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A New Neodiplostomid (Digenea) From the Intestine of Chicks Infected with

Metacercariae From the Grass Snake,Rhabdophis tigrina

Author(s): Eun-Hee Shin, Jae-Lip Kim, Jong-Yil Chai

Source: Journal of Parasitology, 94(6):1379-1384. 2008.

Published By: American Society of Parasitologists

DOI: http://dx.doi.org/10.1645/GE-1499.1

URL: http://www.bioone.org/doi/full/10.1645/GE-1499.1

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http://www.bioone.org/page/terms_of_usehttp://www.bioone.org/http://www.bioone.org/doi/full/10.1645/GE-1499.1http://dx.doi.org/10.1645/GE-1499.1

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1379

J. Parasitol.,94(6), 2008, pp. 13791384

American Society of Parasitologists 2008

A NEW NEODIPLOSTOMID (DIGENEA) FROM THE INTESTINE OF CHICKS INFECTED

WITH METACERCARIAE FROM THE GRASS SNAKE, RHABDOPHIS TIGRINA

Eun-Hee Shin, Jae-Lip Kim, and Jong-Yil Chai*

Department of Parasitology and Tropical Medicine, Seoul National University College of Medicine, and Institute of Endemic Diseases,Seoul National University Medical Research Center, Seoul 110-799, Republic of Korea. e-mail: [email protected]

ABSTRACT: Three species of neodiplostomula are known to inhabit the European grass snake,Rhabdophis tigrina, in the Republicof Korea: Pharyngostomum cordatum (large-sized neodiplostomula), an intestinal trematode of cats; Neodiplostomum seoulense(small-sized neodiplostomula), an intestinal trematode of humans and rodents; and Neodiplostomum leei (small-sized neodiplo-stomula), which migrates to the livers of rodents and is an intestinal trematode of birds. The present study describes a fourthspecies,Neodiplostomum (Conodiplostomum) boryongense n. sp. (Digenea: Neodiplostomidae), based on adult flukes recoveredfrom the small intestines of chicks experimentally infected with small-sized neodiplostomula from the grass snake. The newspecies differs from 13 previously known species. It also differs from N. seoulense in its larger body size, severely bilobed testes,and smaller genital atrium, and from N. leeiin its larger body size, smaller ventral sucker, presence of a genital cone, and vitellinefollicles distributed chiefly in the forebody. The new species does not migrate to the livers of rodents nor does it develop toadulthood in the rodent intestines. However, the neodiplostomula of the new species are indistinguishable from those of the other2 species. Results show that at least 4 species of neodiplostomula inhabit the grass snake in the Republic of Korea.

In the European grass snake, Rhabdophis tigrina (Boie,

1826), in the Republic of Korea, 3 species of neodiplostomula,

i.e., 1 large-sized and 2 small-sized species, are known to exist(Chai et al., 1990; Chai and Shin, 2002). The large-sized spe-

cies, having numerous excretory granules, is Pharyngostomum

cordatum(Diesing, 1850); it develops into an adult fluke in the

small intestines of cats (Chai et al., 1990) and migrates to the

lungs, intercostal muscles, heart, and brain in rodent interme-

diate hosts (Shin et al., 2001). The small-sized neodiplostomula,

with smaller numbers of excretory granules, are divided into at

least 2 species, i.e., Neodiplostomum seoulense (Seo et al.,

1964), which develops into an adult in the small intestines of

mice and rats (Seo et al., 1988; Seo, 1990); and Neodiplosto-

mum leei, which develops into an adult in the small intestines

of chicks and migrates to the livers of rodents without maturing

(Chai and Shin, 2002; Shin et al., 2006). Natural human intes-

tinal infections with N. seoulense have been detailed (Seo etal., 1982; Hong et al., 1984, 1986). However, N. leei infection

has been reported only in the intestine of experimentally in-

fected chicks; its larval infections were detected in the livers

of mice and rats, as well as in the viscera of the grass snake

(Chai and Shin, 2002).

