Eduved International Journal of Interdisciplinary Research

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1 MORPHOLOGICAL DESCRIPTION OF SOME PARASITIC PROTOZOANS INFECTING FRESH WATER FISHES OF RIVER ASAN (MURENA), M.P. MANISHA DESHPANDE Department of Zoology, R.B.S. College, Agra ABSTRACT In the present thesis, an effort has been made to study the various species of Myxobolus in fresh water fishes. Total 695 fishes belonging to different genera and 8 species are investigated for this purpose. Nine species of Myxobolus have been described. Their incidence, Morphology and taxonomy are studied and compared. An attempt has been made to study the pathological effects of parasites on different organs viz. skin, muscles and gills. Effect of parasites on the growth of fishes is also noticed. Infection in Muscles is caused by Myxobolus cultus, Myxobolus dujardini, Myxobolus cycloid, Myxobolus oviforme, Myxobolus cognati, Myxobolus ellipsoideus, Myxobolus cerebralis. The infection in Muscles is reported in Channa striatus, Clarias batrachus, Heteropneustes fossilis, Labeo rohita, Catla catla, Cirrhinus mrigala and Wallago attu. Gills are infected by Myxobolus oviforme in Clarias batrachus. Myxosporidian infection is noticed in gill lamellae only. Skin and muscles are infected by Myxobolus oviforme in Clarias batrachus and the infection is reported in melanophores. Calcification is reported in gills only while necrosis is only noticed in skin. INTRODUCTION The great majority of the infectious diseases of fishes are mostly caused either by bacteria or by Protozoans. Among the Protozoans the sporozoans are the largest in number. These Protozoans are endoparasites occur in the skin, muscle and gills and are causative agent of various diseases in fresh water fishes. Among the sporozoan parasites Myxobolus is an important parasitic Protozoan, its many species are pathogenic in nature, often causing fatal diseases or even death to host fish. Myxobolus parasitic Protozoans affect fish population by causing mortality, reduction in growth, weight loss, and suppression of reproductive activity. The significance of recognizing these parasites increases with the development of aquaculture. The life cycle of Myxobolus parasite is not uniform. Infection of the host occurred by spores, two haploid nuclei after fusion become diploid zygote with mononucleus. This zygote grows up in the infected organ of the host and divide by multiple nuclear fission. Vegetative stages produced as trophozoite and they also multiply themselves further by fission. The growth and reproductive phases of trophozoite follow the formation of spores. The spores of Myxozoans are characterized by two or some time more than two rod shaped shells which have two polar coiled filaments. It is reported that spores in the host fish are ingested through mouth, the spores then shoots off the polar capsule in digestive tract and fasten firmly to the intestinal wall. The amoeboid young which presumably hatch from spores in the intestine, penetrate lymph vessel and blood and reach to different body parts. In the present study, an attempt has been made to study infection of Myxobolus parasitic Protozoans in some edible fresh water fishes of river Asan of Murena district. The work also provides an attempt to 136 Vol. 2 issue 05 May 2015

