phenotype results from piRNA variation that is dependent on the function of the piRNA-associated argonaute prg-1. We identified candidate piRNAs causal to the resistance phenotype and putative genes targeted for silencing by downstream 22G RNAs. Our results indicate that small RNAs require consideration in drug resistance mechanisms in nematodes, because the piRNA pathway and related small RNA pathways are conserved in many medically and agriculturally important parasitic nematodes. Importantly, this resistance mechanism could be mediated through the heritable transgenerational effects of small RNAs. This finding has significant implications for parasite control and the management of drug resistance in other phyla and systems.

Crombie, Tim et al. (2021) International Worm Meeting "Repeated Sampling of Caenorhabditis elegans Across the Hawaiian Islands Reveals Spatiotemporal Patterns of Genetic Diversity"

Stevens, Lewis, Buchanan, Claire, Andersen, Erik, Stinson, Loraina, Dilks, Clayton, Tanny, Robyn, Lu, Dan, Zhang, Gaotian, Evans, Kathryn, Zdraljevic, Stefan, Crombie, Tim, Lee, Daehan, Roberto, Nicole, Wang, Ye, Cook, Daniel

[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?

Yohann Duverger et al. (2006) European Worm Meeting "A French Functional Genomics Platform"

Yohann Duverger, Jonathan Ewbank, Jerome Reboul, Daniel Wong

[European Worm Meeting, 2006]

Yohann Duverger1, Jrme Reboul2, Daniel Wong1 and Jonathan Ewbank1 For the last three years, we have offered a service of worm sorting based around the Union Biometrica COPAS platform. The COPAS machine is equipped with a Zymark twister robot, allowing automated analysis of multiple 96 well plates. We recently upgraded the machine to include the Profiler II that generates >1000 individual measurements per worm simultaneously for up to 4 channels (including 2 fluorescent). This equipment has been applied to a wide range of biological questions. They include quantifying the level of fluorescent reporter gene expression, large-scale RNAi and genetic screens and combinatorial library drug screening. Examples of each will be presented.. This platform is part of a fully integrated functional genomics facility open to the academic community (see http://www.ciml.univ-mrs.fr/EWBANK_jonathan /RIO.html). Other resources include the ORFeome (developed in Marc Vidals laboratory), and Julie Ahringers RNAi library, together with whole-genome microarrays. For the latter, through a collaboration with the Genome Sequencing Center at Washington, and the transcriptome platform at Nice, we have spotted the Illumina long oligo set onto glass slides and provide microarrays free of charge to the French C.elegans community and at cost price to academic researchers in Europe.. This French functional genomics platform has been made possible through funding from the National Genopole network, Marseille-Nice genopole, the CNRS and support from Union Biometrica.

Attila L Kovcs et al. (2005) International Worm Meeting "Electron microscopy of autophagy in Caenorhabditis elegans"

Judit Fehr, Tibor Vellai, Krisztina Takcs-Vellai, Zsolt Plfia, Jnos Kovcs, Attila L Kovcs

[International Worm Meeting, 2005]

Cellular autophagy is a process for the degradation of cytoplasmic constituents in eukaryotic cells. Since its discovery in 1957 in the epithelial cells of kidney of the newborn mice (1) electron microscopy has been and still remains an indispensable method for studying autophagy. One of the main reasons of the late start of autophagy research in C. elegans is the relative difficulty of performing transmission electron microscopy with worm samples. Recently we have developed a technique by which autophagic processes of the worm become accessible for systematic morphological and, in three major tissue types, for morphometric analysis by transmission electron microscopy (2). Our poster introduces the method, presents the criteria for the identification and morphological analysis of various types of autophagic vacuoles in all major cell types, and shows the latest morphometric data on autophagy in hypodermal, gut epithelial and body wall muscle cells during postembryonic development including all four larval, as well as the predauer, dauer and postdauer stages. Our results indicate that the cells of continuously feeding worms are practically devoid of autophagic vacuoles. Significant increase in the quantity of autophagic vacuoles can be observed at the end of each larval stage when the lethargus is reactivated. Systematic measurements on Daf-c mutants in the predauer period show that preparation for the dauer stage does not involve constitutive autophagic activity. (1) Clark S.L. (1957) Cellular differentiation in the kidneys of newborn mice studied with the electron microscope, J. Biophysic. Biochem. Cytol. 3, 349 (2) Kovacs AL, Vellai T, Muller F (2004) Autophagy in Caenorhabditis elegans. In: "Autophagy" Ed. Daniel J. Klionsky, Landes Bioscience 2004, Chapter 17, pp 216-223

