Until now, 4 species belonging to Microphallidae have been described in Korea (Microphalloides japonicus from Helice tridens tridens, Levinseniella spp. from Macrophthalmus japonicus, and Gynaecotyla squatarolae and Microphallus koreana from Macrophthalmus dilatatus) [6-9]. Maritrema species have not been previously reported in Korea, but the metacercariae were found for the first time during a survey on shore crabs of the western Korea coastal areas. In this study, the metacercariae were shown to be those of Maritrema spp., and identified as a new species by experimental infection in mice. Although the suitable host of Maritrema was known to be the bird, but mice were used in this study due to unavailability of chicks. For the adult worms recovered from mice, we assigned the name Maritrema jebuensis n. sp. Maritrema is the fifth microphallid having been found in Korea.

In October 2009, the Asian shore crabs, Hemigrapsus sanguineus, were caught in the mud-flats of Jebu-do (island), Seosinmyeon, Hwaseong-si, Gyeonggi-do during low tides. The crabs were mainly found beneath stones, and well-differentiated from Macrophthalmus spp. by morphology (Fig. 1A). After transportation to the laboratory, 40 of 50 crabs were ground in a mortar with a pestle, and filtered through a series of nets ranging from 60-500 µm in size. Then, the crab homogenates were washed several times with PBS, and the metacercariae of Maritrema sp. were collected from the sediments using a stereomicroscope. Some of the metacercariae were fixed in 10% neutral formalin, and were used for morphological investigation after acetocarmine staining. The rests were used for experimental infection.

Then, specific pathogen-free (SPF) ICR mice, 5-week-old, were purchased from Koatech Co. Ltd. (Pyeongtaek, Korea) and maintained under SPF conditions at the Animal Facility of Dankook University. To obtain adult worms, 20 mice were orally infected each with 100 metacercariae, and 4 mice were sacrificed on days 1, 3, 5, 7, and 9 post-infection (PI). The small intestines were removed, and the microphallid worms were recovered from the intestinal contents under a stereomicroscope. The collected flukes were counted and fixed in 10% formalin. After staining with Semichon's acetocarmine, the flukes were observed under a light microscope for species identification. Measurements were done on all of the recovered worms (in µm).

The mean number of Maritrema n. sp. metacercariae per Asian shore crabs (H. sanguineus) was 30.5 (range 0-102). The metacercariae were mainly found in the muscles, especially near the legs. The metacercaria of Maritrema n. sp. was round and 300 × 300 µm in size. The wall was thick and composed of 2 layers. In outer layer, numerous striations were observed, and a ring-shaped vitellarium inside it (Fig. 1B). In addition to Maritrema n. sp., the metacercariae of Gynaecotyla squatarolae were also recovered (mean 32.6; range 0-125).

The average recovery rate of Maritrema n. sp. adult worms was 4.3% on day 1 PI, 1.3% on day 3 PI, 3.0% on day 5 PI, 1.0% on day 7 PI, and 0.0% on day 9 PI. The 1-day-old worm already had the ovary and testes (Fig. 1C), and the intrauterine eggs appeared on day 3 PI (Fig. 1D). The length of adult worms was the highest on day 3 PI, 681 µm (614-762), continuously decreased to 530 µm (515-545) on day 7 PI. The size of intrauterine eggs was 22 × 11 µm. The morphological characteristics of 3-day worms were as follows: Body linguiform, 681 µm long by 250 (228-287) µm wide at ventral sucker level. Oral sucker subterminal, 57 (53-64) µm × 56 (53-61) µm. Anterior border of ventral sucker is located at midline of the body, 79 (66-86) µm × 79 (77-86) µm. Sucker ratio (oral sucker to ventral sucker) was 0.72. Prepharynx 51 (26-88) µm long, pharynx 31 (24-33) µm long, and esophagus 147 (132-156) µm long. Ceca reached middle of ventral sucker. Both testes were symmetrical and same in size, 61 × 61 µm. Cirrus sac intercecal, arcuate with thin wall, 103 (92-112) µm long by 29 (26-31) µm wide. The right end of cirrus sac reached the left border of ovary, not the right margin of body, and the left end reached at level of genital pore. Seminal vesicle and prostatic cells inside the cirrus sac, the latter surrounding curled ejaculatory duct. Invaginated cirrus slightly developed. Genital pore conspicuous, sinistrolateral to ventral sucker. Ovary dextral at level of ventral sucker, unilobed, 57 (46-70) µm × 76 (66-86) µm. Uterus occupied the majority of hindbody, from anterior border of testis to the end of body. Metraterm thick walled, sinistral to genital pore. Vitellarium formed a complete ring, reaching close to margins of hindbody, surrounding uterine coils and testes.

