Pakistan J. Parasitol., 68; December 2019: 33-38

HISTOPATHOLOGY AND OTHER ASPECTS OF THE CLINOSTOMUM

COMPLANATUM INFECTION IN THE FRESHWATER FISH, PSEUDOSPHROMENUS

CUPANUS FROM THE SOUTH WESTERN GHATS

Jithila, P.J. and Prasadan, P.K.*

Ecological Parasitology and Tropical Biodiversity Laboratory, Department of Zoology,

Kannur University, Mananthavady Campus, Wayanad–670645, Kerala, India

*Corresponding author: prasadanpk@kannuruniv.ac.in

Abstract

The pathological effects of Clinostomum complanatum on the operculum and head muscles of the freshwater fish

Pseudosphromenus cupanus (P. cupanus) and occurrence, intensity and mean abundance of the parasitic infection were

studied. Host specimens were collected from small rivulets in the Wayanad region of the Western Ghats. The

histopathology of operculum and head muscles showed heavy necrosis and fibrosis of tissues and infiltration of

immune cells at the site of infection. The study reports the prevalence (44%), intensity (8.77) and mean abundance

(3.86) of C. complanatum infection in P. cupanus. The parasite is probably pathogenic to the host and the fish bears

heavy burden of the parasites.

Keywords: Histopathology, Clinostomum complanatum, Pseudosphromenus cupanus, Western Ghats, Wayanad.

INTRODUCTION

Fishes are hosts to taxonomically diverse helminth parasites and infections can significantly affect behaviour,

fecundity, metabolism, health and survival of fishes (Barber et al., 2000; Lafferty, 2008; Seppanen et al., 2009). The

genus Clinostomum with its broad spectrum of species has a wide geographic distribution with several species of

freshwater fishes and amphibians acting as second intermediate hosts and a wide range of piscivorous birds as

definitive hosts.

The metacercaria of C. complanatum infects skin, head, muscles, mesenteries, viscera and fins of freshwater

fishes (Ukoli, 1966; Dias et al., 2003; Shareef and Abidi, 2012; Jyrwa et al., 2016). It causes yellow grub disease in

fishes (Kagei et al., 1984) and an earlier study documented yellow grub disease in the ornamental fish Poecilia

reticulata of Wayanad (Prasadan and Devi, 2007). The fishes act as potential second intermediate hosts of

Clinostomum spp. and may result in the occurrence of various pathological conditions in fishes (Kagei et al., 1984;

Belló et al., 2000). Apart from the threat posed to the fish culture operations, C. complanatum has been recognized

as an important zoonotic agent. Human infections were reported from various geographical areas (Yamashita, 1938;

Cameron, 1945; Hirai et al., 1987; Park et al., 2009).

The paper describes the pathological effects of C. complanatum on the operculum and head muscles of the

freshwater fish P. cupanus. The paper also reports the prevalence, intensity and mean abundance of C. complanatum

infection in P. cupanus.

MATERIALS AND METHODS

Epidemiological study: Sweep net were used in the Wayanad region in order to collect host specimens from small

rivulets. The fishes were brought alive to the laboratory in suitable containers. The fishes were investigated for the

presence of metacercarial cysts, sacrificed by cervical rupture and were observed under a Labomed (Luxeo 4Z)

stereozoom microscope for larval digeneans. Metacercariae, when present, were studied under Nikon ECLIPSE Ni-

U phase contrast research microscope (Nikon, Japan) with or without vital staining. The excysted larvae were

examined by permanent whole mounts fixed in 5% Formalin under slight cover glass pressure and staining with

Acetocarmine (Cantwell, 1981).

Prevalence, intensity and mean abundance of infection were measured following Bush et al., (1997). Prevalence

is the number of hosts infected with one or more individuals of a particular parasite species (or taxonomic group)

divided by the number of hosts examined for that parasite species. It is commonly expressed as percentage. Intensity

of infection is the total number of parasites of a particular species found in a sample divided by the number of hosts

infected with that parasite. Mean abundance is the total number of individuals of a particular parasite species in a

Jithila and Prasadan 34

sample of a particular host species divided by the total number of hosts of that species examined (including both

infected and uninfected hosts).