Goldman, Gal et al. (2021) International Worm Meeting "The role of network topology in the performance of the circuit for nociceptive behaviors in C. elegans"

Pechuk, Vladyslava, Schneidman, Elad, Oren-Suissa, Meital, Goldman, Gal

[International Worm Meeting, 2021]

The nature of the relationship between neural circuits and the resulting animal behavior is a key question in neurobiology. It was previously established that the specific synaptic and cellular properties of neural networks can be widely disparate, yet maintain similar function (Prinz et al., 2004). It is therefore clear that some features of a network's structure are important to retain certain functional features. The sensitivity of such networks to changes in the topology have not been characterized. To explore the contribution of topology to the network's performance, we focused here on the circuit for nociceptive behaviors in C. elegans. The neurons of the circuit are shared between the two sexes, but their connectivity is different (Cook et al., 2019). The behaviors that result from these circuits are sexually dimorphic as well. The distinct network topologies and behavioral outputs make this circuit a good example for exploring the relationship between structure and function in neural networks. We simulated the response of the nociceptive circuits to external stimuli, in males and in hermaphrodites, using a wide range of realistic values for the circuit's biophysical parameters (synaptic strengths, conductivity, membrane time constants, etc.). We then searched for the parameters' space in which the activity of the motor output neurons in the simulation would match the worms' behaviors in experimental observations. We found an overlap between the sexes in terms of the synaptic and cellular parameters that allow for the correct behavior of the network. Moreover, our results suggest that the connectivity alone might be sufficient to explain the behavioral differences between the sexes. Notably, more stringent requirements of the models' performance suggests that the connections in this network cannot be all excitatory, as has been commonly assumed, or that additional inhibitory neurons must play an important role in shaping the circuit's response to tail stimulation. Future analysis will further explore the relations between the network's topology and the joint activity patterns of the neurons as measured by calcium imaging.

Jarrell, Travis A. et al. (2013) International Worm Meeting "The connectome of the anterior nervous system of the C. elegans adult male."

Nguyen, Kenneth, Bloniarz, Adam E., Brittin, Christopher A., Cook, Steven J., Hall, David H., Emmons, Scott W., Xu, Meng, Jarrell, Travis A., Wang, Yi

[International Worm Meeting, 2013]

The innate behavioral repertoires of the two sexes of a species are guided by differing reproductive priorities. C. elegans male copulation is controlled by a neural network in the tail in which a majority of the neurons and muscles are specific to the male. But known differences in olfactory preferences and exploratory tendencies emanate from behaviors controlled by circuits in the head, where the complement of neurons is nearly identical in the two sexes. We determined connectivity in the anterior nervous system of the adult male from a 1,500 section-long thin section EM series extending from near the tip of the nose, through the nerve ring, and part way into the retrovesicular ganglion. This region contains the bulk of the synapses, excluding ventral cord nmj's. To make a comparison to the hermaphrodite, we re-reconstructed legacy Cambridge micrographs using our software, which allows us to score synaptic weights (see abstract by Cook et al). While our analysis is at an early stage, we can already see the essential result: in the adjacency matrices that display the connectivity, it is difficult to spot differences that appear greater than would be expected given the inherent variability of neuronal wiring. Known circuits in navigation and other responses are conserved. Thus behavioral differences likely emerge from differing circuit properties rather than differing connectivity. There are two possible exceptions: AIM synapses onto AIB and RIA synapses onto RIB in the male only. One set of male-specific synapses expected involves the male-specific head CEM sensory neurons, and the tail EF interneurons, which receive extensive input from the copulatory circuits in the tail and extend processes through the ventral nerve cord into the nerve ring. Both of these neuron classes have as their strongest targets the AVB command interneurons for forward locomotion. This suggests one of their functions may be to inhibit forward locomotion when a hermaphrodite is sensed or during copulation. They make additional connections to be further explored.