2 describe the pathological effects of Myxobolous on different organs of infected fishes viz. skin, muscles and gills. Effect of Myxobolous parasites on the growth of fishes were also studied from all localities. MATERIALS AND METHODS The present experimental work was started in the month of January 2009 and observations were made round the year. Local fisherman of Asan river were contacted and fishes in tin container brought in laboratory. The fishes were collected alive from different study sites. The following sites were selected for collection of fishes- Chonda gaon - Site A Jaroni gaon - Site B Karua gaon - Site C Girgoni gaon - Site D Kutwal gaon - Site E Silata gaon - Site F A total of 500 fishes of different species i.e. Labeo rohita, Catla catla, Heteropneustes fossilis, Clarius batrachus, Wallago attu, Mystus seenghala, Cirrhina mrigala, Cyprninus carpio, Puntius ticto and Channa striatus were collected and bring in laboratory. After quarantine for one day they selected of equal size 15-25cm length for examine the presence of Protozoan parasite. In each aquarium 25 fishes of different species maintained. The artificial fish food of floating type pellets which is available in the market was provided to the fishes till the investigation lasted. In this food crude protein 35%, crude fat 3%, crude fibre 4% and moisture was 10%. The fishes were used for study after 7 days of collection. RESULTS AND DISCUSSION MYXOBOLUS CULTUS This Protozoan during study obtained in Channa striatus, a fresh water fish. Out of 153 examined fishes only 3 fishes were reported, infected by M. cultus. The spores were present posterior to gills, penetrated in muscles. These infected fishes catched from Asan river site of Silata gaon. The spores are oval in shape and very smooth. The size of spores measure with the help of compound microscopic scale as 2.3µm-2.5 µm in length and 1.2 µm-1.5 µm in width. Each spore consist of two polar capsule, each of nearly equal in size and shape. Each polar capsule present at the anterior of the spore cavity. Polar filaments are coiled 3-5 turns. Polar capsule values are symmetrical and notched anteriorly. Sporoplasm present to the posterior of spore cavity and an iodinophilous vacuole present in sporoplasm, which is some what oval. Table-1 Measurement of Myxobolus cultus parasite Experimental findings Measurement Previous findings 2.3µm-2.5 µm Length of spore 10.2 µm 1.2 µm-1.5 µm Width of spore 6.0 µm 0.6 µm-0.8 µm Length of polar capsule 2.0 µm-2.5 µm 0.4 µm-0.6 µm Width of polar capsule 1.2 µm-1.5 µm 137 Vol. 2 issue 05 May 2015

3 Taxonomic identification The present species Myxobolus cultus reported by Yokoyama et al. (1995) from Goldfish Carassius auratus. Polar capsules are present at the anterior side of the spore cavity and polar filaments are coiled (3-5 turns) in both investigations. The number and structure of polar capsules are similar in both cases. Iodinophilous vacuoles also present. In fresh water fish (Channa striatus) this parasite observed in muscles while previously reported in cartilage of marine fish. On the basis of the foresaid morphology the present specimens of Myxobolous are identified as Myxobolus cultus (Yokoyama et al., 1995). In present study: Host - Channa striatus Site of infection - Muscles Morphometric of spore and polar capsules Spore Length µm-2.5 µm Width µm-1.5 µm Polar capsule Length µm-0.7 µm Width µm-0.6 µm MYXOBOLUS DUJARDINI, THELOHAN, 1892 Channa striatus (Ham.) a fresh water fish is the host of this species of Myxobolus. A few infection of Myxobolus dujardini is obtained and the intensity of infection is rather poor. Only 2-4 parasites are observed in a slide. Incidence- a low infection of Myxobolus dujardini is observed only 5 fishes out of 62 fishes examined are found to be infected. Morphology Spores are ellipsoidal in shape and spore valves are relatively thin, symmetrical and smooth. Spores are 1.9µm-2.5µm in length and 1.3µm -1.8µm in width. There are two polar capsules which are pyriform in shape and equal in size. Size of polar capsule is 1.0µm-1.5µm in length and 0.7µm-1.0µm in width, occupying almost half length of spore. Polar capsules are located at the anterior part of the spore cavity, both capsules lie very close to each other. Polar capsule valves are symmetrical, medium size nucleus occur towards posterior of polar capsule. A sporoplasm occupies the posterior half of the spore cavity. A round iodinophilus vacuole is present in sporoplasm. Taxonomical identification The present species is identical to the species, reported by Thelohan (1892) from Rutikus rutilus host. Mature spores are ellipsoidal in shape, nucleated pyriform polar capsule which are very close to each other, a rounded iodinophilous vacuole present at the posterior side in sporoplasm and a small nuclei present at posterior end of polar capsula. The parasites reported in Channa striatus collected from Kali nadi and Silata gaon. In Rutilus rutilus, Thelohen (1892) reported size of spore as 12µm-13µm in length and 7µm-8µm as width but he has not reported the size of polar capsule, while in present studies spores are 1.9µm Taxonomic study- Host- Channa striatus Site of infection- in gills Locality- Silata gaon (site F) 138 Vol. 2 issue 05 May 2015

