Chemotactic migration of newly excysted juvenile Clonorchis sinensis is suppressed by neuro-antagonists

Abstract

The metacercariae of the Clonorchis sinensis liver fluke excyst in the duodenum of mammalian hosts, and the newly excysted juveniles (CsNEJs) migrate along the bile duct via bile chemotaxis. Cholic acid is a major component of bile that induces this migration. We investigated the neuronal control of chemotactic behavior of CsNEJs toward cholic acid. The migration of CsNEJs was strongly inhibited at sub-micromolar concentration by dopamine D1 (LE-300 and SKF-83566), D2 (spiramide, nemonapride, and sulpiride), and D3 (GR-103691 and NGB-2904) receptor antagonists, as well as a dopamine reuptake inhibitor (BTCP). Neuropeptides, FMRFamide, peptide YY, and neuropeptide Y were also potent inhibitors of chemotaxis. Meanwhile, serotonergic, glutamatergic, and cholinergic inhibitors did not affect chemotaxis, with the exception of fluoxetine and CNQX. Confocal immunofluorescence analysis indicated that dopaminergic and cholinergic neurons were colocalized in the somatic muscle tissues of adult C. sinensis. Our findings suggest that dopaminergic neurons and neuropeptides play a major role in the chemotactic migration of CsNEJs to bile, and their inhibitors or modulators could be utilized to prevent their migration from the bile duct.

Fig 1. Inhibition of chemotactic migration of CsNEJs to cholic acid by dopaminergic inhibitors.

Percent inhibition of chemotactic migration of CsNEJs toward 50 mM cholic acid by dopamine D₁ (A), D₂ (B), and D₃ (C) receptor antagonists, and a dopamine reuptake inhibitor (D). CsNEJs were incubated in a solution containing a test compound at 0.01–100 μM for 10 min, after which 50 mM cholic acid was placed at the positive end to induce chemotaxis. Migration distance of CsNEJs over 60 min in the presence of each compound was subtracted from the control migration distance, and the ratio is presented as percent inhibition.

Fig 2. Effects of serotonergic inhibitors on the cholic acid-induced chemotaxis of CsNEJs.

CsNEJs were stimulated with 50 mM cholic acid (CA) in the presence of fluoxetine (A), spiroxatrine (B), ritanserin (C), or Y-25130 (D), and migration distance was measured at 60 min. Asterisk * indicates P < 0.05 compared to 50 mM cholic acid only.

Fig 3. Effects of glutamatergic inhibitors on the cholic acid-induced chemotaxis of CsNEJs.

CsNEJs were stimulated with 50 mM cholic acid (CA) in the presence of CNQX (A), NBQX (B), MK-801 (C), or cyclothiazide (D), and migration distance was measured at 60 min. Asterisk * indicates P < 0.05 compared to 50 mM cholic acid only.

Fig 4. Effects of cholinergic inhibitors on the cholic acid-induced chemotaxis of CsNEJs.

CsNEJs were stimulated with 50 mM cholic acid (CA) in the presence of pirenzepine (A) or benzoquinonium (B), and migration distance was measured at 60 min. Asterisk * indicates P < 0.05 compared to 50 mM cholic acid only.

Fig 5. Effects of neuropeptides on the cholic acid-induced chemotaxis of CsNEJs.

CsNEJs were stimulated with 50 mM cholic acid (CA) in the presence of FMRFamide (A), peptide YY (B), or neuropeptide Y (C), and migration distance was measured at 60 min. Asterisks indicate * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to 50 mM cholic acid only.

Fig 6. Confocal immunofluorescent micrographs of adult C. sinensis.

Double-immunostaining with antibodies to tyrosine hydroxylase (TH) (A, D), choline acetyltransferase (ChAT) (B, E), and images merged (C, F). Upper panels show the lateral margin to the level of the ventral sucker, and lower panels show the area between the esophagus and ventral sucker.