In this study, the presence of Maritrema sp. has been demonstrated for the first time in Korea, and the Asian shore crab, H. sanguineus, has been shown to be the second intermediate host. Although the metacercariae of M. novaezealandensis were found in Macrophthalmus crabs [10], the survey on M. japonicus and M. dilatatus revealed no metacercariae of Maritrema spp. in Korea [11]. Since the tidal currents help to mix the cercariae population from a large number of snails, the presumed 1st intermediate hosts, it was suggested that the crabs may commonly be infected by numerous kinds of cercariae [10].

The basis for identification of the present specimen as a new species was as follows: Above all, the vitelline follicles formed a complete ring in the present specimen. M. pacifica, M. orensensis, M. neomi, M. patulus, M. magdalenense, M. borneoense, M. minuta, M. macropharynga, and M. jilinensis were excluded due to the incomplete ring of vitellaria [12-15]. Ceca reached the lateral wall at the anterior border of the ovary in the specimen, but at the level of vitellaria in M. arenaria and in M. paracadiae [13,16] and the testicular level in M. bonaerensis [17]. Posteromedial curving is present in M. obstipum, and ovary posterior to ventral sucker in M. novaezealandensis and M. madryensis [12,18,19]. Ventral sucker is larger than oral sucker in the present specimen, differentiating M. pyrenaica and M. feluie [15,20]. Ovary is trilobed in M. spinosulum and M. eroliae [14]. Ovary is more median in M. ovata, M. gratiosum, and M. acadiae [16]. The prominent metraterm is the characteristic feature shared by M. bonaerensis and M. orensensis [14, 17]. Hence, it was concluded that the present specimen did not belong to the pre-existing Maritrema spp., and we suggested this new species of Maritrema as M. jebuensis.

However, the adults of Maritrema spp. are very similar in morphology, rendering separation of the species identification difficult. This difficulty is further complicated by the variation in structures generally used as taxonomic criteria [1]. For example, the disposition of the vitellaria, uterus, and sucker sizes showed considerable variation in Maritrema obstipum [18]. Genetic analysis is a powerful method to differentiate species, but few molecular studies have been performed on Maritrema spp., including M. novaezealandensis [10]. More information should be accumulated for molecular differentiation of Maritrema spp. Interestingly, the metacercariae of Maritrema n. sp. were mainly found in the muscles of crabs. This finding contrasts with those of Gynaecotyla squatarolae, being located in the hepatopancreas [8]. In addition, it was reported that the metacercariae of M. novaezealandensis were found encysted in the body cavity of the crustacean host [12]. These findings might help the differentiation of microphallid metacercariae during the research.

In case of M. obstipum, the infection in ducklings lasted 28 days, 24 days in chicks, 5 days in mice, and 2 days in hamsters [18]. It seems to be reasonable since Maritrema spp. utilize shorebirds as definitive hosts. In this study, the infection lasted only 7 days along with the low worm recovery rate due to the usage of mice. Compared to Microphallus koreana, the adult worms of which were recovered from only chicks [9], the host specificity of Maritrema n. sp. might be low. From this study, the presence of Maritrema sp., named as M. jebuensis, was demonstrated in Korea for the first time. Because Maritrema is a parasite of birds, further study on birds is needed.