The similar larval morphologies of strigeid flukes (Sudari-

kov, 1971) create difficulties in their species diagnosis when

recovered from intermediate hosts, which indicates the possible

presence of mixed infections with different species in the same

host. Until the present, all small-sized neodiplostomula in the

European grass snake inhabiting the Republic of Korea had

been regarded as N. seoulense due to their uniform morpholo-

gies (Hong et al., 1982, 1983). However, they recently turned

out to be a mixture of at least 2 species, N. seoulense and N.leei (Chai and Shin, 2002).

The present study was undertaken to determine whether a

third species is present among the small-sized neodiplostomula

that inhabit the grass snake in the Republic of Korea. We dis-

covered a new species of Neodiplostomum that develops into

an adult in the small intestine of an avian host. When the new

species was used to infect rats and mice, the neodiplostomula

Received 6 October 2008; revised 18 April 2008; accepted 18 April2008.

* To whom correspondence should be addressed.

did not migrate to the liver, but instead they remained as larvae

in the small intestine and were expelled around day 10 post-

infection (PI). The new species differed morphologically fromN. seoulense, N. leei, and other previously reported species of

Neodiplostomum (Conodiplostomum).

MATERIALS AND METHODS

Experimental animals

Eight-week-old male BALB/c mice, which were raised under specif-ic-pathogen-free (SPF) conditions, were purchased from the Korean An-imal Center (Jinju, Republic of Korea). Newborn broilers were pur-chased from a hatchery in Gyonggi-do (Province), Republic of Korea.Animals were maintained under SPF conditions at the animal facilityof the Seoul National University College of Medicine, Seoul, Republicof Korea. Experiments were carried out in accordance with the guide-lines issued by our Institutional Animal Care and User Committee.

Source and isolation of small-sized neodiplostomula

European grass snakes,R. tigrina, were purchased from various localenzootic areas: Gyonggi-do Province (Ansung and Pyungtaek counties),Chungcheongnam-do Province (Chunan, Asan, Onyang, Yeasan, Bor-yong, and Hongsung counties), and Gangwon-do Province (Hongcheonand Hoeongsung counties). Snakes were kept hibernated at 4 C in acold room until use. Small-sized neodiplostomula (Fig. 1) were col-lected by a previously described digestion technique (Chai and Shin,2002). In brief, snakes were killed under ether anesthesia and thenskinned. The entire visceral mass and carcasses were removed andchopped before incubation in artificial gastric juice containing 0.5%pepsin (1:10,000; Sigma Chemical Co., St. Louis, Missouri) and 0.8%HCl. The digested mixture was incubated for 90 min at 37 C and waswashed several times by filtering through coarse and fine stainless steelmeshes (U.S. standard testing sieves; W. S. Tyler Inc., Mentor, Ohio).After final filtration, the mixture was transferred to a Petri dish, and

neodiplostomula were collected using a stereomicroscope. The wholeprocedure was performed under cold conditions to maintain the viabilityof neodiplostomula. Large-sized neodiplostomula ofP. cordatum (Fig.1) were collected and removed.

Experimental infection of animals and worm recovery

Experimental infection of newborn chicks and mice with small-sizedneodiplostomula was performed as described previously (Chai and Shin,2002). In brief, the animals that had been infected orally with small-sized neodiplostomula were killed by cervical dislocation under etheranesthesia at days 7, 14, and 21 PI. Their small intestines were removedand longitudinally opened in a Petri dish containing saline, and thepresence of adult flukes was examined using a stereomicroscope. Theliver, lungs, brain, heart, and kidneys were also resected, cut into small

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1380 THE JOURNAL OF PARASITOLOGY, VOL. 94, NO. 6, DECEMBER 2008

FIGURE1. (A) Large- (Pharyngostomum cordatum) and small-sized neodiplostomula (arrows) (mixture ofNeodiplostomum seoulense, N. leei,and N. boryongense n. sp.) collected from the mesentery and omentum of the grass snake, Rhabdophis tigrina, in the Republic of Korea. Bar 0.45 mm. (B) An acetocarmine-stained adult specimen of N. boryongense n. sp. (ventral view), recovered from the small intestine of an experi-mentally infected newborn chick at day 14 PI. Bar 0.3 mm.

pieces, compressed between 2 glass slides, and examined for the pres-ence of worms using a stereomicroscope.

