Authors

Discussion

Benzimidazole (BZ) resistance in Parascaris univalens, a major equine parasite, poses an emerging threat to parasite control. Unlike strongyle nematodes, Parascaris spp. lack the canonical β-tubulin mutations (F167Y, E198A, F200Y) associated with BZ resistance in other parasite species. To further investigate the molecular mechanisms behind BZ-resistance in Parascaris spp. two complementary strategies were applied in this study. First, all seven β-tubulin isotypes (A–G) using pooled eggs from a BZ-resistant (45% efficacy) and fully susceptible farm sequenced. Deep amplicon sequencing generated 2.3 million PacBio HiFi reads spanning complete gene loci. Read- and cluster-based pipelines identified variants, phased haplotypes, and selection signatures. Second, to distinguish genetic from transcriptional mechanisms, β-tubulin expression was analyzed in adult worms following 24-hour in vitro thiabendazole exposure using RNA-seq. None of the canonical BZ-resistance SNPs were detected at codons 167, 198, or 200. Instead, three novel amino acid substitutions showed strong resistance associations in the resistant isolate: V315M (OR > 8400, p < 0.001) and F167S (OR > 2100, p < 0.001) in Pun-bt-B, and Q447P (OR = 3.94, p < 0.001) in Pun-bt-C. Pun-bt-B exhibited an extreme selective sweep with 9.5-fold haplotype diversity reduction (n₉₅: 2 vs 19) and near fixation of a single resistant haplotype, representing a selective sweep. Pun-bt-C showed moderate consolidation (2.3-fold) with enrichment of two resistant meta-haplotypes, while Pun-bt-E displayed significant depletion (30-fold) of susceptible-associated haplotypes. Expression analysis confirmed high basal transcription of Pun-bt-A and Pun-bt-B, but no consistent BZ-induced changes, indicating genetically encoded resistance rather than transcriptionally regulated. It can therefore be concluded that BZ resistance in P. univalens is defined by selective sweeps and novel β-tubulin mutations (V315M, Q447P) rather than canonical SNPs. These findings established distinct ascarid-specific resistance mechanisms and identify candidate molecular markers for future diagnostics and surveillance. These findings indicate that a broader genome-based monitoring approaches are recommended over traditional single-site genotyping.