The cysteine proteases of metacercariae are involved in metacercarial excystment host immune modulation and possibly in tissue penetration. the secretory function of metacercarial cysteine proteases in addition to its role as a route for eliminating waste products. also exert pivotal roles in metacercarial excystment (Chung et al. 1995 host IgG degradation (Chung et al. 1997 and evasion from IgG-dependent eosinophil cytotoxicity (Shin et al. 2001 In the process of excystment metacercariae secrete two distinct cysteine proteases with molecular masses of 28 and 27 kDa respectively which cleave macromolecules of extracellular matrices (Chung et al. 1995 Although we understand part of the functional roles of the enzymes the origin of the 28 and 27 kDa enzymes has not been clarified in the metacercariae. To determine the tissue origin of the enzymes we purified the enzymes prepared the polyclonal antibodies reacting to the enzymes and performed immunolocalization studies. MATERIALS AND METHODS Metacercarial excystment and preparation of excretory-secretory product (ESP) Metacercariae of were obtained from naturally infected freshwater crayfish metacercarial ESP. metacercariae was localized in epithelia and luminal contents of intestine and suggested that the protease might play an important role in nutrient digestion. In this study however polyclonal antibodies to the 28 and 27 kDa cysteine proteases reacted against the epithelium of the excretory bladders and excretory concretions of the metacercariae. We could not confirm in this study however whether the thiol protease of metacercariae (Hamajima et al. 1985 is the same with the 28 and 27 kDa cysteine proteases. It has been suggested that the syncytial epithelium of trematodes excretory duct and bladder are involved in a number of physiological BNS-22 activities other than serving as a route for the elimination of metabolic wastes (Smyth and Halton 1983 According to Orido (1990) the syncytial cells of the excretory bladder of metacercariae contained a large quantity of excretory materials and were filled with Golgi complexes. These structural characteristics suggested that the excretory bladder in metacercariae exerts active secretory function. The secretion from epithelium of the excretory BNS-22 bladder should contain metacercarial 28 and 27 kDa cysteine proteases for the following two reasons. As shown in this study polyclonal antibodies against 28 and 27 kDa cysteine proteases reacted against the luminal linings of the excretory bladder and outer linings of the excretory concretions of metacercariae. In addition ESP of newly excysted metacercariae contained highly active 28 and 27 kDa cysteine proteases (Chung et al. 1995 After excystment calcareous concretions of the excretory bladder of metacercariae disappeared within 7-10 days in a rat definitive host (Orido 1990 The concretions of disappeared in the same period. In this connection the specific cysteine proteases decline dramatically during maturation stages in the definitive hosts. This phenomenon seems to be mainly due to the decreased secretion of highly active 28 and 27 kDa cysteine proteases and increased secretion of other cysteine proteases of weak activity. The developmentally modulated secretion of cysteine protease may be closely correlated with penetration and migration of the metacercariae within their definitive host (Chung et al. 1997 Recently cruzipain-like 28 kDa cysteine protease was reported as a developmentally regulated cysteine protease of (Yun et al. 2000 In situ hybridization study showed that the cruzipain-like protease was localized only DP2 in the intestinal epithelium of adult worms. Despite having the same molecular mass the cysteine protease of metacercariae is distinct BNS-22 from that of an adult worm. In conclusion we demonstrated that both 28 and BNS-22 27 kDa cysteine proteases of newly excysted metacercariae come from the excretory bladder. Footnotes This study was supported by a grant from the Cheju National University Medical Research Fund.