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 β-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 β-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 β-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 β-tubulin gene. Furthermore, we used genome-editing to show that the most common parasitic nematode β-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 β-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.

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.

32Benzimidazoles (BZ) are essential components of the limited chemotherapeutic arsenal available to 33 control the global burden of parasitic nematodes. The emerging threat of BZ resistance among nearly all 34 nematode species necessitates the development of novel strategies to identify genetic and molecular 35 mechanisms underlying this resistance. All detection of parasitic helminth resistance to BZ is focused on 36 the genotyping of three variant sites in the orthologs of the β-tubulin gene found to confer resistance in the 37 free-living nematode Caenorhabditis elegans. Because of the limitations of laboratory and field 38 experiments in parasitic nematodes, it is difficult to look beyond these three sites, and additional BZ 39 resistance is observed in the field. Here, we took an unbiased genome-wide mapping approach in the 40 free-living nematode species C. elegans to identify the genetic underpinnings of natural resistance to the 41 commonly used BZ, albendazole (ABZ). We found a wide range of natural variation in ABZ resistance in 42 natural C. elegans populations. In agreement with known mechanisms of BZ resistance in parasites, we 43 find that a majority of the variation in ABZ resistance among wild C. elegans strains is caused by variation 44 in the β-tubulin gene ben-1. This result shows empirically that resistance to ABZ naturally exists and 45 segregates within the C. elegans population, suggesting that selection in natural niches could enrich for 46 resistant alleles. We identified 25 distinct ben-1 alleles that are segregating at low frequencies within the 47 C. elegans population, including many novel molecular variants. Population genetic analyses indicate that 48ben-1 variation arose multiple times during the evolutionary history of C. elegans and provide evidence 49 that these alleles likely occurred recently because of local selective pressures. Additionally, we find 50 purifying selection at all five β-tubulin genes, despite predicted loss-of-function resistants variants in ben-1, 51indicating that BZ resistance in natural niches is a stronger selective pressure than loss of one β-tubulin 52 gene. Furthermore, we use genome-editing to show that the most common parasitic nematode β-tubulin 53 allele that confers BZ resistance, F200Y, confers resistance in C. elegans. Importantly, we identified a 54 novel genomic region that is correlated with ABZ resistance in the C. elegans population but independent 55 of ben-1 and the other β-tubulin loci, suggesting that there are multiple mechanisms underlying BZ 56 resistance. Taken together, our results establish a population-level resource of nematode natural diversity 57 as an important model for the study of mechanisms that give rise to BZ resistance. 58 . CC-BY 4.0 International license It is made available under a was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint (which . http://dx.doi.org/10.1101/372623 doi: bioRxiv preprint first p...

Parasitic nematodes cause a massive worldwide burden on human health along with a loss of livestock and agriculture productivity. Anthelmintics have been widely successful in treating parasitic nematodes. However, resistance is increasing, and little is known about the molecular and genetic causes of resistance for most of these drugs. The free-living roundworm Caenorhabditis elegans provides a tractable model to identify genes that underlie resistance. Unlike parasitic nematodes, C. elegans is easy to maintain in the laboratory, has a complete and well annotated genome, and has many genetic tools. Using a combination of wild isolates and a panel of recombinant inbred lines constructed from crosses of two genetically and phenotypically divergent strains, we identified three genomic regions on chromosome V that underlie natural differences in response to the macrocyclic lactone (ML) abamectin. One locus was identified previously and encodes an alpha subunit of a glutamate-gated chloride channel (glc-1). Here, we validate and narrow two novel loci using near-isogenic lines. Additionally, we generate a list of prioritized candidate genes identified in C. elegans and in the parasite Haemonchus contortus by comparison of ML resistance loci. These genes could represent previously unidentified resistance genes shared across nematode species and should be evaluated in the future. Our work highlights the advantages of using C. elegans as a model to better understand ML resistance in parasitic nematodes.

37 Recent efforts to understand the natural niche of the keystone model organism Caenorhabditis elegans have 38 suggested that this species is cosmopolitan and associated with rotting vegetation and fruits. However, most 39 of the strains isolated from nature have low genetic diversity likely because recent chromosome-scale 40 selective sweeps contain alleles that increase fitness in human-associated habitats. Strains from the Hawaii 41 Islands are highly divergent from non-Hawaiian strains. This result suggests that Hawaiian strains might 42contain ancestral genetic diversity that was purged from most non-Hawaiian strains by the selective sweeps. 43To characterize the genetic diversity and niche of Hawaiian C. elegans, we sampled across the Hawaiian 44Islands and isolated 100 new C. elegans strains. We found that C. elegans strains are not associated with 45any one substrate but are found in cooler climates at high elevations. These Hawaiian strains are highly 46 Page 2 of 23 diverged compared to the rest of the global population. Admixture analysis identified 11 global populations, 47 four of which are from Hawaii. Surprisingly, one of the Hawaiian populations shares recent ancestry with non-48Hawaiian populations, including portions of globally swept haplotypes. This discovery provides the first 49 evidence of gene flow between Hawaiian and non-Hawaiian populations. Most importantly, the high levels of 50 diversity observed in Hawaiian strains might represent the complex patterns of ancestral genetic diversity in 51 the C. elegans species before human influence. 52 53Introduction 54Over the last 50 years, the nematode Caenorhabditis elegans has been central to many important 55 discoveries in the fields of developmental, cellular, and molecular biology. The vast majority of these insights 56 came from the study of a single laboratory-adapted strain collected in Bristol, England known as N2 (Brenner, 57