Histopathological study: For histopathological studies infected tissues were washed thoroughly with water and

preserved in buffered Formalin for about 24 hours. After thorough washing the specimens were dehydrated in Ethyl

Alcohol series (50%, 70%, 90% and 100%) and Acetone followed by clearing in Methyl Benzoate. Paraffin blocks

were prepared with molten wax maintained at 58oC. Serial sections having 7 µm thickness were cut using the

Microtec rotatory microtome (Germany) and stained with Heidenhain’s Haematoxylin and Eosin to study the

histopathology and infiltration of cells at the site of attachment. The DPX mounted sections were studied under

Nikon ECLIPSE Ni-U phase contrast research microscope and photographed with the Nikon Y-TV55 camera

attached to the microscope.

RESULTS

Epidemiology

Out of the 150 specimens of P. cupanus examined, 66 were infected with C. complanatum (Fig. 1). The average

length of P. cupanus examined was 5.72 cm. Both excysted and encysted metacercariae were obtained. The

encysted metacercariae were found attached to the operculum, muscles in the head region and mesenteries; the

excysted metacercariae were found free in the body cavity. The body surface of the infected fishes seem to be

normal with no signs of yellow grub (Fig. 2) were noticed. The abdominal region showed swelling due to heavy

infestation. The infected P. cupanus showed sluggish movement.

Prevalence of infection: Sixty six of 150 P. cupanus screened were infected and the prevalence of infection was

44%.

Intensity of infection: Five hundred and seventy nine C. complanatum were recovered from 66 infected P. cupanus

and hence the intensity was 8.77.

Mean abundance: Five hundred and seventy nine C. complanatum were recovered from 150 P. cupanus examined

and therefore, the mean abundance was 3.86.

Fig. 1: The excysted metacercaria of C. complanatum. A. Anterior part B. Posterior part

HISTOPATHOLOGY AND OTHER ASPECTS OF THE CLINOSTOMUM COMPLANATUM INFECTION 35

Fig. 2: P. cupanus (Valenciennes)

Histopathology

The histopathology showed that the opercular epithelia surrounding the metacercariae were necrotic (Fig. 3). There

is heavy infiltration of lymphocytes and eosinophils in the site of infection and its surrounding tissues (Figs. 4 and 5). The number of lymphocytes seems to be high in the site of infection, whereas that of eosinophils were high at the surrounding tissue. The opercular muscle tissue surrounding the metacercariae showed heavy necrosis and fibrosis (Fig. 6).

The histopathology revealed that the head muscle tissue surrounding the metacercarial cyst was necrotic and fibrotic, whereas the underlying tissue seemed to be normal. The erosion of epithelia surrounding the cyst was evident (Fig. 7). There is severe tissue damage and infiltration of immune cells at the region of infection.

DISCUSSION

The genus Clinostomum Leidy, 1856 was established for Rudolphi’s worm Distoma complanatum (1819). The

Clinostomum complanatum is designated as its type species (Braun, 1899) and various studies reported the metacercaria of C. complanatum (Rudolphi, 1819) Braun, 1899 from freshwater fishes of India (Siddiqui and Nizami, 1982; Jyrwa et al., 2016). Metacercariae of Clinostomum species described from freshwater fishes of India included C. piscidium Southwell and Prashad, 1918; C. prashadi Bhalerao, 1942; C. dasi Bhalerao, 1942; C. gideoni Bhalerao, 1942; C. indicum Bhalerao, 1943; Clinostomum sp. Srivastava, 1950; C. schizothoraxi Kaw, 1950; C. macrosomum Jaiswal, 1957; C. mastacembeli Jaiswal, 1957; C. giganticum Agarwal, 1959; C. orientale Mukherjee, 1967 and metacercaria of Clinostomum sp. Rekharani and Madhavi, 1985.

Earlier studies showed that several freshwater fishes, including edible and ornamental fishes act as potential intermediate hosts of C. complanatum. More than 25 species of fishes have been reported from different localities of the world as the second intermediate host for C. complanatum (Aohagi et al., 1992; Malek and Mobedi, 2001; Vianna et al., 2005; Thapa et al., 2008; Aghlmandi et al., 2018). C. complanatum is of zoonotic importance as there are reports of laryngopharyngitis in humans due to C. complanatum infection (Hirai et al., 1987; Chung et al., 1995; Lee et al., 2017). Thus studies on the pathological effects on infected fishes have considerable importance.

We found the prevalence of infection was 44%. An earlier study pointed out that the size of fish was not related to the intensity of infection with C. complanatum and they also observed high prevalence of infection (66.7%) (Silva-Souza and Ludwig, 2005). The present work agrees with the above report as the size of fishes did not have any correlation with the intensity of infection.

