Hahnel SR et al. (2020) Int J Parasitol Drugs Drug Resist "Caenorhabditis elegans in anthelmintic research - Old model, new perspectives."

Heisler I, Hahnel SR, Andersen EC, Kulke D, Dilks CM

[Int J Parasitol Drugs Drug Resist, 2020]

For more than four decades, the free-living nematode Caenorhabditis elegans has been extensively used in anthelmintic research. Classic genetic screens and heterologous expression in the C. elegans model enormously contributed to the identification and characterization of molecular targets of all major anthelmintic drug classes. Although these findings provided substantial insights into common anthelmintic mechanisms, a breakthrough in the treatment and control of parasitic nematodes is still not in sight. Instead, we are facing increasing evidence that the enormous diversity within the phylum Nematoda cannot be recapitulated by any single free-living or parasitic species and the development of novel broad-spectrum anthelmintics is not be a simple goal. In the present review, we summarize certain milestones and challenges of the C. elegans model with focus on drug target identification, anthelmintic drug discovery and identification of resistance mechanisms. Furthermore, we present new perspectives and strategies on how current progress in C. elegans research will support future anthelmintic research.

Hahnel SR et al. (2018) PLoS Pathog "Extreme allelic heterogeneity at a Caenorhabditis elegans beta-tubulin locus explains natural resistance to benzimidazoles."

McGrath PT, Andersen EC, Rodriguez BC, Hahnel SR, Zdraljevic S, Zhao Y

[PLoS Pathog, 2018]

Benzimidazoles (BZ) are essential components of the limited chemotherapeutic arsenal available to control the global burden of parasitic nematodes. The emerging threat of BZ resistance among multiple nematode species necessitates the development of novel strategies to identify genetic and molecular mechanisms underlying this resistance. All detection of parasitic helminth resistance to BZ is focused on the genotyping of three variant sites in the orthologs of the -tubulin gene found to confer resistance in the free-living nematode Caenorhabditis elegans. Because of the limitations of laboratory and field experiments in parasitic nematodes, it is difficult to look beyond these three sites to identify additional mechanisms that might contribute to BZ resistance in the field. Here, we took an unbiased genome-wide mapping approach in the free-living nematode species C. elegans to identify the genetic underpinnings of natural resistance to the commonly used BZ, albendazole (ABZ). We found a wide range of natural variation in ABZ resistance in natural C. elegans populations. In agreement with known mechanisms of BZ resistance in parasites, we find that a majority of the variation in ABZ resistance among wild C. elegans strains is caused by variation in the -tubulin gene ben-1. This result shows empirically that resistance to ABZ naturally exists and segregates within the C. elegans population, suggesting that selection in natural niches could enrich for resistant alleles. We identified 25 distinct ben-1 alleles that are segregating at low frequencies within the C. elegans population, including many novel molecular variants. Population genetic analyses indicate that ben-1 variation arose multiple times during the evolutionary history of C. elegans and provide evidence that these alleles likely occurred recently because of local selective pressures. Additionally, we find purifying selection at all five -tubulin genes, despite predicted loss-of-function variants in ben-1, indicating that BZ resistance in natural niches is a stronger selective pressure than loss of one -tubulin gene. Furthermore, we use genome-editing to show that the most common parasitic nematode -tubulin allele that confers BZ resistance, F200Y, confers resistance in C. elegans. Importantly, we identified a novel genomic region that is correlated with ABZ resistance in the C. elegans population but independent of ben-1 and the other -tubulin loci, suggesting that there are multiple mechanisms underlying BZ resistance. Taken together, our results establish a population-level resource of nematode natural diversity as an important model for the study of mechanisms that give rise to BZ resistance.

Gibson, Sophia B et al. (2021) MicroPubl Biol

Harper, Clare S, Gibson, Sophia B, Lackner, Laura L, Andersen, Erik C

[MicroPubl Biol, 2021]

Parasitic nematode infections continue to have an enormous impact on human and livestock health worldwide (Hotez et al., 2014; Kaplan & Vidyashankar, 2012). A limited arsenal of anthelmintic drugs are available to combat these infections. One of the most widely used classes is benzimidazoles (BZ), and resistance against this class is widespread (Kaplan & Vidyashankar, 2012). Previous studies to understand parasitic nematode resistance using the free-living model organism Caenorhabditis elegans showed that variation in the C. elegans beta-tubulin gene ben-1, an ortholog of beta-tubulins in parasitic nematodes, confers resistance to BZ drugs (Dilks et al., 2020; Driscoll et al., 1989; Hahnel et al., 2018). The most common missense mutation resistance alleles are F167Y, E198A, and F200Y (Avramenko et al., 2019; Mohammedsalih et al., 2020). Although computational models have predicted that these amino acids are involved in the binding of BZ compounds to beta-tubulins, the binding remains to be investigated empirically at the structural level because nematode-specific beta-tubulin structures have not been created (Aguayo-Ortiz et al., 2013; Hahnel et al., 2018). To better understand the mechanisms of resistance, we sought to obtain those crystallographic structures.

