Praziquantel inhibits Caenorhabditis elegans development and species-wide differences might be cct-8-dependent.

Anthelmintic drugs are used to treat parasitic roundworm and flatworm infections in humans and other animals. Caenorhabditis elegans is an established model to investigate anthelmintics used to treat roundworms. In this study, we use C. elegans to examine the mode of action and the mechanisms of resistance against the flatworm anthelmintic drug praziquantel (PZQ), used to treat trematode and cestode infections. We found that PZQ inhibited development and that this developmental delay varies by genetic background. Interestingly, both enantiomers of PZQ are equally effective against C. elegans, but the right-handed PZQ (R-PZQ) is most effective against schistosome infections. We conducted a genome-wide association mapping with 74 wild C. elegans strains to identify a region on chromosome IV that is correlated with differential PZQ susceptibility. Five candidate genes in this region: cct-8, znf-782, Y104H12D.4, Y104H12D.2, and cox-18, might underlie this variation. The gene cct-8, a subunit of the protein folding complex TRiC, has variation that causes a putative protein coding change (G226V), which is correlated with reduced developmental delay. Gene expression analysis suggests that this variant correlates with slightly increased expression of both cct-8 and hsp-70. Acute exposure to PZQ caused increased expression of hsp-70, indicating that altered TRiC function might be involved in PZQ responses. To test if this variant affects development upon exposure to PZQ, we used CRISPR-Cas9 genome editing to introduce the V226 allele into the N2 genetic background (G226) and the G226 allele into the JU775 genetic background (V226). These experiments revealed that this variant was not sufficient to explain the effects of PZQ on development. Nevertheless, this study shows that C. elegans can be used to study PZQ mode of action and resistance mechanisms. Additionally, we show that the TRiC complex requires further evaluation for PZQ responses in C. elegans.

Heritable Cas9-induced nonhomologous recombination in C. elegans.

Identification of the genetic basis of phenotypic variation within species remains challenging. In species with low recombination rates, such as Caenorhabditis elegans , genomic regions linked to a phenotype of interest by genetic mapping studies are often large, making it difficult to identify the specific genes and DNA sequence variants that underlie phenotypic differences. Here, we introduce a method that enables researchers to induce heritable targeted recombination in C. elegans with Cas9. We demonstrate that high rates of targeted nonhomologous recombination can be induced by Cas9 in a genomic region in which naturally occurring meiotic recombination events are exceedingly rare. We anticipate that Cas9-induced nonhomologous recombination (CINR) will greatly facilitate high-resolution genetic mapping in this species.

Cas9-induced nonhomologous recombination in C. elegans.

Identification of the genetic basis of phenotypic variation within species remains challenging. In species with low recombination rates, such as Caenorhabditis elegans , genomic regions linked to a phenotype of interest by genetic mapping studies are often large, making it difficult to identify the specific genes and DNA sequence variants that underlie phenotypic differences. Here, we introduce a method that enables researchers to induce targeted recombination in C. elegans with Cas9. We demonstrate that high rates of targeted recombination can be induced by Cas9 in a genomic region in which naturally occurring recombination events are exceedingly rare. We anticipate that Cas9-induced nonhomologous recombination (CINR) will greatly facilitate high-resolution genetic mapping in this species.

C. elegans as a model for inter-individual variation in metabolism.

Individuals can exhibit differences in metabolism that are caused by the interplay of genetic background, nutritional input, microbiota and other environmental factors1-4. It is difficult to connect differences in metabolism to genomic variation and derive underlying molecular mechanisms in humans, owing to differences in diet and lifestyle, among others. Here we use the nematode Caenorhabditis elegans as a model to study inter-individual variation in metabolism. By comparing three wild strains and the commonly used N2 laboratory strain, we find differences in the abundances of both known metabolites and those that have not to our knowledge been previously described. The latter metabolites include conjugates between 3-hydroxypropionate (3HP) and several amino acids (3HP-AAs), which are much higher in abundance in one of the wild strains. 3HP is an intermediate in the propionate shunt pathway, which is activated when flux through the canonical, vitamin-B12-dependent propionate breakdown pathway is perturbed5. We show that increased accumulation of 3HP-AAs is caused by genetic variation in HPHD-1, for which 3HP is a substrate. Our results suggest that the production of 3HP-AAs represents a 'shunt-within-a-shunt' pathway to accommodate a reduction-of-function allele in hphd-1. This study provides a step towards the development of metabolic network models that capture individual-specific differences of metabolism and more closely represent the diversity that is found in entire species.

