The miracidia of N. seoulense were obtained from eggs as described by earlier studies (Chung et al., 1996, 2002). Each laboratory-reared Segmentina hemisphaerula snail was exposed to 10-20 miracidia in a 24-well plastic plate (SPL Lifesciences, Pocheon, Korea) containing aerated tap water. After 2 weeks, the cercarial release was examined in infected snails at 700 lux illumination. Cercariae were first detected from the 19^th to the 32^th day after miracidial challenge. Cercariae were washed 5 times with sterile distilled water containing 100 units/ml of penicillin and 100 µg/ml of streptomycin (Sigma Co., St. Louis, Missouri, USA), collected (Fig. 1A), and promptly transferred to culture media for in vitro cultivation.

Medium 199 was used as the basic culture medium. In our preliminary studies, this medium was confirmed to be the most suitable medium for this purpose (data not shown). Penicillin (100 units/ml) and streptomycin (100 µg/ml) were added to medium to prevent contamination. All cultivation reagents were purchased from Sigma Co. A 5.4 ml volume of medium 199 was placed in each well of a 6-well culture plate (SPL Lifesciences) for in vitro cultivation, and a total of 100 cercariae of N. seoulense were added to each well, followed by 0.6 ml (10% v/v) of serum, i.e., FBS, horse serum, chicken serum, human serum, or African clawed frog (Xenopus laevis) serum. FBS, horse serum, and chicken serum were purchased from Sigma Co., human serum and African clawed frog serum from a normal healthy man and a laboratory-bred adult female frog. Cultivation was performed at 30 ± 1℃ under 5% CO₂, and the media were changed daily. Cultivations were continued until all cercariae died, and all cultivations were repeated twice. Morphologic changes in cercariae were examined during culture under an inverted microscope.

Serum was preheated at 56℃ for 30 min to inactivate complement. The chelator EDTA was used to block the classical and alternative complement pathways, and another chelator ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to block the classical complement pathway (Nelson et al., 2000). These agents were prepared as 200 mM stock solutions and stored at 4℃ until use (Fine et al., 1972). To block the complement pathways, stock solutions were added to medium to a final concentration of 20 mM. MgCl₂ solution was used to restore the classical and alternative pathways blocked by EDTA. To evaluate the complement pathways related to cercarial tail loss, 50 N. seoulense cercariae were cultured in medium 199 alone, or in medium 199 containing heat inactivated serum, 20 mM EDTA added serum, 20 mM EGTA added serum, or 20 mM EDTA plus 20 mM MgCl₂ added serum. All cultures were repeated twice.

Cercariae added to culture media (Fig. 1A) remained motionless for 30 min, and then slowly recovered from 30 min to 1 hr after being added to media. In all media added 10 % sera, rolling up of cercarial tail tips (Fig. 1B) was observed between 1 and 2 hr after addition to media, and cercariae were observed to simultaneously show a vigorous shaking movement. From about 3 hr, tail degradation (Fig. 1C) was evident in some cercariae, in combination with a continuous shaking movement. However, all cercariae died within 24 hr in media containing chicken serum, human serum, and African clawed frog serum, and within 48 hr in media containing horse serum. In fact, they only survived in media containing FBS, in which separation of the cercarial body and degraded tail was clearly observed from about 4 hr to 72 hr. The tail degradation and separation times tended to vary for each cercaria, but the sequence adopted was identical for all, as follows; initially, the bifurcated tail tips of cercariae rolled up and degraded from their terminal portions (Fig. 1B), followed by tail stem degradation (Fig. 1C), the formation of a bubble between the cercarial body and tail stem (Fig. 1D), and finally cercarial body and degraded tail separation (Fig. 1E). The survival rate of the worms in media containing FBS was 86.3% at 72 hr, when the separation of body and tail occurred in 19.3% of the cultured 300 cercariae and 67.0 % were alive with a degraded tail. Some cercariae died during tail degradation (Fig. 1F). After 3 days, further separation of cecarial bodies and tails ceased, and worm death accelerated. In total 13.3% of 300 cercariae survived until day 7, but all were dead at day 12.

Cercariae cultured in medium 199 showed no changes, all died within 5 days. For cercariae in medium 199 containing 10% heat inactivated FBS and the medium 199 containing 10% FBS plus 20 mM EDTA, rolling up changes of bifurcated tail terminals were observed in some cercariae on day 3, but no tail degradation was observed. The addition of 20 mM EGTA caused 14.2% of the cultured 155 cercariae to lose their tails on day 3, when 45.8% remained alive with a degraded tail. In 10% FBS contained 20 mM EDTA plus 20 mM MgCl₂, 12.8% of the cultured 149 cercariae had lost their tails on day 3 and 36.2% remained alive with a degraded tail.