Morphological observation of adult flukes

Adult flukes collected from the small intestines of chicks werewashed several times with saline, fixed in 10% neutral formalin, andstained with Semichons acetocarmine. The flukes were dehydrated ina graded series of ethanol and mounted in Canada balsam. Some of thedehydrated specimens were embedded in paraffin, cut into 5-m sec-tions, and stained with hematoxylin and eosin. Measurements were per-formed on 20 fixed specimens obtained at day 14 PI. Unless otherwisestated, measurements are given in micrometers (m), with a range fol-lowed by the mean in parentheses.

RESULTS

Neodiplostomula without further development were found in

the livers of infected mice at days 721 PI; they were confirmed

to be N. leei (Chai and Shin, 2002). Apart from these, adult

flukes were found in the small intestines of infected mice at

days 721 PI, and all of them were identified as N. seoulense

(Seo et al., 1964). However, neodiplostomula without further

development were also found in the small intestines of infected

mice between day 7 and day 12 PI (data not shown). These

neodiplostomula were harvested and administered orally to new

mice and chicks to see whether they would develop. In mice,

these neodiplostomula did not migrate to the liver, lungs, brain,

heart, or kidneys, and they did not mature into adults in the

small intestines from day 7 to day 21 PI. However, in chicks,

adult neodiplostomes that morphologically differed fromN. leei

were harvested from the small intestines at days 714 PI. These

neodiplostomes are reported here as a new species.

Another experiment to obtain the adult flukes of the new

species was performed. In brief, the small-sized neodiplosto-

mula in the grass snakes were harvested and used to directly

infect newborn chicks. Adult neodiplostomes were subsequent-

ly harvested at days 721 PI, and they consisted of 2 different

species. One species was confirmed to be N. leei, and the other

species was morphologically identical to the new species, which

was harvested from chicks given neodiplostomula retained in

the small intestines of mice at days 712 PI.

DESCRIPTION

Neodiplostomum(Conodiplostomum)boryongensen. sp.(Figs. 13; Table I)

Diagnosis: Body bisegmented tapering anteriorly, 2,0751,725 (avg.1,838) long and 7251,100 (883) wide at middle portion of forebody.Covered with numerous scale-like spines chiefly on forebody. Forebodyspoon-shaped, with well developed ventral cancavity, 730875 (807)long, 7251,100 (903) wide. Hindbody broadly cylindrical, tapering

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SHIN ET AL.NEODIPLOSTOMUM BORYONGENSEN. SP. 1381

FIGURE 2. (A) Neodiplostomum (Conodiplostomum) boryongense n. sp. (holotype) from an experimentally infected chick, day 14 PI. Bar 0.25 mm. (B) Diagram of the reproductive system of N. (C.) boryongense n. sp., lateral view. OV, ovary; LC, Laurers canal; T, testis; VR,vitelline reservoir; SV, seminal vesicle; IC, intestinal cecum; GC, genital cone; HD, hermaphroditic duct; SR, seminal reservoir; MG, Mehlisgland; UT, uterus; VT, vitelline follicles. Bar 0.1 mm.