Evaluating the power and limitations of genome-wide association studies in Caenorhabditis elegans.

Quantitative genetics in Caenorhabditis elegans seeks to identify naturally segregating genetic variants that underlie complex traits. Genome-wide association studies scan the genome for individual genetic variants that are significantly correlated with phenotypic variation in a population, or quantitative trait loci. Genome-wide association studies are a popular choice for quantitative genetic analyses because the quantitative trait loci that are discovered segregate in natural populations. Despite numerous successful mapping experiments, the empirical performance of genome-wide association study has not, to date, been formally evaluated in C. elegans. We developed an open-source genome-wide association study pipeline called NemaScan and used a simulation-based approach to provide benchmarks of mapping performance in collections of wild C. elegans strains. Simulated trait heritability and complexity determined the spectrum of quantitative trait loci detected by genome-wide association studies. Power to detect smaller-effect quantitative trait loci increased with the number of strains sampled from the C. elegans Natural Diversity Resource. Population structure was a major driver of variation in mapping performance, with populations shaped by recent selection exhibiting significantly lower false discovery rates than populations composed of more divergent strains. We also recapitulated previous genome-wide association studies of experimentally validated quantitative trait variants. Our simulation-based evaluation of performance provides the community with critical context to pursue quantitative genetic studies using the C. elegans Natural Diversity Resource to elucidate the genetic basis of complex traits in C. elegans natural populations.

Local adaptation and spatiotemporal patterns of genetic diversity revealed by repeated sampling of Caenorhabditis elegans across the Hawaiian Islands.

The nematode Caenorhabditis elegans is among the most widely studied organisms, but relatively little is known about its natural ecology. Genetic diversity is low across much of the globe but high in the Hawaiian Islands and across the Pacific Rim. To characterize the niche and genetic diversity of C. elegans on the Hawaiian Islands and to explore how genetic diversity might be influenced by local adaptation, we repeatedly sampled nematodes over a three-year period, measured various environmental parameters at each sampling site, and whole-genome sequenced the C. elegans isolates that we identified. We found that the typical Hawaiian C. elegans niche comprises moderately moist native forests at high elevations (500-1,500 m) where ambient air temperatures are cool (15-20°C). Compared to other Caenorhabditis species found on the Hawaiian Islands (e.g., Caenorhabditis briggsae and Caenorhabditis tropicalis), we found that C. elegans were enriched in native habitats. We measured levels of genetic diversity and differentiation among Hawaiian C. elegans and found evidence of seven genetically distinct groups distributed across the islands. Then, we scanned these genomes for signatures of local adaptation and identified 18 distinct regions that overlap with hyper-divergent regions, which may be maintained by balancing selection and are enriched for genes related to environmental sensing, xenobiotic detoxification, and pathogen resistance. These results provide strong evidence of local adaptation among Hawaiian C. elegans and contribute to our understanding of the forces that shape genetic diversity on the most remote volcanic archipelago in the world.

The distribution of mutational effects on fitness in Caenorhabditis elegans inferred from standing genetic variation.

The distribution of fitness effects (DFE) for new mutations is one of the most theoretically important but difficult to estimate properties in population genetics. A crucial challenge to inferring the DFE from natural genetic variation is the sensitivity of the site frequency spectrum to factors like population size change, population substructure, genome structure, and nonrandom mating. Although inference methods aim to control for population size changes, the influence of nonrandom mating remains incompletely understood, despite being a common feature of many species. We report the DFE estimated from 326 genomes of Caenorhabditis elegans, a nematode roundworm with a high rate of self-fertilization. We evaluate the robustness of DFE inferences using simulated data that mimics the genomic structure and reproductive life history of C. elegans. Our observations demonstrate how the combined influence of self-fertilization, genome structure, and natural selection on linked sites can conspire to compromise estimates of the DFE from extant polymorphisms with existing methods. These factors together tend to bias inferences toward weakly deleterious mutations, making it challenging to have full confidence in the inferred DFE of new mutations as deduced from standing genetic variation in species like C. elegans. Improved methods for inferring the DFE are needed to appropriately handle strong linked selection and selfing. These results highlight the importance of understanding the combined effects of processes that can bias our interpretations of evolution in natural populations.

Balancing selection maintains hyper-divergent haplotypes in Caenorhabditis elegans.

Across diverse taxa, selfing species have evolved independently from outcrossing species thousands of times. The transition from outcrossing to selfing decreases the effective population size, effective recombination rate and heterozygosity within a species. These changes lead to a reduction in genetic diversity, and therefore adaptive potential, by intensifying the effects of random genetic drift and linked selection. Within the nematode genus Caenorhabditis, selfing has evolved at least three times, and all three species, including the model organism Caenorhabditis elegans, show substantially reduced genetic diversity relative to outcrossing species. Selfing and outcrossing Caenorhabditis species are often found in the same niches, but we still do not know how selfing species with limited genetic diversity can adapt to these environments. Here, we examine the whole-genome sequences from 609 wild C. elegans strains isolated worldwide and show that genetic variation is concentrated in punctuated hyper-divergent regions that cover 20% of the C. elegans reference genome. These regions are enriched in environmental response genes that mediate sensory perception, pathogen response and xenobiotic stress response. Population genomic evidence suggests that genetic diversity in these regions has been maintained by long-term balancing selection. Using long-read genome assemblies for 15 wild strains, we show that hyper-divergent haplotypes contain unique sets of genes and show levels of divergence comparable to levels found between Caenorhabditis species that diverged millions of years ago. These results provide an example of how species can avoid the evolutionary dead end associated with selfing.

Whole-organism eQTL mapping at cellular resolution with single-cell sequencing.

Genetic regulation of gene expression underlies variation in disease risk and other complex traits. The effect of expression quantitative trait loci (eQTLs) varies across cell types; however, the complexity of mammalian tissues makes studying cell-type eQTLs highly challenging. We developed a novel approach in the model nematode Caenorhabditis elegans that uses single-cell RNA sequencing to map eQTLs at cellular resolution in a single one-pot experiment. We mapped eQTLs across cell types in an extremely large population of genetically distinct C. elegans individuals. We found cell-type-specific trans eQTL hotspots that affect the expression of core pathways in the relevant cell types. Finally, we found single-cell-specific eQTL effects in the nervous system, including an eQTL with opposite effects in two individual neurons. Our results show that eQTL effects can be specific down to the level of single cells.

DNA sequences that differ between individuals often change the activation pattern of genes. That is, they change how, when, or why genes switch on and off. We call such DNA sequences 'expression quantitative trait loci', or eQTLs for short. Many of these eQTLs affect biological traits, but their effects are not always easy to predict. In fact, these effects can change with time, with different stress levels, and even in different types of cells. This makes studying eQTLs challenging, especially in organisms with many different types of cells. Standard methods of studying eQTLs involve separately measuring which genes switch on or off under every condition and in each cell. However, a technology called single cell sequencing makes it possible to profile many cells simultaneously, determining which genes are switched on or off in each one. Applying this technology to eQTL discovery could make a challenging problem solvable with a straightforward experiment. To test this idea, Ben-David et al. worked with the nematode worm Caenorhabditis elegans, a frequently-used experimental animal which has a small number of cells with well-defined types. The experiment included tens of thousands of cells from tens of thousands of genetically distinct worms. Using single cell sequencing, Ben-David et al. were able to find eQTLs across all the different cell types in the worms. These included many eQTLs already identified using traditional approaches, confirming that the new method worked. To understand the effects of some of these eQTLs in more detail, Ben-David et al. took a closer look at the cells of the nervous system. This revealed that eQTL effects not only differ between cell types, but also between individual cells. In one example, an eQTL changed the expression of the same gene in opposite directions in two different nerve cells. There is great interest in studying eQTLs because they provide a common mechanism by which changes in DNA can affect biological traits, including diseases. These experiments highlight the need to compare eQTLs in all conditions and tissues of interest, and the new technique provides a simpler way to do so. As single-cell technology matures and enables profiling of larger numbers of cells, it should become possible to analyze more complex organisms. This could one day include mammals.

Natural variation in the sequestosome-related gene, sqst-5, underlies zinc homeostasis in Caenorhabditis elegans.

