Abstract
Maritrema spp. (Digenea: Microphallidae) are parasites of birds, but have not been found in the Republic of Korea. In this study, metacercariae of Maritrema sp. were discovered in the Asian shore crab, Hemigrapsus sanguineus, caught in the mud-flats of Jebu-do, Hwasung-gun, Gyeonggi-do, and the adult flukes were confirmed by experimental infection into mice. Based on the symmetric ribbon-like vitellarium, adult flukes of Maritrema sp. were identified, but did not belong to previously described species in terms of the following morphologic characteristics: ceca reaching to the lateral wall at the anterior border of the ovary; ventral sucker larger than oral sucker; a prominent metraterm; and vitellarium forming a complete ring. Hence, we named this microphallid M. jebuensis n. sp. after the island where the second intermediate hosts were collected. From this study, it has been shown that Maritrema sp. is distributed in Korea and transmitted by the Asian shore crab, H. sanguineus.
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Key words: Maritrema jebuensis, intestinal fluke, Asian shore crab, Jebu-do
Species of the genus
Maritrema Nicoll, 1907 (Microphallidae) occur in widely separated parts of the world, a finding which is consistent with birds serving as the definitive hosts of
Maritrema spp. [
1].
Maritrema spp. are found in the intestines of birds, causing enteritis and ulceration [
2,
3]. The characteristic organ of
Maritrema spp. is the vitellarium, which appears as a symmetric ribbon reaching close to the margins of hind body, surrounding the uterine coils and testes [
4]. The life cycle involves gastropods as first intermediate hosts, and crustaceans as second intermediate hosts [
5].
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.
Taxonomic summary
Experimental host: mouse
Locality: Jebudo (island), Seosin-myeon, Hwaseong-si, Gyeonggi-do, Republic of Korea
2nd intermediate host: Asian Shore Crabs, Hemigrapsus sanguineus
Site of infection: small intestine
Etymology: The specific name is derived from the name of the island where the second intermediate hosts were collected.
Holotype: Adult fluke is deposited in Department of Parasitology, College of Medicine, Dankook University, Cheonan-si, Chungnam 330-714, Republic of Korea.
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.
References
- 1. Coil WH. Notes on the Maritrema Nicoll, 1907 (Trematoda: Microphallidae) with the description of two new species. J Parasitol 1955;41:533-537.
- 2. Swales WE. On Streptovitella acadiae (gen et spec. nov.), a trematode of the family Heterophyidae from the black duck (Anas rubripes). J Parasitol 1933;11:115-118.
- 3. Hoeve J, Scott ME. Ecological studies on Cyathocotyle bushiensis (Digenea) and Sphaeridiotrema globulus (Digenea), possible pathogens of dabbling ducks in southern Québec. J Wildl Dis 1988;24:407-421.
- 4. Bray RA, Gibson DI, Jones A. Keys to the Trematoda. Natural History Museum. 2008, Vol 3:London, UK. CABI Publishing Co; pp 460-464.
- 5. DeBlock S. Contribution a l'etude des Microphallidae Travassos, 1920. XXIV.-Tentative de phylogenie et de taxonomic. Bulletin du Museum d'Historie Naturelle 1971;7:353-468.
- 6. Seo BS, Yoon JS, Lee CW, Lee SH. Studies on Microphalloides japonicus. Korean J Parasitol 1964;2:41-51.
- 7. Choi DW, Lee SW, Kim JM, Kim JK. Studies on the larval trematodes from brackish water crabs. Studies on a kind of Levinseniella. Korean J Parasitol 1965;3:31-38.
- 8. Seo M, Guk SM, Lee SH, Chai JY. Gynaecotyla squatarolae (Digenea: Microphallidae) from rats experimentally infected with metacercariae from the shore crab, Macrophthalmus dilatatus. Korean J Parasitol 2007;45:199-204.
- 9. Guk SM, Chai JY, Sohn WM, Kim YM, Sim S, Seo M. Microphallus koreana n. sp. (Trematoda: Microphallidae) transmitted by a marine crab, Macrophthalmus dilatatus. Korean J Parasitol 2008;46:165-169.
- 10. Keeney DB, Waters JM, Poulin R. Clonal diversity of the marine trematode Maritrema novaezealandensis within intermediate hosts: the molecular ecology of parasite life cycles. Mol Ecol 2007;16:431-439.
- 11. Lee HJ, Chai JY, Lee JW, Jin H, Min KH, Cho YJ, Seo M. Surveys of Gynaecotyla squatarolae and Microphallus koreana (Digenea: Microphallidae) metacercariae in two species of estuarine crabs in western coastal areas, Korea. Korean J Parasitol 2010;48:81-83.
- 12. Martorelli SR, Fredensborg BL, Mouritsen K, Poulin R. Description and proposed life cycle of Maritrema novaezealandensis n. sp. (Microphallidae) parasitic in red-billed gulls, Larus novaehollandiae scopulinus, from Otago Harbor, South Island, New Zealand. J Parasitol 2004;90:272-277.
- 13. Ching HL. Two new species of Maritrema (Trematoda: Microphallidae) from the Pacific Coast of North America. Can J Zool 1974;52:865-869.
- 14. Cremonte F, Martorelli SR. Description of a new species of Maritrema (Digenea: Microphallidae) from Larus dominicanus (Aves: Laridae) in Buenos Aires coast, Argentina. Folia Parasitol 1998;45:230-232.
- 15. Tkach VV. Maritrema neomi n. sp. (Digenea: Microphallidae) from water shrews (neomys). J Parasitol 1998;84:846-849.
- 16. Hadley CE, Castle RM. Description of a new species of Maritrema Nicoll 1907, Maritrema arenaria, with studies of the life history. Biol Bull 1940;78:338-348.
- 17. Etchegoin JA, Martorelli SR. Description of a new species of Maritrema (Digenea: Microphallidae) from Mar Chiquita coastal lagoon (Buenos Aires, Argentina) with notes on its life cycle. J Parasitol 1997;83:709-713.
- 18. Etges FJ. Studies on the life histories of Maritrema obstipum (Van Cleave and Mueller, 1932) and Levinseniella amnicolae n. sp. (Trematoda: Microphallidae). J Parasitol 1953;39:643-662.
- 19. Diaz JI, Cremonte F. Development from metacercaria to adult of a new species of Maritrema (Digenea: Microphallidae) parasitic in the kelp gull, Larus dominicanus, from the Patagonian coast, Argentina. J Parasitol 2010;96:740-745.
- 20. Gracenea M, Montoliu I, Deblock S. Contribution a l'etude des Microphallidae Travassos, 1920 (Trematoda). XLV. Description de Maritrema feliui n. sp., parasite de musaraignes (mammiferes) en Espagne. Ann Parasitol Hum Comp 1993;68:76-81.
Fig. 1(A) Asian shore crab, Hemigrapsus sanguineus, caught in the mud-flats of Jebu-do, Hwasung-gun, Gyeonggi-do, Korea. (B) A metacercaria of Maritrema n. sp. recovered from the Asian shore crab. The oral sucker (arrow) and the vitellarium (arrowhead) are indicated. Bar = 30 µm. (C) A 1-day old worm of Maritrema n. sp. recovered from a mouse. Note that the vitellarine follicles appeared as a ring (arrow) in posterior part of the body. VS, ventral sucker; OV, ovary; T, testis. Bar = 20 µm. (D) A 5-day old worm of Maritrema n. sp. The hindbody was filled with the intrauterine eggs, surrounded by the vitellarium. VS, ventral sucker; CS, cirrus sac; OV, ovary; T, testis. Bar = 20 µm.