posteriorly, 9501,200 (1,031) long, 625725 (670) wide. Oral suckersubterminal, 6888 (80) long, 5388 (70) wide. Prepharynx short. Pseu-dosucker absent. Pharynx well developed, 5573 (64) long, 3063 (51)wide. Esophagus extremely short or absent. Ceca relatively wide, extendto near posterior end of body. Ventral sucker transversely elliptical,larger than oral sucker, 60 88 (72) long, 88113 (102) wide, on midlineof body just anterior to tribocytic organ. Tribocytic organ large, 310400 (370) long, 300380 (341) wide, round to elliptical with a medianlongitudinal slit. Testes 2, tandem in hindbody, transversely long, se-verely bilobed with a prominent median constriction, forming an eye-glass shape; anterior testis 250420 (336) long, 610720 (658) wide(left lobe 231338 [285] long, 226320 [273] wide; right lobe 220316[268] long, 229303 [266] wide); posterior testis 250430 (362) long,560660 (612) wide (left lobe 305371 [338] long, 214270 [242]wide; right lobe 312396 [354] long, 220266 [243] wide). Vas efferensfrom 2 testes connected to bi- or trilobed seminal reservoir at ventralside of ovary and then to vas deferens and seminal vesicle. Seminalvesicle tubular, highly folded, posterior to posterior testis, median, withmuscular wall at its distal end, giving rise to ejaculatory duct withoutejaculatory pouch. Ejaculatory duct slender, runs postero-ventrally to

join uterus and hermaphroditic duct. Prostatic cells and cirrus lacking.Ovary transversely elliptical, located at median or slightly right side ofbody near the junction between fore- and hindbody, 100168 (129)long, 250330 (283) wide. Oviduct runs postero-laterally to connectwith vitelline reservoir, Mehlis gland, and ootype between testes. Laur-ers canal opens dorsally in the midline. Seminal receptacle lacking.Vitelline reservoir intertesticular. Vitelline follicles distributed denselyfrom level of ventral sucker to anterior end of anterior testis, whichbecome remarkably sparse and confined medially beyond anterior testis.Genital atrium subterminal, opened dorsally, 3550 (44) long, 5575

(63) wide. Genital cone present, without prepuce, protruded into genitalatrium, as seen in longitudinal sections (Fig. 3). Uterus occupying mid-dle field of hindbody, with 1242 (26) eggs, and connected to her-maphroditic duct and genital atrium. Eggs ovoid, immature, operculate,golden yellow, 8890 (89) long, 5360 (56) wide, with a thin, trans-parent, and refractile shell.

Taxonomic summary

Type host: Newborn chicks, Gallus domesticus Linnaeus (experimen-tal).

Site of infection: Small intestine.Intermediate host (paratenic host): European grass snake Rhabdophis

tigrina (Boie, 1826).Site of infection in intermediate host (paratenic host): Visceral wall,

omentum.

Locality: Chungcheongnam-do Province (Boryong, Chunan, Asan,Onyang, Yeasan, and Hongsung counties), Republic of Korea.

Specimens deposited: Holotype, SNU (Seoul National University,Seoul, Republic of Korea) Helm. Coll. (no. 011001); paratypes, U.S.National Parasite Collection (Beltsville, Maryland) (no. 100085).

Etymology:The specific name is originated from the name of a localarea where the grass snake, i.e., a paratenic host, was collected.

Remarks

Among the genera of Neodiplostomidae Shoop, 1989, which have nopseudosuckers, some have a genital cone, whereas others do not. Thegenera that have a genital cone include Neodiplostomum (Conodiplo-stomum) Dubois, 1937, Posthodiplostomum Dubois, 1936, Mesoopho-rodiplostomum Dubois, 1936, and Ornithodiplostomum Dubois 1936,

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FIGURE 3. (A) A longitudinal section ofNeodiplostomum (Conodiplostomum) boryongense n. sp., showing the internal organs, including theovary (OV), seminal reservoir (SR), uterus (UT), testes (T), vitelline reservoir (VR), seminal vesicle (SV), and genital atrium with a genital cone(GC). Bar 0.25 mm. (B) A longitudinal section ofN. (C.) boryongense n. sp., showing the internal organs, including the intestinal cecum (IC),seminal vesicle (SV), uterus (UT) with an egg (E), hermaphroditic duct (HD), excretory pore (EP), and genital atrium with a genital cone (GC).Bar 0.04 mm.

TABLE I. Comparative measurements of Neodiplostomum boryongense n. sp., N. leei, and N. seoulense adult flukes.