Pathophysiology of clinostome metacercarial infection in fishes has been extensively studied and the results indicated that the infection causes severe damage in various tissues (Kalantan et al., 1987; Eiras et al., 1999; Vankara and Vijayalakshmi, 2009). In the present study the histopathology showed heavy necrosis and fibrosis of tissue and immune cells were infiltrated at the site of infestation. The erosion of epithelial cells was evident. The

Jithila and Prasadan 36

results are in agreement with that of the previous studies (Lo et al., 1981; Adeyemo and Agbede, 2008; Shareef and Abidi, 2012) as the infection causes severe damage in the musculature of fish.

The immune cells were infiltrated at the site of infestation was also noted in an earlier study (Shareef and Abidi, 2012). According to Secombes and Chappell (1996) the helminth infections may significantly alter the numbers of leucocytes in the circulation and recruit leucocytes to the site of infestation. The presence of lymphocytes and eosinophils in the infected part may be the result of an early immune response (García et al., 1993). Innate and adaptive defence mechanisms support the fishes in minimizing the impact of parasitic infection (Jones, 2001). Further studies are required to throw ample light to the immunohistochemistry of C. complanatum infection. .

Fig. 3: The cross section of operculum of infected P.

cupanus.

Fig. 4: Necrotic and fibrotic muscle tissues surrounding the

parasite, infiltration of immune cells. Metacercaria (arrow

head), tissue damage (arrow) and infiltration of immune

cells (asterisk).

Fig. 5: Infiltration of immune cells.

Fig. 6: The excysted metacercaria on the operculum, necrotic

and fibrotic muscle tissue surrounding it. Metacercariae (arrow

head) and tissue damage (arrow).

HISTOPATHOLOGY AND OTHER ASPECTS OF THE CLINOSTOMUM COMPLANATUM INFECTION 37

Fig. 7: Severe tissue damage and degeneration of cells. Metacercaria (arrow head), tissue damage (arrow) and

degeneration of cells (asterisk).

ACKNOWLEDGEMENTS

The authors are grateful to Kannur University for providing financial support for this work. One of the authors

(Jithila, P.J.) is thankful to Kerala State Council for Science Technology and Environment for the KSCSTE

Fellowship.

LITERATURE CITED

Adeyemo, A.O. and Agbede, S.A. 2008. Histopathology of Tilapia tissues harbouring Clinostomum tilapiae parasites. Afr. J.

Biomed. Res., 11: 115-118.

Agarwal, S.M. 1959. Studies on the morphology, systematics and life history of Clinostomum giganticum n. sp. (Trematoda:

Clinostomatidae). Indian J. Helminth., 11: 75-115.

Aghlmandi, F., Habibi, F., Afraii, M.A., Abdoli, A. and Shamsi, S. 2018. Infection with metacercaria of Clinostomum

complanatum (Trematoda: Clinostomidae) in freshwater fishes from Southern Caspian Sea Basin. Revue. Méd. Vét., 169:

147-151.

Aohagi, V., Shibahara, T., Machlda, N., Vamaga, V. and Kagota, K. 1992. Clinostomum complanatum (Trematoda:

Clinostomatidae) in Five New Fish Hosts in Japan. J. Wildl. Dis., 28(3): 467-469.

Barber, I., Hoare, D. and Krause, J. 2000. Effects of parasites on fish behaviour: a review and evolutionary perspective. Rev. Fish

Biol. Fisher., 10: 131-165.

Belló, A.R.R., Fortes, E., Belló-Klein, A., Belló, A.A., Llesuy, S.F., Robaldo, R.B. and Bianchini, A. 2000. Lipid peroxidation

induced by Clinostomum detruncatum in muscle of the freshwater fish Rhamdia quelen. Dis. Aquat. Organ., 42: 233-236.

Bhalerao, G.D. 1942. Some metacercarial forms of Clinostomatidae (Trematoda) from India. Proc. Indian Acad. Sci. (Section

B), 16: 67-71.

Bhalerao, G.D. 1943. On two trematodes from fishes in India. Proc. Indian Acad. Sci. (Section B), 18: 119-124.

Braun, M. 1899. Uber Clinostomum Leidy. Zool. Anz., 22: 489-493.