4 Discussion- of the various species of Myxobolus reported so far, the present species conforms with Myxobolus dujardini. The species reported from Scarolinius erythrophthalmus and Rutilus rutilus by Thelohan (1892). Experimental findings show resemblance to previous report of Myxobolus dujardini. Mature spores are ellipsoidal in shape, nucleated, pyriform polar capsule lying very close together, a round iodinophilous vacuole present at the posterior side in sporoplasm and a small nuclei present at posterior end of polar capsule. Thelohan (1892) had measured the length of spore 12µm-13µm and the width of spores 7µm-8µm. but he had not reported the length and width of polar capsule, while in the present observation spores is 1.9µm-2.5µm in length and 1.3µm-1.8µm in width. The size of polar capsule are 1.0µm-1.5µm in length and 0.7µm-1.0µm in width. He reported this species from the gills of marine fish in the same way in the present study this species also reported. However, differences are shown in size of spore, host and locality of host. But on the basis of similarities in morphology specially of polar capsule, the present specimen of Myxobolus are identified as Myxobolus dujardini (Thelohan, 1892). Table-2 Measurement of Myxobolus dujardini parasite in fresh water fish and previous findings in marine fish Range Experimental findings in fresh water fishes Previous findings in marine fishes Length of spore 1.9µm-2.5µm 12.0µm-13.0µm Width of spore 1.3µm-1.8µm 7.0µm-8.0µm Length of polar capsule 1.0µm-1.5µm No report Width of polar capsule 0.7µm-1.0µm No report MYXOBOLUS CEREBRALIS, HOFER, 1903 The host of this species of Myxobolus are Heteropneustes fossilis and Channa striatus. Fishes ranging 15cm-19cm are found to be infected. The intensity is very high, spores of Myxobolus cerebralis are seen in a slide. Infection site of Myxobolus cerebralis in host fishes are as- Host Site of infection Locality Channa striatus Muscles of kidney Silata gaon (site F) Heteropneustes fossilis Muscles of kidney Chonda gaon (site A) Incidence- a fairly high incidence is recorded 25 out of 62 Channa striatus and 3 out of 16 Heteropneustes fossilis are found to be infected. Morphology Spore valves are relatively thin symmetrical and smooth. Ellipsoidal shape in frontal view and disc shape in lateral view. Spores are 2.1µm-2.5µm in length, 1.2µm-1.7µm in width. Polar capsule are two in number and pyriform in shape. Polar capsule is 0.5µm-0.8µm in length and 0.3µm-0.5µm in width. Polar filaments are closely coiled with 4 turns in the polar capsule and occur perpendicularly to the longitudinal axis of the capsule. Iodinophilous vacuole present in sporoplasm. Taxonomical identification The present species of Myxobolus is confirm with Myxobolus cerebralis from salmonid fish Salmo fontinalis, Hofer (1903). Hofer (1903) had mentioned the length of spore 7.0µm-9.0µm and a thickness of 5.0µm, whereas in the present study the length of spore is 2.1µm-2.5µm and the width is 1.2µm- 1.7µm. Hofer (1903) reported this species from cartilage but present species is reported from muscles. 139 Vol. 2 issue 05 May 2015

