Lack of correlation between parasite burden and key weight metrics in poultry infected with intestinal ascarids.

Ascaridia galli and Ascaridia dissimilis are the most common and economically impactful nematode parasites of commercial poultry. These infections rarely cause clinical disease, but reduction in feed conversion efficiency is detected. To determine if feed conversion efficiency reductions correlate with any physiological measures independent of clinical disease, we determined if ascarid infections correlate with changes in the weights of the small intestine, liver, or total animal weight (quantitative measures of animal health). No correlation between parasite burden and these metrics were observed, supporting the concept that feed conversion is the only production metric impacted by ascarid infections.

Direct inference of the distribution of fitness effects of spontaneous mutations from recombinant inbred C. elegans mutation accumulation lines.

The distribution of fitness effects (DFE) of new mutations plays a central role in evolutionary biology. Estimates of the DFE from experimental Mutation Accumulation (MA) lines are compromised by the complete linkage disequilibrium (LD) between mutations in different lines. To reduce LD, we constructed two sets of recombinant inbred lines from a cross of two C. elegans MA lines. One set of lines ("RIAILs") was intercrossed for ten generations prior to ten generations of selfing; the second set of lines ("RILs") omitted the intercrossing. Residual LD in the RIAILs is much less than in the RILs, which affects the inferred DFE when the sets of lines are analyzed separately. The best-fit model estimated from all lines (RIAILs + RILs) infers a large fraction of mutations with positive effects (∼40%); models that constrain mutations to have negative effects fit much worse. The conclusion is the same using only the RILs. For the RIAILs, however, models that constrain mutations to have negative effects fit nearly as well as models that allow positive effects. When mutations in high LD are pooled into haplotypes, the inferred DFE becomes increasingly negative-skewed and leptokurtic. We conclude that the conventional wisdom - most mutations have effects near zero, a handful of mutations have effects that are substantially negative and mutations with positive effects are very rare - is likely correct, and that unless it can be shown otherwise, estimates of the DFE that infer a substantial fraction of mutations with positive effects are likely confounded by LD.

Quantitative tests of albendazole resistance in Caenorhabditis elegans beta-tubulin mutants.

Benzimidazole (BZ) anthelmintics are among the most important treatments for parasitic nematode infections in the developing world. Widespread BZ resistance in veterinary parasites and emerging resistance in human parasites raise major concerns for the continued use of BZs. Knowledge of the mechanisms of resistance is necessary to make informed treatment decisions and circumvent resistance. Benzimidazole resistance has traditionally been associated with mutations and natural variants in the C. elegans beta-tubulin gene ben-1 and orthologs in parasitic species. However, variants in ben-1 alone do not explain the differences in BZ responses across parasite populations. Here, we examined the roles of five C. elegans beta-tubulin genes (tbb-1, mec-7, tbb-4, ben-1, and tbb-6) in the BZ response as well as to determine if another beta-tubulin acts redundantly with ben-1. We generated C. elegans strains with a loss of each beta-tubulin gene, as well as strains with a loss of tbb-1, mec-7, tbb-4, or tbb-6 in a genetic background that also lacks ben-1. We found that the loss of ben-1 conferred the maximum level of resistance following exposure to a single concentration of albendazole, and the loss of a second beta-tubulin gene did not alter the level of resistance. However, additional traits other than larval development could be affected by the loss of additional beta-tubulins, and the roles of other beta-tubulin genes might be revealed at different albendazole concentrations. Therefore, further work is needed to fully define the possible roles of other beta-tubulin genes in the BZ response.

Glial expression of a steroidogenic enzyme underlies natural variation in hitchhiking behavior.

Phoresy is an interspecies interaction that facilitates spatial dispersal by attaching to a more mobile species. Hitchhiking species have evolved specific traits for physical contact and successful phoresy, but the regulatory mechanisms involved in such traits and their evolution are largely unexplored. The nematode Caenorhabditis elegans displays a hitchhiking behavior known as nictation during its stress-induced developmental stage. Dauer-specific nictation behavior has an important role in natural C. elegans populations, which experience boom-and-bust population dynamics. In this study, we investigated the nictation behavior of 137 wild C. elegans strains sampled throughout the world. We identified species-wide natural variation in nictation and performed a genome-wide association mapping. We show that the variants in the promoter of nta-1, encoding a putative steroidogenic enzyme, underlie differences in nictation. This difference is due to the changes in nta-1 expression in glial cells, which implies that glial steroid metabolism regulates phoretic behavior. Population genetic analysis and geographic distribution patterns suggest that balancing selection maintained two nta-1 haplotypes that existed in ancestral C. elegans populations. Our findings contribute to further understanding of the molecular mechanism of species interaction and the maintenance of genetic diversity within natural populations.