Crombie, T.A. et al. (2019) International Worm Meeting "Massive sampling of Caenorhabditis elegans across the Hawaiian Islands reveals remarkable genetic diversity on the islands and admixture with European populations."

Andersen, E.C., Evans, K.S., Zdraljevic, S., Cook, D.E., Hahnel, S.R., Rodriguez, B.C., Zhang, G., Crombie, T.A., Tanny, R.E., van der Zwaag, J., Brady, S.C., Lee, D., Wang, Y.

[International Worm Meeting, 2019]

Global surveys of Caenorhabditis elegans genetic diversity suggest that large-scale selective sweeps on chromosomes I, IV, V, and X are associated with reduced diversity in some regions of the world. These sweeps are thought to be caused by adaptation to human-associated environments, so swept strains might not represent the natural diversity present before human influence. Interestingly, strains isolated from the Hawaiian Islands are generally highly diverged from the rest of the global population and contain little evidence of these sweeps. Therefore, we sought to better characterize the natural diversity of the species by collecting C. elegans from the Hawaiian islands. At two different times, we used a collection protocol to sample nematodes from a total of 3,264 sites across five Hawaiian islands. Among the 4,558 nematodes we isolated, we identified five Caenorhabditis species including C. elegans, C. briggsae, C. tropicalis, C. kamaaina, and a new species, C. oiwi. On the first collection trip, we discovered that C. elegans was generally found in cooler environments at higher elevations than other members of the genus. These data enabled improvements to our collection protocols that increased our success rate for isolating C. elegans from 1.8% to 4.1% on the second collection trip. We identified 26 distinct C. elegans strains from our first collection trip, increasing the total number of Hawaiian strains to 43. The mean genome-wide nucleotide diversity of these 43 Hawaiian strains is three-fold higher than the 233 non-Hawaiian strains from around the globe. We used ADMIXTURE to investigate the ancestry of all 276 strains and identified six distinct ancestral populations. Surprisingly, we saw evidence of admixture between some of the Hawaiian strains and one predominantly swept European population. Taken together, these findings confirm that divergent strains can be collected reliably from the Hawaiian Islands but raise new questions about the direction, magnitude, and timing of gene flow among swept and non-swept populations that has contributed to the global pattern of diversity in the species.

Shawn Lockery et al. (2001) Worm Breeder's Gazette "Tight-seal whole-cell patch clamping of C. elegans neurons: erratum"

Miriam Goodman, Shawn Lockery

[Worm Breeder's Gazette, 2001]

We regret to inform the C. elegans community that the published recipe for internal saline for whole-cell recordings[1,2] from neurons was incorrect. The published recipe was (in mM): KGluconate 125, KCl 18, NaCl 0, CaCl2 0.7, MgCl2 1, HEPES 10, EGTA 10. The recipe actually used was (in mM): KGluconate 125, KCl 18, NaCl 4, CaCl2 0.6, MgCl2 1, HEPES 10, EGTA 10. The main effect of this error resides in the difference in NaCl concentration. The correct saline will produce a predicted Na reversal potential of 90 mV with the published external saline, while the erroneous published saline has an undefined ENa. Because C. elegans lacks voltage-gated Na channels, this difference in salines may have little or no effect on recordings of voltage-gated currents. It may, however, affect measurements of currents carried by ligand-gated currents and currents carried by DEG/ENaC channels. We apologize for any inconvenience this error may have caused. 1. Goodman, M.B., Hall, D.H., Avery, L., and Lockery, S.R. (1998) Active Currents Regulate Sensitivity and Dynamic Range in C. elegans Neurons. Neuron 20:763-772. 2. Lockery, S.R. and Goodman, M.B. (1998) Tight-seal whole-cell patch clamping of C. elegans neurons. Methods in Enzymology 295:201-217.