5 El-Mansy et al. (1999) reported this species in the oligochaete alternate host Tubifex tubifex but not described its morphology. There are no marked differences between the two reports. The structure of spore and polar capsule are identical in both investigations. Iodinophilous vacuole is present in both reported cases. The polar capsule reported 2.1µm-2.5µm in length and 1.2µm-1.7µm in width. While in present study the length of polar capsule is 0.5µm-0.8µm and width is 0.3µm-0.5µm. The parasite is collected from the different fresh water fishes Channa striatus and Heteropneustes fossilis. On the basis of the above similarities, the present specimens of Myxobolus are identical as Myxobolus cerebralis (Hofer, 1903). Table-3 Measurement of Myxobolus cerebralis in all fresh water fishes are as follows- Range Host Heteropneustes fossilis Channa striatus Length of spore 2.1µm-2.4µm 2.0µm-2.5µm Width of spore 1.3µm-1.6µm 1.2µm-1.5µm Length of polar capsule 0.5µm-0.7µm 0.5µm-0.8µm Width of polar capsule 0.3µm-0.5µm 0.3µm-0.5µm Table-4 Measurement of Myxobolus cerebralis in fresh water fish are as follows Range Experimental findings Previous findings Length of spore 2.1µm-2.5µm 7.0µm-9.0µm Width of spore 1.2µm-1.7µm 5.0µm Length of polar capsule 0.5µm-0.8µm 2.0µm-2.5µm Width of polar capsule 0.3µm-0.5µm 1.2µm-1.5µm Discussion Myxobolus cerebralis conforms from salmonid fish Salmo fontinalis, Hofer (1903) and Plehn (1905) who mentioned the length of spore 7.0µm-9.0µm and width 5.0µm, whereas in the present case, the length of spore is 2.1µm-2.5µm and the width 1.2µm-1.7µm. They reported this species from cartilage, while present species is reported from muscles. Hoffman (1990) and El-Mansy et al. (1998) reported it in the oligochaete alternate host Tubifex tubifex but did not describe its morphology. There are marked differences between the two reports. The number and structure of polar capsule in both investigation. Iodinophilous vacuole is also present in both cases. The polar capsules present as 2.0µm-2.5µm in length and 1.2µm-1.5µm in width, while in present case the length of polar capsule if 0.5µm-0.75µm and width is 0.3µm-0.5µm. The parasite is collected from Heteropneustes fossilis and Channa striatus. On the basis of above identity, the present specimens are identified as Myxobolus cerebralis (Hofer, 1903). MYXOBOLUS OVIFORME, PARISI, 1912 Channa striatus, Heteropneustes fossilis, Clarias batrachus, Labeo rohita and Wallago attu, the fresh water fishes are the host of these species of Myxobolus. The intensity of infection is much more than other Myxobolus species, parasite are visible in one slide. Heavy infection of Myxobolus oviforme is observed in these fishes. 140 Vol. 2 issue 05 May 2015

6 Table-5 Specific site of infection of Myxobolus oviforme parasite in each host fishes Host Site of infection Locality Channa striatus Lamellae of gills, skin and muscles Chonda gaon (site A) Heteropneustes fossilis Lamella of gills Silata gaon (site F) Clarias batrachus Lamellae of gills, skin Silata gaon (site F) Labeo rohita Skin Chonda gaon (site A) Wallogo attu Lamellae of gills Jaroni gaon (site B) Incidence- 29 fishes out of 155 are examined as infected by Myxobolus oviforme. Morphology: Spore valves are smooth and symmetrical as well as spores are pear shape and elongated. Spore length is 3.2µm-4.2µm and width 2µm-3.1µm. polar capsule are two in number and oval in shape, both are equal in size. In polar capsule nuclei present in the centre. Size of capsule are 1.3µm-1.7µm in length and 1.1µm-1.2µm in width. Sporoplasm are triangular. A vacuole is present in the sporoplasm. Taxonomy Host- Channa striatus, Heteropneustes fossilis, Clarias batrachus, Labeo rohita and Wallago attu Site of infection- infected site observed in muscles, lamellae of gills and skin. Locality- Chonda gaon, Jaroni gaon and Silata gaon. Discussion- identification and description of Myxobolus oviforme in various fishes reported by many researchers, Parisi (1912), Auerbach (1912), Kudo (1920), Jameson (1929, 1931), Shulman and Shulman, Aibova (1953), Shulman (1962, 1966), Barisheva and Bauer (1957), Walliker (1968),Kennedy (1974) and Molnar (2000) reported presence of Myxobolus oviforme in liver, kidney and gills of Apogen rexmullorum, Coris julis,cymatoyster aggregates, Damalichthys argyrosmus, Godus callaris, Gadus morhaa, Hypsurus cargi, Onchorhynchus garbuscha, Onchorhynchus keta, Onchorhynchus nerka, Onchorhynchus tsahawytseha, Salmo gairdneri, Salmo irideus, Salmo salar, Salmo trutta and Alburnus albrunus. They had mentioned the length of spore 9µm-11µm and the width of spores 6.5µm-8.0µm. in the present observation the Myxobolus oviforme measure as 3.5µm-4.1µm in length and 2.3µm-3.1µm in width. They reported the length of polar capsules 3.0µm-4.5µm but nobody reported width of polar capsuler while in the present study, the length of polar capsule is 1.3µm-1.7µm and 1.1µm-1.2µm in width. Two polar capsule are equal in size. A vacuole is present in sporoplasm in both investigations. Parisi (1912) and other workers reported elongated oval spores with rounded extremities in gills. In present case, the spore is elongated in the muscle of Channa striatus, Heteropneustes fossilis and Labeo rohita, slightly pointed and longitudinal in muscles and gills of Clarias batrachus and Wallago attu while only slightly pointed in skin of Clarias batrachus as already described. Moreover, the present material is obtained from the different host and different collecting sites, hence, on the basis of the 141 Vol. 2 issue 05 May 2015