Astrid Kleinert et al. (2006) European Worm Meeting "Analysis of Insulin-Like Peptides in C. elegans by Mass Spectrometry"

Astrid Kleinert, Daniel Hess, Joy Alcedo, Jan Hofsteenge

[European Worm Meeting, 2006]

Astrid Kleinert, Daniel Hess, Jan Hofsteenge and Joy Alcedo. Genetic analyses in C. elegans have implicated an endocrine signaling pathway, the insulin/IGF-1 pathway, in regulating lifespan. Mutations in the gene daf-2, which encodes an insulin/IGF-1 receptor homologue, can increase worm lifespan by more than 100% (1). In addition, the insulin and IGF-1 pathways have been shown to influence fly and mouse lifespan (2-5).. Although downstream components of the DAF-2 pathway have been studied in detail, far less is known about the putative DAF-2 ligands predicted to be encoded by 38 insulin-like genes in the worm. It remains unknown which of the predicted insulin-like peptides are present in wild type or in longevity mutants. In order to determine this, we have initiated a project to analyze the polypeptide subfractions of the C. elegans proteome by mass spectrometry. Worm lysates will be prepared from mixed stage, wild-type worms, excluding membrane-bound proteins. Acid-ethanol extraction and gel filtration will be tested as methods to obtain a subfraction enriched in small polypeptides. Peptide identification will be done by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Data analyses will be carried out using a MASCOT search database, which contains a subset of the UNIPROT database and a database generated of relevant C. elegans polypeptides. Finally, using the SILAC method for quantification (6), we plan to compare differences in the levels of polypeptides, e.g., insulin-like peptides, between wild-type worms and longevity mutants. . References: (1) Kenyon et al., 1993. Nature 366: 461-4. (2) Clancy et al., 2001. Science 292: 104-6. (3) Tatar et al., 2001. Science 292: 107-110. (4) Holzenberger et al., 2003. Nature 421: 182-7. (5) Blher et al., 2003. Science 299:572-4. (6) Krijgsveld et al., 2003. Nature Biotech. 21: 927-931.

Pechuk, Vladyslava et al. (2021) International Worm Meeting "Sexually-dimorphic responses to noxious stimuli in C. elegans result from differences in interneuron connectivity rather than in sensory processing"

Pechuk, Vladyslava, Salzberg, Yehuda, Oren-Suissa, Meital

[International Worm Meeting, 2021]

Sexually reproducing animals display sexually dimorphic behaviors, geared towards reproductive success. Are there differences in the way the two sexes interpret and respond to the same aversive input? To address this question we analyzed the worm's avoidance responses to hazardous conditions. C. elegans generates an escape response to aversive stimuli by integrating sensory information from the polymodal nociceptive ASH head neurons and tail neurons, and conveying it to the main reversal interneuron AVA. The recent full mapping of the male connectome (Cook et al. 2019) suggests that the sex-shared neurons in the avoidance circuit are dimorphically connected, e.g. ASH to AVA connection is predicted to exist only in hermaphrodites. We measured the response of both sexes to the aversive stimuli SDS and glycerol using a behavioral tail-drop assay. We found that the two sexes exhibit dose-dependent sexually dimorphic responses to the aversive stimuli - across multiple nociceptive modalities, hermaphrodites exhibited a lower pain threshold than males. The behavioral differences and the suggested anatomical maps prompted us to functionally deconstruct the avoidance circuit. To examine potential sexual dimorphism at the sensory level, we compared ASH receptor expression levels (OCR-2, OSM-9, OSM10, QUI-1, ODR-3, GPA-3), ASH glutamatergic secretion by imaging the pHluorin sensor, and neuronal activation by calcium imaging in both sexes. We found that the ASH sensory neuron is non-dimorphic for all these parameters and responds similarly in the two sexes. Furthermore, we activated ASH optogenetically, thus bypassing the sensory input level, and found that hermaphrodites responded with a reversal at a lower LED intensity compared to males, in agreement with the tail-drop assay. Lastly, imaging of the downstream AVA interneuron revealed a stronger and longer response to the stimulus in hermaphrodites compared to males, further pointing to the connectivity and interneuron levels as the key sources for dimorphism in the circuit. Together, our results suggest that dimorphic responses to noxious cues arise due to neuronal circuit dimorphism downstream of sensory processing. We hypothesize that differences in circuit connectivity, rather than sensory perception per se, allow for sex-specific behavioral adaptation.