Several local collections of three lymnaeid snail species were mainly used in the culture studies. The snails were maintained mainly in conventional aquaria. Initially, tap water was conditioned before use simply by adding 4-5 drops of 5% sodium thiosulfate solution to each aquarium for dechlorination (pH, 6.5-7.0). This water was aerated for one week prior to use. The aquaria were placed on shelves in a shelving unit and exposed alternately to 12 hr of artificial light provided by a 15-watt cool, white fluorescent tube and 12 hr of darkness. An automatic timer controlled the light cycle. The water of all the glass aquaria or the plastic trays for the juvenile snails was continuously aerated by bubbling air from a air compressor through the water. The water temperature for the aquaria ranged from 25-27℃. Lettuce and Tetra SML flakes (Tetra SML®, Tetra Co., D-452 Melle, West Germany) were supplied to the lymnaeid snails twice a week (Chung, 1984).

Three species of lymnaeid snails collected from various local areas were first examined for the emergence of trematode cercariae. For the observation of trematode cercariae from snails, each snail was placed in a well of 24-well plastic plate (Emerald Biostructures Inc., Bainbridge Island, WA, USA) containing the appropriate amount of aerated tap water, kept under a fluorescent light for 2 hr and examined for the presence of shedding cercariae. Later, the snails were crushed for further examination of cercariae inside the snails.

Three species of laboratory-bred lymnaeid snails infected with cercariae, were examined in order to obtain metacercariae of E. cinetorchis. A total of 50 cercariae shed from lymnaeid snails were exposed to each experimental snail of S. hemisphaerula, known as the second intermediate host (Chung et al., 2001) and the infected specimens were examined for the detection of metacercariae. After cracking the snail shell and removing the pieces of shell, the animal bodies were dissected and digested in artificial gastric juice for 1 hr at 37℃. The metacercariae were collected from snails every week from two weeks after the cercarial challenge. The number of Echinostoma metacercariae per known volume was counted under the low power (×100) of a light microscope.

A total of 50 metacercariae was fed orally using a tuberculin syringe connected with a plastic tube to each rat (Sprague-Dawley strain, 120 g of body weight). Rat feces was examined daily for eggs of E. cinetorchis. Immediately after finding the eggs, the rats were killed by cervical dislocation and dissected. The small intestines of the rats were removed. The worms were collected under the dissecting microscope and their numbers were determined. Worms were fixed with 10% formalin under the cover slip pressure and stained with Semichon's acetocarmine. The stained worms were observed for morphological comparisons with the standard parasitic specimens.

Parasite-free snails were reared in the laboratory to obtain their laboratory-bred offsprings. The snail eggs of each species were collected and cultured to get laboratory-bred adult snails in a batch which were employed for the susceptibility experiments to the target parasite E. cinetorchis. The infected rats were used as the source of eggs for the susceptibility studies of snails. For the susceptibility experiments of E. cinetorchis miracidia to the laboratory-bred snails, eggs (Fig. 1B) were collected by tearing the worms. The eggs were rinsed 3 times with distilled water and transferred to the petri dishes containing conditioned water with a few drops of Fungizon solution (Gibco Life Technologies Inc., Grand Island, New York). The eggs were incubated in the conditioned water at 26℃ in a dark incubator with aeration. Some hatched miracidia were used to challenge the three lymnaeid snail species targeted in this study. Each snail was exposed to a dose of twenty miracidia hatched from the eggs of E. cinetorchis. Twenty days after miracidial infection, the release of cercariae was examined from the snails kept under the fluorescent illumination (700 Lux) for 2 hr. The cercariae released from laboratory-bred snails were also exposed to the three lymnaeid species targeted for observing the metacercariae of E. cinetorchis as described before.

Natural infections of E. cinetorchis in the three lymnaeid snail species collected from several local areas are shown in Table 1. In field-collected lymnaeid snails, only A. ollula (Fig. 1A) was found to be infected with cercariae of E. cinetorchis (infection rate: 0.7%), while none of R. auricularia coreana and F. truncatula were found shedding the cercariae.

The metacercariae of E. cinetorchis were recovered in all S. hemisphaerula (100%) experimentally infected with echinostome cercariae from field-collected Austropeplea snails.

Adult worms were also recovered from the ileo-cecal region of the rats infected with the metacercariae from experimental S. hemisphaerula (Fig. 1F). The recovery rate of 20% was recorded in the rats infected with metacercariae three weeks after metacercarial infection.

When the petri dishes containing embryonated eggs were placed under the light sources (17,000 Lux), almost 90% of the miracidia hatched on the 21-22nd day after incubation at 26℃. Miracidia (Fig. 1C) hatched from eggs of E. cinetorchis were exposed to the laboratory-bred snails of three lymnaeid species suspected as the first intermediate hosts in this study.

Out of 20 snails of each species infected with miracidia, 8 A. ollula and 5 F. truncatula (infection rates, 40% and 25%, respectively) shed a large number of echinostome cercariae (Fig. 1D). The snails of R. auricularia coreana were not infected experimentally with miracidia of E. cinetorchis (Table 2). An average of 1,249 cercariae per each A. ollula snail infected with E. cinetorchis miracidia were released during the 19-day period of observation after the 21st day after miracidial infection (Table 3).

The metacercariae in three laboratory-bred lymnaeid snail species infected with E. cinetorchis cercariae from experimentally infected A. ollula and F. truncatula were also found at the 14th day after cercarial challenge. All three experimental lymnaeid snail species became infected with infection rate of 40%, 100% and 60%, respectively (Table 4 and Fig. 1E).