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Additional file 1: Table S1. The present table shows the IC90 values obtained from the set-up and verification of in vitro assays in H. contortus: exsheathed third-stage (xL3) automated motility assay and xL3 development to fourth-stage (L4). Table S2. xL3 motility in the medium and DMSO control groups at 24 h of incubation. Statistical analysis and study of the existence of significant differences between the medium control group and DMSO control group for each experiment. Table S3. xL3 motility in the medium and DMSO control groups at 48 h of incubation. Statistical analysis and study of the existence of significant differences between the medium control group and the DMSO control group for each experiment. Table S4. xL3 motility in the medium and DMSO control groups at 72 h of incubation. Statistical analysis and study of the existence of significant differences between the medium control group and the DMSO control group for each experiment. Table S5. xL3 % of development in the medium and DMSO control groups at 7 days of incubation. Statistical analysis and study of the existence of significant differences between the medium control group and the DMSO control group for each experiment. Table S6. Commercial anthelmintic drugs and their activity at different concentrations for each H. contortus assay. Bold numbers indicate that the drug was active at the concentration tested. Table S7. Activity of a novel series of benzimidazole derivatives (at 25 µM) for each H. contortus assay (incubation time of 72 h for xL3 or adult stage motility test and 7 days for L4 development test). Bold numbers indicate that the compound was considered active. Figure S1. The present figure shows the morphological differences between xL3 and L4 stages of H. contortus, where the most notable distinction is the well-developed pharynx and the presence of a complete buccal capsule in L4 stage. Figure S2. Albendazole sulfoxide dose-response curve in xL3 automated motility assay at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM of three different experiments with six replicates for each concentration. Figure S3. Monepantel dose-response curve in xL3 automated motility assay at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM of three different experiments, with six replicates for each concentration. Figure S4. Ivermectin dose-response curve in xL3 automated motility assay at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM of three different experiments with six replicates for each concentration. Figure S5. Levamisole dose-response curve in xL3 automated motility assay at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM of three different experiments with six replicates for each concentration. Figure S6. Albendazole sulfoxide dose-response curve in xL3 to L4 development assay. Each data point represents mean ± SEM, of three different experiments, with three replicates for each concentration. Figure S7. Monepantel dose-response curve in xL3 to L4 development assay. Each data point represents mean ± SEM of three different experiments with three replicates for each concentration. Figure S8. Ivermectin dose-response curve in xL3 to L4 development assay. Each data point represents mean ± SEM of three different experiments with three replicates for each concentration. Figure S9. Albendazole sulfoxide effect on motility of H. contortus adult stage at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM motility score of three different experiments with six replicates for each concentration. Red dotted line indicates a motility score of 1.5, a value at which a product is considered active. Figure S10. Monepantel effect on motility of H. contortus adult stage at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM motility score of three different experiments with six replicates for each concentration. Red dotted line indicates a motility score of 1.5, a value at which a product is considered active. Figure S11. Ivermectin effect on motility of H. contortus adult stage at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM motility score of three different experiments with six replicates for each concentration. Red dotted line indicates a motility score of 1.5, a value at which a product is considered active. Figure S12. Levamisole's effect on motility of H. contortus adult stage at 24 h (dotted line), 48 h (dashed line) and 72 h (solid line). Each data point represents mean ± SEM motility score of three different experiments with six replicates for each concentration. Red dotted line indicates a motility score of 1.5, a value at which a product is considered active.