0µm 2.6µm- 3.0µm 1.4µm- 1.8µm 1.1µm- 1.2µm Heteropneustes fossilis Clarias batrachus Labeo rohita 3.3µm-3.8µm 3.3µm-4.1µm 3.3µm- 3.7µm 2.2µm-2.8µm 2.4µm-3.0µm 2.1µm- 2.9µm 1.3µm-1.6µm 1.3µm-1.7µm 1.

7 above similarities, the present specimens of Myxobolus are identified as Myxobolus oviforme (Parisi, 1912). Table-6 Measurement of Myxobolus oviforme in all fresh water fishes are as follows- Range Length spore Width spore Length polar capsule Width polar capsule of of of of Host Channa striatus 3.5µm- 4.0µm 2.6µm- 3.0µm 1.4µm- 1.8µm 1.1µm- 1.2µm Heteropneustes fossilis Clarias batrachus Labeo rohita 3.3µm-3.8µm 3.3µm-4.1µm 3.3µm- 3.7µm 2.2µm-2.8µm 2.4µm-3.0µm 2.1µm- 2.9µm 1.3µm-1.6µm 1.3µm-1.7µm 1.3µm- 1.8µm 1.0µm-1.2µm 1.2µm-1.3µm 1.1µm- 1.2µm Wallago attu 3.2µm- 3.9µm 2.3µm- 3.0µm 1.3µm- 1.7µm 1.1µm- 1.2µm Table-7 Measurement of Myxobolus oviforme parasite in fresh water fishes are as follows- Measurement Experimental findings Previous findings Length of spore 3.5µm-4.1µm 9µm-11µm Width of spore 2.3µm-3.1µm 6.5µm-8.0µm Length of polar capsule 1.3µm-1.7µm 3.0µm-4.5µm Width of polar capsule 1.1µm-1.2µm No reported Hand drawing Camera lucida drawing SV Spore valve PC Polar capsule S Sporoplasm IV Iodinophilous vacuole 142 Vol. 2 issue 05 May 2015

Fig. 1: Drawings of Myxobolus cultus Hand

8 Fig. 1: Drawings of Myxobolus cultus Hand drawing Camera lucida drawing SV Spore valve PC Polar capsule S Sporoplasm IV Iodinophilous vacuole N Nuclei Fig. 2: Drawings of Myxobolus dujardini Hand drawing Camera lucida drawing SV Spore valve PC Polar capsule S Sporoplasm IV Iodinophilous vacuole PF Polar filaments 143 Vol. 2 issue 05 May 2015

Fig. 3: Drawings of Myxobolus cerebralis Hand drawing Camera lucida drawing SV Spore valve PC Polar capsule S Sporoplasm V Vacuole Fig. 4: Drawings of Myxobolus oviforme REFERENCES 1. Bandyopadhyay P.