Body organs

N. boryongense n. sp.*

Length Width

N. leei*

Length Width

N. seoulense

Length Width

Whole body 1,838 113 883 127 1,446 115 829 74 1,170 71 578 42

Forebody 807 58 903 115 643 54 829 74 568 37 561 47Hindbody 1,031 75 670 36 804 77 626 52 602 60 539 40

Oral sucker 80 7 70 11 76 7 76 3 62 3 58 3

Pharynx 64 5 51 12 55 2 53 8 55 3 37 4

Ventral sucker 72 9 102 7 85 8 124 9 61 2 73 5

Tribocytic organ 370 27 341 25 226 16 294 50 275 29 245 23

Anterior testis 336 52 658 45 191 39 238 49 166 18 480 26

Posterior testis 362 55 612 31 260 48 235 30 220 25 455 29

Ovary 129 21 283 35 119 23 274 29 93 9 198 19

Genital atrium 44 6 63 6 38 21 68 27 78 13 116 20

Eggs 89 1 56 2 92 4 53 3 93 3 60 2

* Recovered from experimental chicks at day 14 PI. Mean SD in micrometers (n 20).

Recovered from experimental mice at day 14 PI. Mean SD in micrometers (n 20).

whereas those lacking a genital cone include Neodiplostomum(Neodip-lostomum) Dubois, 1937, FibricolaDubois, 1932, HysteromorphaLutz,1931, andDiplostomumvon Nordmann, 1832 (Sudarikov, 1960; Schell,1985; Niewiadomska, 2002). Our specimens have a genital cone butwithout a prepuce; therefore,Posthodiplostomum Dubois, 1936 can beruled out.MesoophorodiplostomumDubois, 1936 has an ovary betweenthe 2 tandem testes, and Ornithodiplostomum Dubois, 1936 has an ova-ry at the lateral side of the anterior testis. However, in our specimens,the ovary is anterior to the anterior testis near the junction between thefore- and hindbody. Therefore, we determined our specimens to be anew species ofNeodiplostomum ( Conodiplostomum) Dubois, 1937.

In Neodiplostomum (Conodiplostomum) or Conodiplostomum (raisedto a generic level by Sudarikov, 1971), 13 species in total have beendescribed previously, i.e., 12 from birds (Sudarikov, 1960; Yamaguti,1971) and 1 from mammals (Dronen et al., 1995). Neodiplostomumseoulense was originally described as Fibricola seoulensis (Seo et al.,1964) but was later transferred to Neodiplostomum (Hong and Shoop,1994, 1995), and it has been described to have a genital cone. Hence,we propose placing N. seoulense in subgenus Neodiplostomum ( Cono-diplostomum). The 12 bird-infecting species (Yamaguti, 1971) differfrom the new species in terms of the body size and shape, egg size,relative size of oral and ventral suckers, size and shape of the tribocytic

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SHIN ET AL.NEODIPLOSTOMUM BORYONGENSEN. SP. 1383

organ, distribution of vitelline follicles, and shape of testes. These spe-cies have an encysted metacercariae stage, the so-called neascus type(black spot-like), under the skin of the fish host (Sudarikov, 1960,1971; Shoop, 1989; Dronen et al., 1995), but the new species has anunencysted neodiplostomulum type of metacercaria in the mesenteryor omentum of the reptile host. The metacercariae of species infectingmammals,C. assymmetricum, are yet unknown; it has a small, unlobed,asymmetric anterior testis (Dronen et al., 1995), whereas the new spe-

cies infects birds, and has a large, bilobed, symmetric anterior testis.The new species differs from N. seoulense in the relative size of thegenital atrium versus the total body length; smaller in the new speciesthan in N. seoulense (Seo, 1990). The new species also differs from N.seoulense in the shape of the 2 testes; the testes of the former areseverely bilobed, with a prominent median constriction, forming an eye-glass shape, but those of the latter are less bilobed, resembling a but-terfly-shaped cufflink (Seo, 1990). The new species sexually matures inchicks, but N. seoulense uses rodents and humans as its definitive hosts(Seo, 1990). Neodiplostomum (Neodiplostomum) leei is also morpho-logically similar to the new species. However, N. leei has a larger ven-tral sucker and lacks a genital cone (Chai and Shin, 2002). Its vitellinefollicles are extensive, in both the fore- and hindbody, from the levelof the pharynx to near the posterior end of the body (Chai and Shin,2002), whereas those of the new species are less extensive, withoutdistribution between the pharynx and the ventral sucker. The testes ofthe new species are in the shape of an eye glass, but those of N. leei

