Secondly, docking simulations were performed using AutoDock Vina  to evaluate the binding energies of CsMRP4 with nine bile acids (Table 2 and Fig. 4). All the bile acids tested bound favorably to the inner cavity of CsMRP4 (Fig. 4a). Taurolithocholic acid (TLCA) (Fig. 4b) and LCA (Fig. 4c) showed the highest affinities with CsMRP4, whereas deoxycholic acid (DCA) (Fig. 4i) and cholic acid (CA) (Fig. 4j) revealed moderate affinities. Interestingly, our docking results are in line with previous transport assay data, which indicated that TLCA bound favorably to MRP4 at a low concentration, but other bile acids needed much higher concentrations . TLCA had the highest affinity for MRP4 overexpressed in HEK cells, followed by taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA), taurocholic acid (TCA), glycocholic acid (GCA) and cholic acid (CA). We also then added more primary and secondary bile acids such as LCA, chenodeoxycholic acid (CDCA), and DCA. Among them, LCA at 2–4 μM concentration was reported to have a significant adverse effect on the survival of juvenile C. sinensis . Thus, the high affinity of LCA could be required for removing LCA from the worm’s body for survival. However, these findings remain to be established at the biochemical level, which needs to be studied in the future.Table 2Docking results between CsMRP4 and bile acids using AutoDock VinaBile acidsPubChem IDBinding energy (kcal/mol)No. of configurationsTaurolithocholic acid (TLCA)a SID 103579026-13.43Lithocholic acid (LCA)SID 103542513-12.23Taurochenodeoxycholic acid (TCDCA)a SID 312642451-10.15Chenodeoxycholic acid (CDCA)SID 24875071-9.97Taurodeoxycholic acid (TDCA)a CID 2733768-9.75Taurocholic acid (TCA)a SID 828139-9.36Glycocholic acid (GCA)a SID 177011773-9.27Deoxycholic acid (DCA)CID 222528-8.45Cholic acid (CA)a SID 223730521-8.15 aBinding affinities of bile acids to MRP4 in HEK cells  Fig. 4Best docking conformation of bile acids with CsMRP4. The 3D model of CsMRP4 is visualized as a white ribbon diagram, which is depicted in transparency (90%) to show the bile acid docked deep into the inner cavity (a). The red circle indicates the inner cavity for bile acid binding. The results are arranged according to the order of calculated binding affinities of complex bile acid-CsMRP4, as listed in Table 2: b TLCA, c LCA, d TCDCA, e CDCA, f TDCA, g TCA, h GCA, i DCA, j CA