Cyprocide selectively kills nematodes via cytochrome P450 bioactivation.

Left unchecked, plant-parasitic nematodes have the potential to devastate crops globally. Highly effective but non-selective nematicides are justifiably being phased-out, leaving farmers with limited options for managing nematode infestation. Here, we report our discovery of a 1,3,4-oxadiazole thioether scaffold called Cyprocide that selectively kills nematodes including diverse species of plant-parasitic nematodes. Cyprocide is bioactivated into a lethal reactive electrophilic metabolite by specific nematode cytochrome P450 enzymes. Cyprocide fails to kill organisms beyond nematodes, suggesting that the targeted lethality of this pro-nematicide derives from P450 substrate selectivity. Our findings demonstrate that Cyprocide is a selective nematicidal scaffold with broad-spectrum activity that holds the potential to help safeguard our global food supply.

Genetic variants that modify neuroendocrine gene expression and foraging behavior of C. elegans.

The molecular mechanisms underlying diversity in animal behavior are not well understood. A major experimental challenge is determining the contribution of genetic variants that affect neuronal gene expression to differences in behavioral traits. In Caenorhabditis elegans, the neuroendocrine transforming growth factor-ß ligand, DAF-7, regulates diverse behavioral responses to bacterial food and pathogens. The dynamic neuron-specific expression of daf-7 is modulated by environmental and endogenous bacteria-derived cues. Here, we investigated natural variation in the expression of daf-7 from the ASJ pair of chemosensory neurons. We identified common genetic variants in gap-2, encoding a Ras guanosine triphosphatase (GTPase)-activating protein homologous to mammalian synaptic Ras GTPase-activating protein, which modify daf-7 expression cell nonautonomously and promote exploratory foraging behavior in a partially DAF-7-dependent manner. Our data connect natural variation in neuron-specific gene expression to differences in behavior and suggest that genetic variation in neuroendocrine signaling pathways mediating host-microbe interactions may give rise to diversity in animal behavior.

Natural variation in protein kinase D modifies alcohol sensitivity in Caenorhabditis elegans.

Differences in naïve alcohol sensitivity between individuals are a strong predictor of later life alcohol use disorders (AUD). However, the genetic bases for alcohol sensitivity (beyond ethanol metabolism) and pharmacological approaches to modulate alcohol sensitivity remain poorly understood. We used a high-throughput behavioral screen to measure acute behavioral sensitivity to alcohol, a model of intoxication, in a genetically diverse set of over 150 wild strains of the nematode Caenorhabditis elegans. We performed a genome-wide association study to identify loci that underlie natural variation in alcohol sensitivity. We identified five quantitative trait loci (QTL) and further show that variants in the C. elegans ortholog of protein kinase D, dkf-2, likely underlie the chromosome V QTL. We found that resistance to intoxication was conferred by dkf-2 loss-of-function mutations as well as partly by a PKD inhibitor in a dkf-2-dependent manner. Protein kinase D might represent a conserved, druggable target to modify alcohol sensitivity with application towards AUD.

Quantifying the fitness effects of resistance alleles with and without anthelmintic selection pressure using Caenorhabditis elegans.

Albendazole (a benzimidazole) and ivermectin (a macrocyclic lactone) are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channels (GluCls), but it is unknown whether GluCl genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans, we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of-function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required the loss of two GluCl genes (avr-14 and avr-15). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole conditions and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C. elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance.

Gene duplication and evolutionary plasticity of lin-12/Notch gene function in Caenorhabditis.