Zinc is an essential trace element that acts as a co-factor for many enzymes and transcription factors required for cellular growth and development. Altering intracellular zinc levels can produce dramatic effects ranging from cell proliferation to cell death. To avoid such fates, cells have evolved mechanisms to handle both an excess and a deficiency of zinc. Zinc homeostasis is largely maintained via zinc transporters, permeable channels, and other zinc-binding proteins. Variation in these proteins might affect their ability to interact with zinc, leading to either increased sensitivity or resistance to natural zinc fluctuations in the environment. We can leverage the power of the roundworm nematode Caenorhabditis elegans as a tractable metazoan model for quantitative genetics to identify genes that could underlie variation in responses to zinc. We found that the laboratory-adapted strain (N2) is resistant and a natural isolate from Hawaii (CB4856) is sensitive to micromolar amounts of exogenous zinc supplementation. Using a panel of recombinant inbred lines, we identified two large-effect quantitative trait loci (QTL) on the left arm of chromosome III and the center of chromosome V that are associated with zinc responses. We validated and refined both QTL using near-isogenic lines (NILs) and identified a naturally occurring deletion in sqst-5, a sequestosome-related gene, that is associated with resistance to high exogenous zinc. We found that this deletion is relatively common across strains within the species and that variation in sqst-5 is associated with zinc resistance. Our results offer a possible mechanism for how organisms can respond to naturally high levels of zinc in the environment and how zinc homeostasis varies among individuals.

Natural variation in a glucuronosyltransferase modulates propionate sensitivity in a C. elegans propionic acidemia model.

Mutations in human metabolic genes can lead to rare diseases known as inborn errors of human metabolism. For instance, patients with loss-of-function mutations in either subunit of propionyl-CoA carboxylase suffer from propionic acidemia because they cannot catabolize propionate, leading to its harmful accumulation. Both the penetrance and expressivity of metabolic disorders can be modulated by genetic background. However, modifiers of these diseases are difficult to identify because of the lack of statistical power for rare diseases in human genetics. Here, we use a model of propionic acidemia in the nematode Caenorhabditis elegans to identify genetic modifiers of propionate sensitivity. Using genome-wide association (GWA) mapping across wild strains, we identify several genomic regions correlated with reduced propionate sensitivity. We find that natural variation in the putative glucuronosyltransferase GLCT-3, a homolog of human B3GAT, partly explains differences in propionate sensitivity in one of these genomic intervals. We demonstrate that loss-of-function alleles in glct-3 render the animals less sensitive to propionate. Additionally, we find that C. elegans has an expansion of the glct gene family, suggesting that the number of members of this family could influence sensitivity to excess propionate. Our findings demonstrate that natural variation in genes that are not directly associated with propionate breakdown can modulate propionate sensitivity. Our study provides a framework for using C. elegans to characterize the contributions of genetic background in models of human inborn errors in metabolism.

Deep sampling of Hawaiian Caenorhabditis elegans reveals high genetic diversity and admixture with global populations.

Hawaiian isolates of the nematode species Caenorhabditis elegans have long been known to harbor genetic diversity greater than the rest of the worldwide population, but this observation was supported by only a small number of wild strains. To better characterize the niche and genetic diversity of Hawaiian C. elegans and other Caenorhabditis species, we sampled different substrates and niches across the Hawaiian islands. We identified hundreds of new Caenorhabditis strains from known species and a new species, Caenorhabditis oiwi. Hawaiian C. elegans are found in cooler climates at high elevations but are not associated with any specific substrate, as compared to other Caenorhabditis species. Surprisingly, admixture analysis revealed evidence of shared ancestry between some Hawaiian and non-Hawaiian C. elegans strains. We suggest that the deep diversity we observed in Hawaii might represent patterns of ancestral genetic diversity in the C. elegans species before human influence.

Tightly linked antagonistic-effect loci underlie polygenic phenotypic variation in C. elegans.