Bush, A.O., Lafferty, K.D., Lotz, J.M. and Shostak, A.W. 1997. Parasitology meets ecology on its own terms: Margolis et al.

Revisited. J. Parasitol., 83(4): 575-583.

Cameron, T.W.M. 1945. Fish carried parasites in Canada (1) Parasites carried by freshwater fish. Can. J. Comp. Med., 9: 245-

254.

Cantwell, G.E. 1981. Methods for invertebrates. In: Staining procedures, Clark, G. (ed). Williams and Wilkins, Baltimore. pp.

255-280.

Chung, D.I., Moon, C.H., Kong, H.H., Choi, D.W. and Lim, D.K. 1995. The first human case of Clinostomum complanatum

(Trematoda: Clinostomatidae) infection in Korea. Korean J. Parasitol., 33: 219-223.

Dias, M.L.G.G., Eiras, J.C., Machado, M.H., Souza, G.T.R. and Pavanelli, G.C. 2003. The life cycle of Clinostomum

complanatum Rudolphi, 1814 (Digenea, Clinostomidae) on the floodplain of the high Parana river, Brazil. Parasitol. Res.,

89: 506-508.

Eiras, J.C., Dias, M.L.G.G., Pavanelli, G.C. and Machado, M.H. 1999. Histological studies on the effects of Clinostomum

marginatum (Digenea, Clinostomidae) in its second intermediate host Loricariichthys platymetopon (Osteichthyes,

Loricariidae) of the upper Paraná River, Brazil. Acta. Sci., 21(2): 237-241.

Jithila and Prasadan 38

García, M.L.J., Osorio, S.D. and Constantino, F. 1993. Prevalence of parasites and their histological lesions in tilapia from the

lake of Amela, Tecoman, Colima, Mexico. Vet. Mexico, 24(3): 199-205.

Hirai, H., Ooiso, K., Kifune, T., Kiyoto, T. and Sakaguchi, Y. 1987. Clinostomum complanatum in posterior wall of the pharynx

of a human. Jap. J. Parasitol., 36: 142-144.

Hirai, H., Oolso, H., Kifune, T., Kiyota, T. and Sakaguchi, Y. 1987. Clinostomum cornplanaturn infection in posterior wall of the

pharynx of a human. Japanese Journal of Parasitobogy, 36: 142-144.

Jaiswal, G.P. 1957. Studies on the trematode parasites of fishes and birds found in Hyderabad State. Zool. Jahrb. Abt.

Syst., 85: 1-72.

Jones, S.R.M. 2001. The occurrence and mechanisms of innate immunity against parasites in fish. Dev. Comp. Immunol., 25:

841-852.

Jyrwa, D.B., Thapa, S. and Tandon, V. 2016. Helminth parasite spectrum of fishes in Meghalaya, Northeast India: a checklist. J.

Parasit. Dis., 40(2): 312-329.

Kagei, N., Yanohara, Y., Uchikawa, R. and Sato, A. 1984. On the yellow grubs, metacercaria of Clinostomum complanatum

(Rudolphi, 1819), found in the cultured loach. Jap. J. Parasitol., 33(1): 59-62.

Kalantan, A.M.N., Arfin, M. and Nizami, W.A. 1987. Seasonal incidence and pathogenicity of metacercaria of Clinostomum

complanatum (Trematoda: Digenea) in Aphanius dispar. Jpn. J. Parasitol., 36: 17-23.

Kaw, B.L. 1950. Studies in Helminthology. Helminth parasites of Kashmir. Part-I. Trematodes. Indian J. Helminthol., 2: 67-126.

Lafferty, K.D. 2008. Ecosystem consequences of fish parasites. J. Fish Biol., 73: 2083-2093.

Lee, G.S., Park, S.W., Kim, J., Seo, K.S., You, K.W., Chung, J.H., Moon, H.C. and Hong, G.Y. 2017. A case of endoscopically

treated laryngopharyngitis resulting from Clinostomum complanatum infection. Korean J. Gastroenterol., 69: 177-180.

Leidy, J. 1856. A synopsis of Entozoa and some of the common commercial fishes of India and Pakistan. Rec. Indian Mus., 57:

1-320.

Lo, C.F., Huber, F., Kou, G.H. and Lo, C.J. 1981. Studies of Clinostomum complanatum (Rud., 1819). Fish Pathol., 15: 219-227.