Zhang, Gaotian et al. (2021) International Worm Meeting "The genetic architectures of gene expression variation in wild C. elegans"

Hahnel, Steffen, Andersen, Erik, Roberto, Nicole, Zhang, Gaotian, Lee, Daehan

[International Worm Meeting, 2021]

Natural variation in gene expression is a major source of phenotypic diversity among individuals. Genetic variants underlying gene expression differences are known as expression quantitative trait loci (eQTL). In Caenorhabditis elegans, both local eQTL (located close to the genes they influence) and distant eQTL (located farther away from the genes they influence) have been identified using linkage analysis from a panel of recombinant inbred advanced intercross lines (RIAILs) between the laboratory reference strain, N2, and a wild strain, CB4856. However, the eQTL detected using RIAILs were limited to genetic variants between N2 and CB4856. Here, we investigated the natural variation in gene expression of 205 genetically distinct C. elegans wild strains by performing RNA-sequencing on synchronized young adult hermaphrodites. We obtained reliable expression of 25,896 protein-coding transcripts (16,106 genes). We used genome-wide association (GWA) analysis to identify 3,342 local eQTL for 3,342 transcripts (2,777 genes), and 2,835 distant eQTL for 2,206 transcripts (2,082 genes). We found that most of the narrow-sense heritability for transcript expression variation is explained by detected eQTL. Of the 2,835 distant eQTL, 1,670 eQTL significantly clustered in 54 hotspots across the C. elegans genome. We will further explore causal genes underlying these distant eQTL hotspots and their functions. Additionally, we applied mediation analysis to the eQTL data and other organism-level quantitative traits to elucidate the genetic effects on phenotypic variation mediated by gene expression. For instance, instead of relying on time-consuming investigations of rare genetic variants missed in GWA studies and prior knowledge of the trait as published in our study of C. elegans responses to benzimidazoles (Hahnel et al. 2018), the significant mediating effect of the expression of ben-1, the causal gene, was quickly and successfully identified by mediation analysis. Our results suggest that mediation analysis using expression data facilitates identification of causal genes in GWA studies in C. elegans.

TM Morse et al. (1999) International C. elegans Meeting "Time-series analysis of pirouettes in C. elegans chemotaxis."

TM Morse, JT Pierce, SR Lockery, ML Moravec, TC Ferree, SE Owens, AB Bates

[International C. elegans Meeting, 1999]

Chemotaxis in C. elegans involves a series of abrupt turns (pirouettes) triggered by movement down a gradient of chemical attractant * . Analysis of the time series of concentration change experienced by a worm, together with its pirouette record, suggests a three-stage chemotaxis mechanism in which chemical concentration (C(t)) is differentiated (dC(t)/dt), smoothed (q(t)), and converted into pirouette probability by a nonlinear function (P(q[t])). To test the plausibility of this mechanism, we constructed a computer model in which the smoothing filter and the nonlinearity were estimated from the time series of dC(t)/dt and the pirouette records of real worms. Pirouettes were modeled by sampling randomly, via a Poisson process with probability P(q[t]), from the distribution of direction changes associated with pirouettes in real worms. The average chemotaxis index (time-average of normalized C(t)) of model worms (0.47 +/- 0.05 SD, n = 2000) closely matched the average chemotaxis index of real worms (0.45 +/- 0.04 SD, n = 45), indicating that the three-stage mechanism is quantitatively sufficient to account for C. elegans chemotaxis. To determine the form of the smoothing filter and nonlinearity directly, we have devised a new behavioral assay that subjects unrestrained worms to negative-going impulses in dC/dt as they swim across a sharp border between high and low concentrations of attractant. Preliminary results show that immediately after a border crossing, large impulses make P(t)~= 1, while small impulses make 0 t) >< C. elegans . * Pierce, J.T., and Lockery, S.R., J. Neurosci. (Submitted)

Sreekanth Chalasani et al. (2007) International Worm Meeting "Functional analysis of a neural circuit controlling turning behavior in Caenorhabditis elegans."

Cornelia Bargmann, Jesse Gray, Makoto Tsunozaki, Sreekanth Chalasani, Nikolas Chronis

[International Worm Meeting, 2007]

C. elegans increase its frequency of reversals and turns (jointly termed pirouettes, Pierce-Shimomura et al 1999) after removal of a food stimulus. The AWC and ASK sensory neurons and the AIB interneurons stimulate pirouettes immediately after removal from food, while the AIY and AIA interneurons inhibit pirouettes (Wakabayashi et al 2004, Gray et al 2005). We have found that the sensory neuron AWC releases two neurotransmitters (glutamate and a neuropeptide, NLP-1) when the worm is removed from food. The released glutamate acts to activate AIB and inhibit AIY, promoting reversals. Strains with different reversal frequencies can be generated by manipulating the level of glutamate receptors on interneurons AIB and AIY. Decreasing receptor expression leads to fewer reversals, and increasing receptor expression results in more reversals than in wild-type. The AWC released neuropeptide NLP-1 serves to reduce reversals, suggesting that reversal frequencies are regulated by at least two opposing signaling systems. Consistent with behavioral responses, AWC and AIB respond (by increasing calcium concentration) to removal of stimulus. We plan to extend the imaging studies to other neurons in the circuit. These results provide a plausible molecular explanation that links neurotransmitters, their receptors, and neuronal circuitry to generate behavior. References: Gray, J.M., Hill, J.J., and Bargmann, C.I. (2005). A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191. Pierce-Shimomura, J.T., Morse, T.M., and Lockery, S.R. (1999). The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis. J. Neurosci 19, 9557-9569. Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111.