form a long bridge between the right and left lobes, and they seem moredumbbell-shaped (Chai and Shin, 2002). The new species is larger interms of the size of the whole body and tribocytic organ and possesseseggs that are smaller than those ofN. seoulense and N. leei (Table I).The metacercariae of the new species, N. leei, and N. seoulense areimpossible to tell apart when they are harvested from the grass snake.The neodiplostomula of the new species, when given orally to mice andrats, never migrate to visceral organs; instead, they stay in the smallintestine for some time without development and then disappear by day14 PI.

Because the new species and several others reveal new features inmorphology and life cycle pattern, a modification of the generic andsubgeneric characteristics is warranted. An amended diagnosis is givenas follows.

Neodiplostomum(Conodiplostomum) Dubois, 1937, amend.

Diagnosis: Neodiplostomidae, Neodiplostominae. Body distinctlybisegmented. Vitelline follicles in both fore-and hindbody or confinedto hindbody. Genital cone present, without prepuce. Testes tandem, bi-lobed or unlobed; anterior testis symmetrical or asymmetrical. Ejacu-latory pouch absent. Neascus- or neodiplostomulum-type metacercariaein fish or reptiles; encysted or unencysted. Intestinal parasites of birdsor mammals.

Taxonomic summary

Type species: Neodiplostomum (Conodiplostomum) spathula (Crep-lin, 1829) Dubois, 1937.

Other species: N. (C.) accipitris Dubois and Rausch, 1948; N. (C.)acutum Dubois, 1937; N. (C.) assymmetricum Dronen et al., 1995; N.(C.) australiense Dubois, 1937;N. (C.) banghami Penrod, 1947; N. (C.)brachypterisChatterji, 1942;N. (C.)brachyurum(Nicoll, 1914) Dubois,1937; N. (C.) butasturinum (Tubangui, 1932) Dubois, 1936; N. (C.)

krausei Dubois, 1937; N. (C.) palumbarii Dubois, 1937; N. (C.) per-latum(Ciurea, 1911) Dubois, 1938; N. (C.)sarcorhamphiDubois, 1937.

Species newly assigned: N. (C.) boryongense n. sp. and N. (C.) seou-lense (Seo et al., 1964) Hong and Shoop, 1995.

DISCUSSION

The superfamily Strigeoidea Railliet, 1919 includes 4 fami-

lies, i.e., Strigeidae Railliet, 1919, Diplostomidae Poirier, 1886,

Cyathocotylidae Poche, 1926, and Proterodiplostomidae Du-

bois, 1936, that have a distinct fore- and hindbody and a genital

pore at the posterior end of the body (Schell, 1985). By com-

paring 35 characters and character states of their adult and

metacercaria morphology, Shoop (1989) reconstructed the Stri-

geidae and Diplostomidae through a phylogenetic analysis and

created the Neodiplostomidae for those species without pseu-

dosuckers. Shoop (1989) assigned 2 new subfamilies. Neodip-

lostominae embraces those genera having neodiplostomulum

metacercariae but lacking a genital cone in the adult stage; in-

cluded are species of Neodiplostomum and Fibricola. Crassi-phialinae, in contrast, includes genera that possess a neascus

type metacercariae and a genital cone, i.e., Conodiplostomum.

Our specimens have a neodiplostomulum metacercaria and a

genital cone in adults. They are therefore incompatible with

either Neodiplostomum or Conodiplostomum of Shoop (1989).