Malek, M. and Mobedi, I. 2001. Occurrence of Clinostomum Complanatum (Rudolphi, 1819) (Digenea: Clinostomatidae) in

Capoeta capoeta gracilis (Osteichthys: Cyprinidae) from Shiroud River, Iran. Iranian J. Publ. Health., 30: 95-98.

Mukherjee, R.P. 1967. On a new metacercaria Clinostomum orientale with a brief description of the metacercariae Clinostomum

giganticum Agarwal, 1959. Indian J. Helminth., 19: 183-187.

Park, C.W., Kim, J.S., Joo, H.S. and Kim, J. 2009. A human case of Clinostomum complanatum infection in korea. Korean J.

Parasitol., 47(4): 401-404.

Prasadan, P.K. and Devi, A.R.S. 2007. Yellow grub disease in the ornamental fish, Poecilia reticulate (Poecilidae). J. Exp. Zool.,

10(2): 405-407.

Rekharani, Z. and Madhavi, R. 1985. Digenetic trematodes from mullets of Visakhapatnam (India). Journal of Natural History,

19: 929-951.

Rudolphi, C.A. 1819. Entozoorum synopsis, cui accedunt mantissa duplex et indices locupletimmimi. Augusti Rücker. Berlin. pp.

811.

Secombes, C.J. and Chappell, L.H. 1996. Fish immune responses to experimental and natural infection with helminth parasites.

Annu. Rev. Fish. Dis., 6: 167-177.

Seppanen, E., Kuukka, H., Voutilainen, A., Huuskonen, H. and Peuhkuri, N. 2009. Metabolic depression and spleen and liver

enlargement in juvenile Arctic charr Salvelinus alpinus exposed to chronic parasite infection. J. Fish. Biol., 74: 553-561.

Shareef, P.A.A. and Abidi, S.M.A. 2012. Incidence and histopathology of encysted progenetic metacercaria of Clinostomum

complanatum (Digenea: Clinostomidae) in Channa punctatus and its development in experimental host. Asian Pac. J. Trop.

Biomed., 2(6): 421-426.

Siddiqui, J. and Nizami, W.A. 1982. Seasonal population dynamics of the metacercaria of Clinostomum complanatum in relation

to the sex of the host. Riv. Parasit., 43: 275-279.

Silva-Souza, A.T. and Ludwig, G. 2005. Parasitism of Cichlasoma paranaense Kullander, 1983 and Gymnotus carapo Linnaeus,

1814 by Clinostomum complanatum (Rudolphi, 1814) metacercariae in the Taquari river. Braz. J. Biol., 65(3): 513-519.

Southwell, T. and Prashad B. 1918. Notes from the Bengal fisheries laboratory No. 5. Parasites of Indian fishes with a note on

carcinoma in the climbing Perch. Rec. Indian Mus., 15: 341-355.

Srivastava, N.B. 1950. A preliminary note on the life cycle of Clinostomum sp. and Echinostomum heterostomum (Rud. 1804).

Proc. Nat. Acad. Sci. India, 3: 65-92.

Thapa, S., Jyrwa, D.B. and Tandon, V. 2008. Platyhelminth parasite spectrum in edible freshwater fishes of Meghalaya. In:

Current trends in Parasitology, Tandon, V., Yadav, A.K. and Roy, B. (eds.) Panima Publishing Corporation, New Delhi. pp.

113-125.

Ukoli, F.M.A. 1966. On Clinostotnum tilapiae n. sp., and C. phalacrocoracis Dubois, 1931 from Ghana, and a Discussion of the

Systematics of the Genus Clinostotnum Leidy, 1856. J. Helminthol., 40: 187-214.

Vankara, A.P. and Vijayalakshmi, C. 2009. Histopathological changes of the esophagus of the freshwater eels, Macrognathus

aculeatus (Bl.) and Mastacembelus pancalus (Lac.) due to infection of metacercariae of Clinostomum mastacembeli Jaiswal,

1957. J. Parasit. Dis., 33: 65-69.

Vianna, R.T., Pereira, J.J. and Brandao, D.A. 2005. Clinostomum complanatum (Digenea, Clinostomidae) density in Rhamdia

quelen (Siluriformes, Pimelodidae) from South Brazil. Braz. Arch. Biol. Technol., 48: 635-642.

Yamashita, J. 1938. Clinostomum complanatum, a trematode parasite new to man. Annot. Zool. Japan, 17: 563-566.

(Received in May 2019 and accepted for publication in September 2019)