Characterization of the β-tubulin gene family in Ascaris lumbricoides and Ascaris suum and its implication for the molecular detection of benzimidazole resistance

PLoS Negl Trop Dis. 2021 Sep 27;15(9):e0009777. doi: 10.1371/journal.pntd.0009777. eCollection 2021 Sep.


Background: The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding β-tubulin proteins, the principal targets of BZ drugs.

Methodology and principal findings: First, the β-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different β-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations.

Significance: This study demonstrated the presence of at least seven β-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the β-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of β-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.

Grant support

SR was supported through a Fundamental Research Fellowship (1154819N) of the Research Foundation Flanders ( The two expulsion studies were a part of the Starworms study, supported through a grant from the Bill and Melinda Gates Foundation (OPP1120972, PI is BL). Part of the sequencing work was supported by a travel award of the FWO for SR (K204820N) and a grant from the Bill and Melinda Gates Foundation (OPP1172974, PI is JG). SMJP is funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) PGS-D award (411306230) and Eyes High Recruitment award from UCalgary. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.