Recent work has provided strong empirical support for the classic polygenic model for trait variation. Population-based findings suggest that most regions of genome harbor variation affecting most traits. Here, we use the approach of experimental genetics to show that, indeed, most genomic regions carry variants with detectable effects on growth and reproduction in Caenorhabditis elegans populations sensitized by nickel stress. Nine of 15 adjacent intervals on the X chromosome, each encompassing ∼0.001 of the genome, have significant effects when tested individually in near-isogenic lines (NILs). These intervals have effects that are similar in magnitude to those of genome-wide significant loci that we mapped in a panel of recombinant inbred advanced intercross lines (RIAILs). If NIL-like effects were randomly distributed across the genome, the RIAILs would exhibit phenotypic variance that far exceeds the observed variance. However, the NIL intervals are arranged in a pattern that significantly reduces phenotypic variance relative to a random arrangement; adjacent intervals antagonize one another, cancelling each other's effects. Contrary to the expectation of small additive effects, our findings point to large-effect variants whose effects are masked by epistasis or linkage disequilibrium between alleles of opposing effect.

Selection and gene flow shape niche-associated variation in pheromone response.

From quorum sensing in bacteria to pheromone signalling in social insects, chemical communication mediates interactions among individuals in local populations. In Caenorhabditis elegans, ascaroside pheromones can dictate local population density; high levels of pheromones inhibit the reproductive maturation of individuals. Little is known about how natural genetic diversity affects the pheromone responses of individuals from diverse habitats. Here, we show that a niche-associated variation in pheromone receptor genes contributes to natural differences in pheromone responses. We identified putative loss-of-function deletions that impair duplicated pheromone receptor genes (srg-36 and srg-37), which were previously shown to be lost in population-dense laboratory cultures. A common natural deletion in srg-37 arose recently from a single ancestral population that spread throughout the world; this deletion underlies reduced pheromone sensitivity across the global C. elegans population. We found that many local populations harbour individuals with a wild-type or a deletion allele of srg-37, suggesting that balancing selection has maintained the recent variation in this pheromone receptor gene. The two srg-37 genotypes are associated with niche diversity underlying boom-and-bust population dynamics. We hypothesize that human activities likely contributed to the gene flow and balancing selection of srg-37 variation through facilitating the migration of species and providing a favourable niche for the recently arisen srg-37 deletion.

Natural Variation and Genetic Determinants of Caenorhabditis elegans Sperm Size.

The diversity in sperm shape and size represents a powerful paradigm to understand how selection drives the evolutionary diversification of cell morphology. Experimental work on the sperm biology of the male-hermaphrodite nematode Caenorhabditis elegans has elucidated diverse factors important for sperm fertilization success, including the competitive superiority of larger sperm. Yet despite extensive research, the molecular mechanisms regulating C. elegans sperm size and the genetic basis underlying natural variation in sperm size remain unknown. To address these questions, we quantified male sperm size variation of a worldwide panel of 97 genetically distinct C. elegans strains, allowing us to uncover significant genetic variation in male sperm size. Aiming to characterize the molecular genetic basis of C. elegans male sperm size variation using a genome-wide association study, we did not detect any significant quantitative trait loci. We therefore focused on the genetic analysis of pronounced sperm size differences observed between recently diverged laboratory strains (N2 vs. LSJ1/2). Using mutants and quantitative complementation tests, we demonstrate that variation in the gene nurf-1 underlies the evolution of small sperm in the LSJ lineage. Given the previous discovery that this same nurf-1 variation was central for hermaphrodite laboratory adaptation, the evolution of reduced male sperm size in LSJ strains likely reflects a pleiotropic consequence. Together, our results provide a comprehensive quantification of natural variation in C. elegans sperm size and first insights into the genetic determinants of Caenorhabditis sperm size, pointing at an involvement of the NURF chromatin remodeling complex.

A Novel Gene Underlies Bleomycin-Response Variation in Caenorhabditis elegans.

Bleomycin is a powerful chemotherapeutic drug used to treat a variety of cancers. However, individual patients vary in their responses to bleomycin. The identification of genetic differences that underlie this response variation could improve treatment outcomes by tailoring bleomycin dosages to each patient. We used the model organism Caenorhabditis elegans to identify genetic determinants of bleomycin-response differences by performing linkage mapping on recombinants derived from a cross between the laboratory strain (N2) and a wild strain (CB4856). This approach identified a small genomic region on chromosome V that underlies bleomycin-response variation. Using near-isogenic lines, and strains with CRISPR-Cas9 mediated deletions and allele replacements, we discovered that a novel nematode-specific gene (scb-1) is required for bleomycin resistance. Although the mechanism by which this gene causes variation in bleomycin responses is unknown, we suggest that a rare variant present in the CB4856 strain might cause differences in the potential stress-response function of scb-1 between the N2 and CB4856 strains, thereby leading to differences in bleomycin resistance.