Sreekanth H Chalasani et al. (2005) International Worm Meeting "Molecular analysis of a neural circuit for navigation in Caenorhabditis elegans."

Sreekanth H Chalasani, Nikolas Chronis, Jesse M Gray, Cornelia I Bargmann

[International Worm Meeting, 2005]

Navigation in C.elegans is achieved by sustained forward movement that is interrupted with reversals and turns (jointly termed pirouettes, Pierce-Shimomura et al 1999). We are interested in the neural circuit that controls the frequency of reversals and turns during exploratory behavior. After worms are taken off bacterial food, they exhibit an initial local search with a high frequency of pirouettes. The AWC and ASK sensory neurons and the AIB interneurons stimulate pirouettes immediately after removal from food, while the AIY interneurons inhibit pirouettes. (Tsalik and Hobert 2003, Wakabayashi et al 2004, Gray et al 2005). How is activity transmitted through this neuronal circuit? The neurotransmitters glutamate and dopamine regulate turning frequency (Hills et al 2004). We found that the vesicular glutamate transporter EAT-4 is essential for the generation of pirouettes after removal from food. Using cell-specific rescue of eat-4 mutants, we show that both AWC and ASK sensory neurons can release glutamate to stimulate pirouettes. The released glutamate appears to be sensed by a glutamate-gated chloride channel (GLC-3) that inhibits the AIY interneurons, and the glutamate-gated cation channel GLR-1, which stimulates the AIB interneurons. These results provide a plausible molecular explanation that links neurotransmitters, their receptors, and neuronal circuitry to generate behavior. We are currently attempting to image neuronal activity in these neurons using genetically encoded calcium sensors. References: Gray, J.M., Hill, J.J., and Bargmann, C.I. (2005). A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191. Hills, T., Brockie, P.J., and Maricq, A.V. (2004). Dopamine and glutamate control area-restricted search behavior in Caenorhabditis elegans. J. Neurosci 24, 1217-1225. Pierce-Shimomura, T., Morse, T.M., and Lockery, S.R. (1999). The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis. J. Neurosci 19, 9557-9569. Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111.

Stephen R Wicks et al. (2001) International C. elegans Meeting "A snip-SNP map of the C. elegans genome: Applications to positional cloning."

Robert H Waterston, Ronald H A Plasterk, Stephen R Wicks, Warren R Gish, Raymond T Yeh

[International C. elegans Meeting, 2001]

Single nucleotide polymorphisms (SNPs) are valuable genetic markers of human disease. They also comprise the highest potential density marker set available for mapping experimentally derived mutations in model organisms such as C. elegans . To facilitate the positional cloning of mutations we have identified polymorphisms in CB4856, an isolate from a Hawaiian isle that shows a uniformly high density of polymorphisms compared to the reference Bristol N2 strain. Based on 5.4 Mbp of aligned sequences, we predict 6222 polymorphisms. Furthermore, 3457 of these markers modify restriction enzyme recognition sites (snip-SNPs) and are therefore easily detected as RFLPs. Of these, we have experimentally confirmed 493 by restriction digest to produce a snip-SNP map of the worm genome (ref 1). A mapping strategy using snip-SNPs and bulked segregant analysis (BSA, ref 2) is outlined. CB4856 is crossed into a mutant strain, and exclusion of CB4856 alleles of a subset of snip-SNPs in mutant progeny is assessed with BSA. The proximity of a linked marker to the mutation is estimated by the relative proportion of each form of the biallelic marker in populations of wildtype and mutant genomes. This step bounds the mutation between flanking snip-SNPs. These flanking markers can be used to detect recombination in individual animals, and only recombinant strains need be phenotyped. By mapping the recombination points in individual animals, it is possible to rapidly zoom in on the site of a mutation. The advantages and limitations of this approach will be discussed. References: 1) Wicks, S.R., Yeh, R.T., Gish, W.R., Waterston, R.H. & Plasterk, R.H.A. (in press) Rapid gene mapping in C. elegans using a high density polymorphism map. Nature Genetics . 2) Michelmore, R.W., Paran, I. & Kesseli, R.V. Identification of markers linked to disease-resistance genes by bulked segregant analysis: a rapid method to detect markers in specific genomic regions by using segregating populations. Proc. Natl. Acad. Sci. USA 88, 9828-9832. (1991).