In March 2007, 2 species of brackish water crabs, M. dilatatus (Fig. 1) and M. japonicus, were purchased from a local market in Taean-Eup, Chungcheongnam-do, Republic of Korea. The hepatopancreas of the crabs was separated, ground in a mortar with a pestle, and filtered through a series of nets. Microphallid metacercariae, 106 to 500 µm in size, were collected from the sediments using a stereomicroscope and washed several times with phosphate buffered saline (PBS). The collected metacercariae were used for experimental infection of rats, and some of them were excysted under a cover slip applying slight pressure. The excysted metacercariae were fixed in 10% neutral formalin for microscopic examinations.

In order to obtain adult flukes, 6 Sprague-Dawley rats were experimentally fed 500 metacercariae each. They were sacrificed by cervical dislocation on days 2, 4, and 6 post-infection (PI). The small intestine was resected, divided into 3 portions, and opened longitudinally along the mesenteric border in PBS. The adult flukes were collected from the intestinal content using a stereomicroscope, fixed with 10% neutral formalin under a cover slip pressure, and measured after Semichon's acetocarmine staining. Measurements were done on 10 fixed specimens and given in µm with the mean followed by SD. To observe the eggs, the feces of the rats were examined by the formalinether sedimentation technique.

The brackish water crabs, which were purchased in a market in Taean-Eup, Chungcheongnam-do, consisted of 2 species, i.e. M. dilatatus and M. japonicus. They were different in color and in the morphology of the back. Both species were examined for the presence of metacercariae, and only M. dilatatus was proved to have the metacercariae of G. squatarolae. The mean number of metacercariae per crab was 4.3 (range; 1-11, no. of crabs examined; 850), and they were mainly located in the hepatopancreas of the crab. The metacercarial density increased according to the size of the crab (data not shown).

The average worm recovery rate (WRR) of G. squatarolae adult flukes from rats was 9.4% through days 2-6 PI (Table 1). The WRR was the highest at day 6 PI (13.0%), and the lowest at day 4 PI (3.3%), but statistical difference was not noted. At day 2 PI, 42.7% of the adult flukes were recovered from the middle part of the small intestine, but, at days 4-6 PI, most worms were harvested from the posterior part of the small intestine; 71.7% at day 4 PI and 89.0% at day 6 PI.

Metacercariae (Figs. 2-3): Encysted metacercariae, 391 x 363 (av.), elliptical with a thin wall (Fig. 2), with a small oral sucker and 2 ventral suckers, i.e., larger and smaller ones. Vitelline follicles 2 groups, lobulated, incorporating the excretory bladder between them. Excysted matecercariae, 480 x 270 (av.), and elongated (Fig. 3). Oral sucker subterminal, prepharynx long, and esophagus moderately long. Ceca widely divergent. Larger ventral sucker located at midline of the worm, posterior to the cirrus pouch. Smaller ventral sucker slightly postero-dextral to larger ventral sucker. Cirrus pouch lying transversely anterior to larger ventral sucker, and ending as genital atrium. Ovary between larger acetabulum and left cecal arch. Testes lying beneath each cecal arch, and larger than ovary. Genital atrium antero-dextral to smaller ventral sucker. Vitelline follicles grouped behind each testis, between them located excretory bladder.

Adults (Figs. 4-5): Body ovoid (Fig. 4), with varying size from 561 x 286 to 692 x 361 depending on age of infection (Table 2). General morphology common with excysted metacercariae with some differences. Oral sucker subterminal, prepharynx short, and esophagus moderately long. Ceca widely divergent and short, ending near level of testes. Larger ventral sucker at midline of the worm, equatorial or slightly post-equatorial, posterior to cirrus pouch. Smaller ventral sucker slightly dextral to larger ventral sucker. Cirrus pouch lying transversely anterior to two ventral suckers, containing voluminous seminal vesicle, ejaculatory duct and prostatic gland, and ending in genital atrium medially. Cirrus muscular, lying in genital atrium, composed of 2 spatulated and chitinized projections. Ovary between larger acetabulum and left cecal arch. Seminal receptacle and Mehlis' gland present. Testes larger than ovary, and lying beneath each cecal arch. Genital atrium antero-dextral to smaller ventral sucker. Vitelline follicles extensive, with more than 10 follicles on each side, locating posterior to testes. Uterine coils runs through posterior fields of body, with metraterm ends in genital atrium on median dorsal surface, with distal end reinforced by a heavy cuticular earlobe-shaped plate armed with about 15-20 tiny surface spines. Intrauterine eggs appearing at day 2 PI, occupying half of hindbody at day 4 PI and all available space of hindbody at day 6 PI (Fig. 5). Eggs 21 x 17 (av.), operculated, elliptical, brownish, and thick-shelled.