Effects of acid pepsin pretreatment, bile acids and reductants on the excystation of Clonorchis sinensis (Trematoda: Opisthorchiidae) metacercariae in vitro

Abstract

The effects of acid pepsin pretreatment, bile acids and reductants on the excystation of Clonorchis sinensis metacercariae were studied. The velocities of metacercarial excystation, digestion of the metacercarial outer layer and tryptic BAEE hydrolysis were measured in various media. Pretreatment with acid or acid pepsin accelerated the excystation, but was not required under adequate tryptic activity. Taurocholate and deoxycholate evoked no excystation by themselves. However, in the trypsin medium with taurocholate and deoxycholate, they accelerated both the velocity of metacercarial excystation and of digestion of the outer layer, although the accelerative effect did not increase above 4 −2% deoxycholate. Taurocholate below 1% and deoxycholate below 4 −2% did not affect the rate of tryptic hydrolysis, while this was depressed by deoxycholate over 4 −2%. Addition of deoxycholate over 4 −2% also depressed the final percentage of excystation, as well as worm motility. By themselves, cysteine and 2-mercaptoethanol (MCE) swelled and deformed the cyst wall and evoked metacercarial excystation without trypsin, however, ascorbate and Na dithionite evoked no excystation. Addition of ascorbate to the trypsin medium did not change the velocity of excystation, but this was completely inhibited by the addition of dithionite and was accelerated by the addition of cysteine or MCE. Although excystation was accelerated with an increase of cysteine concentration, this acceleration was depressed at over 0.01 M cysteine. The rate of tryptic hydrolysis was depressed at over 0.002 M cysteine. The present study suggests that the excystation facilitative effects of pretreatment of acid pepsin, bile acids and reductants (cysteine and MCE) in trypsin medium are due to denaturation of protein, detergent action and cleavage of SS-linkage, respectively, on the digestion step of the outer layer of the metacercariae and cessation of the facilitative effect at a high concentration of deoxycholate is related to the rupturing step of the inner layer by larval movement.