Natural variation in C. elegans arsenic toxicity is explained by differences in branched chain amino acid metabolism.

We find that variation in the dbt-1 gene underlies natural differences in Caenorhabditis elegans responses to the toxin arsenic. This gene encodes the E2 subunit of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, a core component of branched-chain amino acid (BCAA) metabolism. We causally linked a non-synonymous variant in the conserved lipoyl domain of DBT-1 to differential arsenic responses. Using targeted metabolomics and chemical supplementation, we demonstrate that differences in responses to arsenic are caused by variation in iso-branched chain fatty acids. Additionally, we show that levels of branched chain fatty acids in human cells are perturbed by arsenic treatment. This finding has broad implications for arsenic toxicity and for arsenic-focused chemotherapeutics across human populations. Our study implicates the BCKDH complex and BCAA metabolism in arsenic responses, demonstrating the power of C. elegans natural genetic diversity to identify novel mechanisms by which environmental toxins affect organismal physiology. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Extreme allelic heterogeneity at a Caenorhabditis elegans beta-tubulin locus explains natural resistance to benzimidazoles.

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 ß-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 found that a majority of the variation in ABZ resistance among wild C. elegans strains is caused by variation in the ß-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 ß-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 ß-tubulin gene. Furthermore, we used genome-editing to show that the most common parasitic nematode ß-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 ß-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.

Single-cell profiling screen identifies microtubule-dependent reduction of variability in signaling.

Populations of isogenic cells often respond coherently to signals, despite differences in protein abundance and cell state. Previously, we uncovered processes in the Saccharomyces cerevisiae pheromone response system (PRS) that reduced cell-to-cell variability in signal strength and cellular response. Here, we screened 1,141 non-essential genes to identify 50 "variability genes". Most had distinct, separable effects on strength and variability of the PRS, defining these quantities as genetically distinct "axes" of system behavior. Three genes affected cytoplasmic microtubule function: BIM1, GIM2, and GIM4 We used genetic and chemical perturbations to show that, without microtubules, PRS output is reduced but variability is unaffected, while, when microtubules are present but their function is perturbed, output is sometimes lowered, but its variability is always high. The increased variability caused by microtubule perturbations required the PRS MAP kinase Fus3 and a process at or upstream of Ste5, the membrane-localized scaffold to which Fus3 must bind to be activated. Visualization of Ste5 localization dynamics demonstrated that perturbing microtubules destabilized Ste5 at the membrane signaling site. The fact that such microtubule perturbations cause aberrant fate and polarity decisions in mammals suggests that microtubule-dependent signal stabilization might also operate throughout metazoans.

Discovery of genomic intervals that underlie nematode responses to benzimidazoles.

Parasitic nematodes impose a debilitating health and economic burden across much of the world. Nematode resistance to anthelmintic drugs threatens parasite control efforts in both human and veterinary medicine. Despite this threat, the genetic landscape of potential resistance mechanisms to these critical drugs remains largely unexplored. Here, we exploit natural variation in the model nematodes Caenorhabditis elegans and Caenorhabditis briggsae to discover quantitative trait loci (QTL) that control sensitivity to benzimidazoles widely used in human and animal medicine. High-throughput phenotyping of albendazole, fenbendazole, mebendazole, and thiabendazole responses in panels of recombinant lines led to the discovery of over 15 QTL in C. elegans and four QTL in C. briggsae associated with divergent responses to these anthelmintics. Many of these QTL are conserved across benzimidazole derivatives, but others show drug and dose specificity. We used near-isogenic lines to recapitulate and narrow the C. elegans albendazole QTL of largest effect and identified candidate variants correlated with the resistance phenotype. These QTL do not overlap with known benzimidazole target resistance genes from parasitic nematodes and present specific new leads for the discovery of novel mechanisms of nematode benzimidazole resistance. Analyses of orthologous genes reveal conservation of candidate benzimidazole resistance genes in medically important parasitic nematodes. These data provide a basis for extending these approaches to other anthelmintic drug classes and a pathway towards validating new markers for anthelmintic resistance that can be deployed to improve parasite disease control.