Independent mechanisms of benzimidazole resistance across Caenorhabditis nematodes

Benzimidazoles (BZs), a widely used class of anthelmintic drugs, target beta-tubulin proteins, disrupt microtubule formation, and cause nematode death. In parasitic nematode species, mutations in beta-tubulin genes ( e.g. , isotype-1 beta-tubulin) are predicted to inhibit BZ binding and are associated with BZ resistance. Similarly, in the free-living nematode Caenorhabditis elegans , mutations in an isotype-1 beta-tubulin ortholog, ben-1 , are the primary drivers of BZ resistance. The recurrent association of BZ resistance with beta-tubulins suggests that BZ resistance is repeatedly caused by mutations in beta-tubulin genes, an example of repeated evolution of drug resistance across nematode species. To evaluate the hypothesis of repeated evolution of BZ resistance mediated by beta-tubulin, we identified predicted resistance alleles in beta-tubulin genes across wild strains from three Caenorhabditis species: C. elegans , Caenorhabditis briggsae , and Caenorhabditis tropicalis . We hypothesized that, if these species experienced similar selective pressures, they would evolve resistance to BZs by mutations in any of three beta-tubulin genes ( ben-1 , tbb-1 , and tbb-2 ). Using high-throughput development assays, we tested the association of predicted beta-tubulin alleles with BZ resistance. We found that a heterogeneous set of variants identified in C. elegans ben-1 were associated with BZ resistance. In C. briggsae , only two variants in ben-1 , predicted to encode a premature stop codon (W21stop) and a missense substitution (Q134H), were associated with BZ resistance. In C. tropicalis , two missense variants were identified in ben-1 , but neither was associated with BZ resistance. C. briggsae and C. tropicalis might have evolved BZ resistance by mutations in other beta-tubulin genes, but we found that variants in tbb-1 or tbb-2 in these species were not associated with BZ resistance. Our findings reveal a lack of repeated evolution of BZ resistance across the three Caenorhabditis species and highlight the importance of defining BZ resistance mechanisms outside of beta-tubulins.