From selection to resistance: Mitochondrial findings in hookworm evolution under drug pressure

Single nucleotide polymorphisms (SNPs) in codons 134, 167, 198, and 200 of the β-tubulin isotype 1 gene are associated with benzimidazole resistance in nematodes. Our group previously selected an Ancylostoma ceylanicum strain resistant to albendazole through drug-induced selective pressure, derived from a wild-type strain maintained under laboratory conditions for over 15 years. This study aimed to investigate mitochondrial genetic variability in the resistant strain compared to its wild-type counterpart. A total of 151 worms from both strains, collected during and 42 months after the selection process, were analyzed for partial sequences of the mitochondrial COI and NAD1 genes. Nucleotide variations were detected exclusively in the resistant strain, with low divergence levels of 0.16 % (1/612 bp) in COI and 0.25 % (1/398 bp) in NAD1. All COI substitutions were synonymous, while NAD1 presented one nonsynonymous mutation. Phylogenetic analyses based on Maximum Likelihood and Bayesian inference revealed strong clustering between resistant and wild-type laboratory samples (COI: 99 % bootstrap; NAD1: 96 %), distinct from field-derived sequences. These findings suggest that genetically homogeneous populations, resulting from isolation and restricted gene flow, may be more susceptible to genetic pressures, including selection, potentially leading to the establishment of resistant parasites. This study underscores the role of population genetics in the evolution of drug resistance and emphasizes the importance of managing genetic diversity to mitigate resistance development.