Gene duplication is an important substrate for the evolution of new gene functions, but the impacts of gene duplicates on their own activities and on the developmental networks in which they act are poorly understood. Here, we use a natural experiment of lin-12/Notch gene duplication within the nematode genus Caenorhabditis, combined with characterization of loss- and gain-of-function mutations, to uncover functional distinctions between the duplicate genes in 1 species (Caenorhabditis briggsae) and their single-copy ortholog in Caenorhabditis elegans. First, using improved genomic sequence and gene model characterization, we confirm that the C. briggsae genome includes 2 complete lin-12 genes, whereas most other genes encoding proteins that participate in the LIN-12 signaling pathway retain a one-to-one orthology with C. elegans. We use CRISPR-mediated genome editing to introduce alleles predicted to cause gain-of-function (gf) or loss-of-function (lf) into each C. briggsae gene and find that the gf mutations uncover functional distinctions not apparent from the lf alleles. Specifically, Cbr-lin-12.1(gf), but not Cbr-lin-12.2(gf), causes developmental defects similar to those observed in Cel-lin-12(gf). In contrast to Cel-lin-12(gf), however, the Cbr-lin-12.1(gf) alleles do not cause dominant phenotypes as compared to the wild type, and the mutant phenotype is observed only when 2 gf alleles are present. Our results demonstrate that gene duplicates can exhibit differential capacities to compensate for each other and to interfere with normal development, and uncover coincident gene duplication and evolution of developmental sensitivity to LIN-12/Notch activity.

Quantitative tests of albendazole resistance in beta-tubulin mutants.

Benzimidazole (BZ) anthelmintics are among the most important treatments for parasitic nematode infections in the developing world. Widespread BZ resistance in veterinary parasites and emerging resistance in human parasites raise major concerns for the continued use of BZs. Knowledge of the mechanisms of resistance is necessary to make informed treatment decisions and circumvent resistance. Benzimidazole resistance has traditionally been associated with mutations and natural variants in the C. elegans beta-tubulin gene ben-1 and orthologs in parasitic species. However, variants in ben-1 alone do not explain the differences in BZ responses across parasite populations. Here, we examine the roles of five C. elegans beta-tubulin genes (tbb-1, mec-7, tbb-4, ben-1, and tbb-6) to identify the role each gene plays in BZ response. We generated C. elegans strains with a loss of each beta-tubulin gene, as well as strains with a loss of tbb-1, mec-7, tbb-4, or tbb-6 in a genetic background that also lacks ben-1 to test beta-tubulin redundancy in BZ response. We found that only the individual loss of ben-1 conferred a substantial level of BZ resistance, although the loss of tbb-1 was found to confer a small benefit in the presence of albendazole (ABZ). The loss of ben-1 was found to confer an almost complete rescue of animal development in the presence of 30 µM ABZ, likely explaining why no additive effects caused by the loss of a second beta-tubulin were observed. We demonstrate that ben-1 is the only beta-tubulin gene in C. elegans where loss confers substantial BZ resistance.

Getting around the roundworms: Identifying knowledge gaps and research priorities for the ascarids.

The ascarids are a large group of parasitic nematodes that infect a wide range of animal species. In humans, they cause neglected diseases of poverty; many animal parasites also cause zoonotic infections in people. Control measures include hygiene and anthelmintic treatments, but they are not always appropriate or effective and this creates a continuing need to search for better ways to reduce the human, welfare and economic costs of these infections. To this end, Le Studium Institute of Advanced Studies organized a two-day conference to identify major gaps in our understanding of ascarid parasites with a view to setting research priorities that would allow for improved control. The participants identified several key areas for future focus, comprising of advances in genomic analysis and the use of model organisms, especially Caenorhabditis elegans, a more thorough appreciation of the complexity of host-parasite (and parasite-parasite) communications, a search for novel anthelmintic drugs and the development of effective vaccines. The participants agreed to try and maintain informal links in the future that could form the basis for collaborative projects, and to co-operate to organize future meetings and workshops to promote ascarid research.

Quantifying the fitness effects of resistance alleles with and without anthelmintic selection pressure using Caenorhabditis elegans.

Albendazole and ivermectin are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channel (GluCl) genes, but it is unknown whether these genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans, we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required loss of two GluCl genes (avr-14 and avr-15) or loss of three GluCl genes (avr-14, avr-15, and glc-1). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C. elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance.