International Journal of Fisheries and Aquatic Studies 2019; 7(4): 334-338
E-ISSN: 2347-5129
P-ISSN: 2394-0506
(ICV-Poland) Impact Value: 5.62
(GIF) Impact Factor: 0.549
IJFAS 2019; 7(4): 334-338
© 2019 IJFAS
www.fisheriesjournal.com
Received: 11-05-2019
Accepted: 15-06-2019
PK Prasadan
Ecological Parasitology and
Tropical Biodiversity
Laboratory, Department of
Zoology, Kannur University,
Mananthavady Campus,
Wayanad, Kerala, India
Sherin Chacko
Ecological Parasitology and
Tropical Biodiversity
Laboratory, Department of
Zoology, Kannur University,
Mananthavady Campus,
Wayanad, Kerala, India
Clinostomum cupani n. sp. metacercaria infecting the
freshwater fish, Pseudosphromenus cupanus from the
Western Ghats, Wayanad region, India
PK Prasadan and Sherin Chacko
Abstract
Clinostomum cupani n. sp. (Digenea, Clinostomidae) from the operculum and body cavity of the
freshwater fish Pseudosphromenus cupanus collected from water bodies in the Wayanad region of the
Western Ghats is described and illustrated. C. cupani n. sp. is new to the genus and is separated from its
congeners on the basis of differences in morphology and morphometry. Detailed comparison of the
present species with C. complanatum Rudolphi, 1814 and C. marginatum Rudolphi, 1819 ismade in this
study. C. cupani n. sp. differs from all other species of Clinostomum Leidy, 1856 in the shape of its
uterus and the presence of pigmented eyespots. It is named after the host P. cupanus. The prevalence of
infection of C. cupani n. sp. is 50%, the intensity of infection is 12.33 and the mean abundance is 6.16.
Keywords: Clinostomum cupani n. sp., digenea, metacercaria, second intermediate host, fish
1. Introduction
The genus Clinostomum Leidy, 1856 (Digenea: Clinostomidae) has heteroxenous life cycle.
Adults are found in the buccal cavity and esophagus of birds, reptiles and mammals [1]. The
freshwater snails are their first intermediate hosts and freshwater fishes or amphibians as their
second intermediate hosts [2, 3]. The metacercariae of Clinostomum are usually known as
yellow grubs. This fish-borne zoonotic trematode causes acute pharyngitis or laryngitis in
those who eat raw fish meat with metacercarial infection [4]. Human infections have been
reported by many workers [4- 9].
Because of the high degree of morphological inconsistency within the same species,
Clinostomum had been subjected to several taxonomic reviews [1, 2, 10-13], of which Ukoli [1] was
one of the pioneer reviewers, who synonymized 20 Clinostomum species with C. complanatum
and documented 13 valid species. Several species were then added to or detracted from the
genus Clinostomum with the support of molecular and conventional taxonomic studies. Now
this cosmopolitan genus encompasses 29 valid species [14].
The present paper describes the morphological features and taxonomic status of a new species
of digenetic trematode metacercaria, C. cupani n. sp. infecting operculum and body cavity of
freshwater fish P. cupanus from the Wayanad region of the Western Ghats.
2. Materials and Methods
2.1 Study area
The study was carried out in the Wayanad region of the Western Ghats, one of the hottest hotspots of biodiversity in the world due to its incredible biological diversity and a high degree of
endemism. The geographical location of Wayanad region is 11° 27' to 15° 58' north latitude
and 75° 47' to 70° 27' east longitude.
Correspondence
PK Prasadan
Ecological Parasitology and
Tropical Biodiversity
Laboratory, Department of
Zoology, Kannur University,
Mananthavady Campus,
Wayanad, Kerala, India
2.2 Methods
Host fish specimens collected from different water bodies using sweep net were kept alive,
brought to the laboratory, maintained in clean glass jars or aquarium and occasionally fed with
cooked rice or fish food. Fish were sacrificed and body surface, fins, abdominal cavity, gill
filaments, eyes and muscles were carefully examined for the presence of metacercariae.
Internal organs were dissected out, placed in separate Petri dishes containing
~ 334 ~
International Journal of Fisheries and Aquatic Studies
0.75% saline, macerated and observed under the binocular
LABOMED Luxeo 4Z stereo zoom microscope. The live
encysted meta cercariae were transferred to 0.75% saline,
excysted by rupturing the cyst wall with a fine needle. Studies
on meta cercariae were carried out on live and fixed
specimens, without supravital staining or staining with neutral
red or methylene blue, under Nikon ECLPSE Ni-U research
microscope. Permanent whole mounts were prepared after
fixing them in 5% formalin under slight cover glass pressure,
and then staining with acetocarmine, following the procedure
outlined by Cantwell [15]. Measurements (in µm) were taken
with the support of Nikon NIS Elements Imaging software.
Sketches were prepared with the Nikon Y- IDT drawing tube
attached to the Nikon ECLPSE Ni-U research microscope and
Photographs were taken with Nikon Y-TV55 camera attached
to a Nikon ECLPSE Ni-U research microscope with the
support of Nikon NIS Elements Imaging Software.
Prevalence, intensity and mean abundance of infection were
measured following Bush et al. [16].
1979.74-2957.25 ×125.52-274.13 (2506 × 181.65), broad at
the postacetabular region, lateral pouches are not prominent,
extending up to the posterior end of the body. Testes deeply
lobed; anterior testis irregular, close to the right margin of left
caecum. 143.36-242.49 × 142.93-301.07 (181.70 × 215.44).
Posterior testis slightly triangular, inter-caecal, 74.24-228.43
× 106.80-366.26 (151.34 × 232.02). Distance between testes
107.11-178.96 (147.23). Cirrus pouch large, slightly ovoid,
113.48-254.90 × 77.05-166.86 (177.88 × 107.75), in the intertesticular space close to the left margin of the right caecum.
Ovary small, slightly oval, 57.81-100.16 × 55.19-91 (78.65 ×
69.01). Ootype at the inter-testicular space between ovary and
the left intestinal caecum. Uterine sac with lateral
evaginations, 209.08- 606.43 × 106.60-324.69 (429.96 ×
218.69) partially covers the intercaecal space in that region.
Excretory bladder V-shaped.
3. Results
3.1 Clinostomum cupani n. sp. (Fig. 1)
Body linguiform, 2188.00-3090.00 (2716.36) long and
648.96-951.24 (815.73) wide. Slightly ovoid anterior part
bears a small sub-terminal oral sucker, 138.14-207.55 ×
149.34-222.57 (171.33 × 195.87), surrounded by oral collar
235.08-319.95 × 372.94-562.15 (270.14 × 443.66). Cuticle
aspinose. Ventral sucker large, well developed, round at the
posterior region, 296.61-551.09 × 306.48-524.04 (397.91 ×
384.13). Distance between suckers 299.19-398.97 (342.04).
Short pre-pharynx, 52.88-84.12 × 11.76-26.77 (69.74 ×
18.32). Pharynx rudimentary, 58.32-85.48 × 40.78-74.68
(69.63 × 59.26).
Fig 2: Eyespots of Clinostomum cupani n. sp. metacercaria marked
with a rectangular box.
Fig 1: Clinostomum cupani n. sp. Metacercaria (Photograph and line
drawing).
Eyespots at the level of the anterior part of pharynx, anterior
to caecal shoulders. Esophageal bulb absent. Intestinal caeca
3.2 Taxonomic summary
Type specimen: Holotype (No. Z-TM 42) deposited in the
helminth parasite collections, Ecological Parasitology and
Tropical Biodiversity Laboratory, Department of Zoology,
Kannur University, Mananthavady Campus, Wayanad670645, Kerala, India.
Type host: P. cupanus ((No. Z-FTM 147) deposited in the
Ichthyology collections, Department of Zoology, Kannur
University, Mananthavady Campus, Wayanad-670645,
Kerala, India.
Type locality: Moolithod, Wayanad District, Kerala, India.
Site of infection: Operculum and body cavity.
Period of collection: June 2018 to January 2019
Prevalence: Three of six fishes (50%) screened were infected.
Intensity: Thirty seven C. cupani n. sp. recovered from three
infected fish (12.33).
Mean abundance: Thirty seven C. cupani n. sp. recovered
from a total of six fishexamined and, therefore, the mean
abundance is 6.16.
Of the six P. Cupanus examined, three were found infected
with a total of 37 parasites. The prevalence of infection was
50%, while the intensity of infection stood at 12.33.
4. Discussion
The present study showing a detailed morphological
description of the metacercaria of C. cupani n. sp.,
~ 335 ~
International Journal of Fisheries and Aquatic Studies
emphasizes the presence of pigmented eyespots (Fig. 2) and
large uterus with lateral pouches. According to Ukoli [1] the
significant characters which are not likely to vary within the
species and which are, therefore, most reliable for the
purposes of classification of Clinostomum species are the
presence of lateral evaginations in the uterine sac and the
anterior extent of the vitellaria. The characters such as the
position of gonads, genital pore & cirrus pouch and the shape
of testes, which vary between meta cercariae and adults and
all other characters that are likely to change due to external
influences.
Table 1: Comparison of morphometric characters (in µm) of metacercariae of the C. marginatum Rudolphi, 1819, C. complanatum Rudolphi,
1814 [11] and C. cupani n. sp.
Clinostomum complanatum Rudolphi,
Clinostomum cupani n. sp. (n = 7)
min–max (mean)
1814 (n = 11) min–max (mean)
Stout, oval, elongate, wider in gonadal
Oval, elongate, wider in gonadal region
Oval, elongate
region
Body L × B
4215.00–6346.00 × 979.00–1532.00
2188.00–3090.00 × 648.96–951.24
4495.00–7874.00 × 1635.00–2434.00
(5402.00 × 1329. 00)
(2716.36 × 815.73)
(5741.00 × 1934.00)
Body L/B
3.05–4.40 (4.03)
2.20–4.37 (2.99)
3.10–3.52 (3.29)
Oral collar B
502.00–874.00 (721.00)
686.00–1030.00 (820.00)
372.94–562.15 (443.66)
Oral sucker L × 238.00–599.00 × 194.00–586.00 (312.00 259.00–337.00 × 284.00–507.00 (294.00 × 138.14–207.55 × 149.34–222.57 (171.33
B
× 290.00)
401.00)
× 195.87)
Oral sucker
0.82–1.05 (0.92)
1.06–1.67 (1.36)
0.22–0.27 (0.23)
B/body B
Ventral sucker
Larger than oral 510.00–757.00 ×
Larger than oral 637.00–910.00 × 766.00–
Larger than oral 296.61–551.09 ×
L×B
571.00–804.00 (669.00 × 708.00)
952.00 (795.00 × 839.00)
306.48–524.04 (397.91 × 384.13)
Ventral sucker
0.99–1.12 (1.06)
1.78–2.69 (2.14)
1.70–2.46 (1.95)
B/oral sucker B
Ventral sucker
0.47–0.62 (0.53)
0.39–0.49 (0.44)
0.42–0.55 (0.465)
B/body B
Distance
between
959.00–1491.00 (1243.00)
860.00–1115.00 (1020.00)
299.19–398.97 (342.04)
suckers
Anterior testis 231.00–413.00 × 264.00–498.00 (307.00 316.00–957.00 × 273.00–559.00 (484.00 × 143.36–242.49 × 142.93–301.07 (181.70
L×B
× 389.00)
412.00)
× 215.44)
Anterior testis
0.68–1.72 (1.29)
0.46–1.22 (0.90)
0.76–1.34 (1.18)
B/L
Posterior testis 233.00–457.00 × 295.00–492.00 (327.00 245.00–441.00 × 408.00–602.00 (328.00 × 74.24–228.43 × 106.80–366.26 (151.34 ×
L×B
× 405.00)
493.00)
232.02)
Posterior testis
0.89–1.52 (1.25)
1.09–1.88 (1.54)
1.19–2.00 (1.52)
B/L
Distance
196.00–393.00 (320.00)
214.00–527.00 (353.00)
107.11–178.96 (147.23)
between testes
85.00–142.00 × 62.00–97.00 (115.00 × 135.00–164.00 × 97.00–178.00 (149.00 × 57.81–100.16 × 55.19–91.00 (78.65 ×
Ovary L × B
80.00)
129.00)
69.01)
Ovary B/L
0.43–0.97 (0.71)
0.59–1.09 (0.87)
0.65–1.05 (0.88)
Well developed, crescent shaped,
Well developed, extending from inter
Large and slightly ovoid, in the intercompressed against left anterior margin of testicular space to posterior right margin of testicular space close to the left margin of
Cirrus sac L× B
anterior testis 172.00–343.00 × 119.00– anterior testis 209.00–405.00 × 124.00–
right caeca 113.48–254.90 × 77.05–
153.00 (256.00 × 137.00)
197.00 (296.00 × 157.00)
166.86 (177.88 × 107.75)
Cirrus sac
0.03–0.06 (0.05)
0.03–0.07 (0.05)
0.05–0.08 (0.06)
L/body L
Located at the level of the anterior part of
Eyespots
Not observed
Not observed
pharynx, anterior tothe caecal shoulders
Large and wider uterus. Uterine sac with
Uterine sac L ×
Tubular proximal uterus. No lateral
Tubular proximal uterus. No lateral
lateral evaginations
B
digitations
digitations
209.08–606.43 × 106.60–324.69 (429.96
× 218.70)
Rudimentary
Pharynx
Not observed
Not observed
58.32–85.48 × 40.78–74.68 (69.63 ×
59.27)
Slightly long 52.88–84.12 ×11.76–26.77
Pre-pharynx
Not observed
Not observed
(69.74 × 18.32)
Oesophagus
Very short. oesophageal bulb present
Oesophageal bulb well developed
Oesophagus and oesophageal bulb absent
Lateral to ventral sucker, characterized by
Lateral pouches in intestinal caeca are not
Lateral to ventral sucker and genital
Intestinal caeca visible intestinal pouches especially in
prominent Caeca more bulbous at
complex, with prominent lateral pouches
postacetabular part
postacetabular region
Characters
Clinostomum marginatum Rudolphi,
1819 (n = 10) min–max (mean)
The present species has some general morphological
similarities with the C. marginatum (Table 1). But it exhibits
some distinct morphological features such as the presence of
pigmented eyespots, large sacculated uterus, pre-pharynx,
rudimentary pharynx; absence of esophagus, positioning of
the cirrus pouch and absence of cuticular spines. The
described species also exhibits changes in morphometry such
as distance between suckers, the ratio between oral sucker
~ 336 ~
International Journal of Fisheries and Aquatic Studies
width and body width and distance between testes.
The present species displays some similarity with C.
complanatum (Table 1). Detailed study revealed that the
species differs from C. complanatum [11] on many
morphological features and morphometry like presence of
pigmented eyespots, pre-pharynx, rudimentary pharynx,
difference in the distance between suckers, ratio between oral
sucker width and body width, size of anterior testis, distance
between testes and shape & size of uterine sac. Neither C.
complanatum nor C. marginatum has lateral digitation of the
uterine sac [17] and presence of pigmented eyespots
distinguishes the described species from these two species.
The species described under the genus Clinostomum from
India documented by Pandey and Agrawal [18] in their work
‘The metacercarial fauna of India’ included C. piscidium
Southwell and Prashad, 1918, C. prashadi Bhalerao, 1942, C.
gideoni Bhalerao, 1942, C. dasi Bhalerao, 1942, C. indicum
Bhalerao, 1943, Clinostomum sp. Srivastava, 1950, C.
schizothoraxi Kaw, 1950, C. microstomum Singh, 1955, C.
giganticum Agarwal, 1955, C. macrosomum Jaiswal, 1957, C.
mastacembeli Jaiswal, 1957, C. progonum Jaiswal, 1957, C.
orientale Mukherjee, 1967, C. lucknowensis Pandey, 1968, C.
trichogasteri Pandey, 1969 and Clinostomum sp. Rekharani
and Madhavi, 1985. The presence of pigmented eyespots and
lateral evaginations in the uterine sac are not reported in any
of these species.
Concerning the other species described, at the metacercarial
stage, the morphological features observed in the present
species differ consistently from C. cutaneum, which has a
unique Y-shaped uterine sac [10], C. phalacrocoracis
metacercaria that bears fan-shaped testes and straight uterus
running from ventral sucker to anterior testis [19]. Although the
presence of lateral digitations in the uterine sac is a common
feature in C. detruncatum and the present species [17] the
former lacks eyespots. The present species varies from C.
tilapiae in which body surface is armed with numerous
cuticular spines and straight uterus running from ventral
sucker to anterior testis [20]. The species described is different
from C. caffarae on the nature of testes, from C. arquus on
the shape of testes & nature of uterine sac and from C.
Cichlidorum on the absence of pharyngeal bulb [14].
5. Acknowledgments
Sherin Chacko is grateful to the Department of Science &
Technology (DST) INSPIRE program for providing Inspire
fellowship to carry out this study.
6. References
1. Ukoli FMA. On Clinostomum tilapiae n. sp., and C.
phalacrocoracis Dubois, 1931 from Ghana, and a
discussion of the systematics of the genus Clinostomum
Leidy, 1856. Journal of Helminthology. 1966; 40:187214.
2. Matthews D, Cribb TH. Digenetic trematodes of the
genus
Clinostomum
Leidy,
1856
(Digenea:
Clinostomidae) from birds of Queensland, Australia,
including C. wilsoni n. sp. from Egretta intermedia.
Systematic Parasitology. 1998; 39:199-208.
3. Dias ML, Eiras JC, Machado MH. The life cycle of
Clinostomum complanatum Rudolphi, 1814 (Digenea,
Clinostomidae) on the floodplain of the high Parani river,
Brazil. Parasitology Research. 2003; 89(6):506-508.
4. Lee GS, Park SW, Kim J, Seo KS, You KW, Chung JH,
et al. A case of endoscopically treated laryngopharyngitis
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
~ 337 ~
resulting from Clinostomum complanatum infection.
Korean Journal of Gastroenterology. 2017; 69:177-180.
Kitagawa N, Oda M, Totoki T, Washizaki S, Oda M,
Kifune T. Lidocaine spray used to capture a live
Clinostomum parasite causing human laryngitis.
American Journal of Otolaryngology. 2003; 24:341-343.
Sohn WM. Fish-borne zoonotic trematode metacercariae
in the Republic of Korea. Korean Journal of Parasitology.
2009; 47:S103-S113.
Hara H, Miyauchi Y, Tahara S, Yamashita H. Human
laryngitis caused by Clinostomum complanatum. Nagoya
Journal of Medical Science. 2014; 76:181-185.
Jung SC, Oh HJ, Kim DM, Park JH. A case of
pharyngitis caused by Clinostomum complanatum.
Korean Journal of Otorhinolaryngology-Head and Neck
Surgery. 2015; 58(1):61-3.
Song HB, Choi MH, Chung EJ. Human Laryngeal
Infection by Clinostomum complanatum. American
Journal of Tropical Medicine and Hygiene. 2018;
98(1):7-8.
Gustinelli A, Caffara M, Florio D, Otachi EO, Wathuta
EM, Fiovaranti ML. First description of the adult stage of
Clinostomum cutaneum Paperna, 1964 (Digenea:
Clinostomidae) from grey herons Ardea cinerea L. and a
redescription of the metacercaria from the Nile tilapia
Oreochromis niloticus niloticus (L.) in Kenya.
Systematic parasitology. 2010; 76:39-51.
Caffara M, Locke SA, Gustinelli A, Marcogliese DJ,
Fiovaranti ML. Morphological and molecular
differentiation of Clinostomum complanatum and
Clinostomum marginatum (Digenea: Clinostomidae)
metacercariae and adults. Journal of parasitology. 2011;
97(5):884-891.
Sereno-Uribe AL, Pinacho-Pinacho CD, García-Varela
M, de León GPP. Using mitochondrial and ribosomal
DNA sequences to test the taxonomic validity of
Clinostomum complanatum Rudolphi, 1814 in fish-eating
birds and freshwater fishes in Mexico, with the
description of a new species. Parasitology research. 2013;
112:2855-2870.
Rosser TG, Alberson NR, Woodyard ET, Cunningham
FL, Pote LM, Griffin MJ. Clinostomum album n. sp. and
Clinostomum marginatum (Rudolphi, 1819), parasites of
the great egret Ardea alba L. from Mississippi, USA.
Systematic Parasitology. 2017; 94:35-49.
Sereno-Uribe AL, García-Varela M, Pinacho-Pinacho
CD, de León GPP. Three new species of Clinostomum
Leidy, 1856 (Trematoda) from Middle American fisheating birds. Parasitology Research. 2018, 1-15.
Cantwell GE. Methods for invertebrates. In: (Eds. Clark
G.) Staining procedures. Baltimore, Williams and
Wilkins, 1981, 255-280.
Bush AO, Lafferty KD, Lotz JM, Shostak AW.
Parasitology meets ecology on its own terms: Margolis et
al. Revisited. Journal of Parasitology. 1997; 83(4):575583.
Acosta AA, Caffara M, Fioravanti ML, Utsunomia R,
Zago AC, Franceschini L et al. Morphological and
molecular characterization of Clinostomum detruncatum
(Trematoda: Clinostomidae) metacercaria infecting
Synbranchus marmoratus. Journal of Parasitology. 2016;
102:151-156.
Pandey KC, Agrawal N. Metacercarial Fauna of India.
Records of Zoological Survey of India, 2013, 117-135.
International Journal of Fisheries and Aquatic Studies
19. Caffara M, Davidovich N, Falk R, Smirnov M, Ofek T,
Cummings D et al. Redescription of Clinostomum
phalacrocoracis metacercariae (Digenea: Clinostomidae)
in cichlids from Lake Kinneret, Israel. Parasite. 2014;
21(32):1-6.
20. Caffara M, Locke SA, Echi PC, Halajian A, Benini D,
Luus-Powell WJ et al. A morphological and molecular
study of Clinostomid metacercariae from African fish
with a redescription of Clinostomum tilapiae.
Parasitology. 2017, 1-11.
~ 338 ~