To cope with this taxonomic problem, we referred to Dubois

(1970), who divided the genus Neodiplostomum Railliet, 1919,

into 2 subgenera: Neodiplostomum (Neodiplostomum) Dubois,

1937, to include those species with an asymmetric anterior tes-

tis but lacking a genital cone, and Neodiplostomum (Conodip-

lostomum) Dubois, 1937, to include those species having a sym-

metric anterior testis and a genital cone. Sudarikov (1971) el-

evated Conodiplostomum to a generic status, a decision sup-

ported by Shoop (1989), D ronen et al. (1995), andNiewiadomska (2002). In contrast, Pearson (1959) and Schell

(1985) retained the subgeneric status of Conodiplostomum, and

Okulewicz et al. (1993) used Neodiplostomum (Conodiplosto-

mum) perlatumfor specimens collected from Poland. Cribb and

Pearson (1993) also proposed to retain the subgeneric status of

Conodiplostomum within Neodiplostomum without using the

subgeneric name. In agreement with Schell (1985) and Cribb

and Pearson (1993), we have retained the subgeneric status of

Neodiplostomum (Conodiplostomum) and proposed an amend-

ment of this subgenus.

The present study confirms that the neodiplostomula inhab-

iting the European grass snake in the Republic of Korea in-

cludes at least 4 species, i.e., P. cordatum (large-sized neodip-

lostomula), N. seoulense (small-sized neodiplostomula), N. leei(small-sized neodiplostomula), and N. (Conodiplostomum) bor-

yongense n. sp. (small-sized neodiplostomula). Although the

life history of N. seoulense has been studied extensively, the

life cycles of the new species and N. leei are poorly known. As

for N. seoulense, the freshwater snail Hippeutis cantori or Seg-

mentina (Polypylis) hemisphaerula and tadpoles and frogs of

Rana nigromaculata are the first and second hosts, respectively

(Seo et al., 1988; Seo, 1990; Chung et al., 1996); the grass

snake, R. tigrina, is a paratenic host (Seo, 1990). Natural in-

fections with N. seoulenseadults have been reported in rats and

mice (Seo et al., 1964; Chai et al., 2007), as well as in humans

(Hong et al., 1984, 1986). Neodiplostomum leei was reported

only from chicks experimentally infected with neodiplostomula

from the viscera of grass snakes or in the livers of rodents (Chaiand Shin, 2002). Tadpoles and frogs of Rana sp. are presumed

to be the second intermediate hosts for N. leei and N. boryon-

gensen. sp.; however, this hypothesis should be verified. Some

avian species are likely to be the definitive hosts for both spe-

cies, but this also needs verification. The grass snake seems to

be a common paratenic host for N. leei, N. seoulense, and N.

boryongensen. sp.; however, N. leei may have other prolonged

paratenic hosts, i.e., mice and rats (Chai and Shin, 2002; Shin

et al., 2006).

The geographic distribution of N. leei, N. seoulense, and N.

boryongensen. sp. in the Korean peninsula overlaps, and mixed

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infections with all 3 species of neodiplostomula in a grass snake

are quite common. However, the sites of infection by each spe-

cies differ. The distribution of N. seoulense is almost nation-

wide, including, in particular, mountainous inland areas of

Gyeonggi-do, Gangwon-do, and Chungcheongbuk-do provinces

(Seo, 1990; Chai et al., 2000). In contrast, N. leei is detected

mainly in the midwestern part of the peninsula, including 7locations, i.e., Ansung and Pyungtaek counties in Gyeonggi-do

Province and Chunan, Asan, Onyang, Yeasan, and Hongsung

counties in Chungcheongnam-do Province (Chai and Shin,

2002). The distribution ofN. boryongense n. sp. has been con-

firmed in Boryong, Chunan, Asan, Onyang, Yeasan, and Hong-

sung counties of Chungcheongnam-do Province, of which 5

counties excluding Boryong overlap with the distribution areas

of N. leei.

ACKNOWLEDGMENTS

This work was supported by BK21 Human Life Sciences, Ministryof Education, Republic of Korea.

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