14(4): 353 356. 2. Chaudhari, S.R. and Chakravarty, M.M. (1970). Studies on the Myxosporidia (Protozoa: Sporozoa) from the food fishes of Bengal. 1.

9 Fig. 3: Drawings of Myxobolus cerebralis Hand drawing Camera lucida drawing SV Spore valve PC Polar capsule S Sporoplasm V Vacuole Fig. 4: Drawings of Myxobolus oviforme REFERENCES 1. Bandyopadhyay P.K., Hemananda T., Mitra A.K., Mohilal N. (2007). Myxobolus dhanachandi sp. n. (Myxozoa, Myxosporea, Bivalvulida) from an Indian freshwater fish Channa orientalis (Bloch-Schneider) Protistology. 14(4): Chaudhari, S.R. and Chakravarty, M.M. (1970). Studies on the Myxosporidia (Protozoa: Sporozoa) from the food fishes of Bengal. 1. Three new species f rom Ophiocephalus punctatus «Bloch); Acta protozool, 8: Cone, D.K, Stickel, R.G, Eck, G.W. and Muzzal, P.M. (1996). Myxobolus cognati n. sp. (Myxosporea) from the opercular integument of Cottus cognatus (Cottidae) in lake Michigan. J. parasitol. 82: Das, B.K. (1991). Pathological studies on fry and fingerlings of rohu, Labeo rohita (Ham.). MF.Sc. Thesis, Orissa University of Agriculture and Technology, Bhubuneswar. 5. Das, B.K. and Mukherjee, S.C. (1998). Pathology of black spot diseuse in fry and finger lings of rohu, Labeo rohita (Ham.). Geobios, 25: EI-Mansy, A., Molnar, K. and Szekely, C. (1998). Development of Myxobolus portucalensis Saraiva et Molnar, 1990 (Myxosporea : Myxobolus in the oligochaete Tubifex tubifex (Muller). Systematic parasitology, 41: Ferguson, J. A., Atkinson, S. D. Whipps, C. M. and Kent M. (2008). Molecular and morphological analysis of Myxobolus spp. of salmonid fishes with the description of a new Myxobolus species. Journal of Parasitology 94: Vol. 2 issue 05 May 2015

10 8. Ferguson, J. A., Atkinson, S. D. Whipps, C. M. and Kent M. (2008). Molecular and morphological analysis of Myxobolus spp. of salmonid fishes with the description of a new Myxobolus species. Journal of Parasitology 94: Fish, F.F. (1939). Observations on Henneguya salminicola, etc. J. Parasit., 25: Gordon, M. (1959). Pigment Cell Biology. Academic Press, New York. 11. Grossheider G. and Korting W. (1992). First evidence that Hoferellus 'I. cyprini (Doflein, 1898) is transmitted by Nais sp. Bulletin of the European Association of fish pathologists, 12: Karamchandani, S.J. (1970). A new species of Myxobolus from the gill cavity of Labeo bata (Hamilton); J. Inland Fish. Soc. India, 2: Kaur H, Singh R. (2009). A new myxosporean species, Myxobolus eirasi sp nov, a known species M. venkateshi Seenappa, Manohar (1981) from Lagler, K.F., Bardach, J.E. and Miller, R.R. (1962). Ichthyology, The study of fishes. John Wiley and sons, Inc., New York and London. pp Kaur H, Singh R. (2011c) Myxobolus harikensis sp. nov. (Myxozoa: Myxobolidae) infecting fins of Cirrhina mrigala(ham.) an Indian major carp in Harike Wetland, Punjab (India). Parasitol Res. doi: /s x. 15. Lalita Kumari, P.S. (1969). Studies on parasitic protozoa (Myxosporidia) of freshwater fishes of Andhra Pradesh, India. Rivista di Parassitologia, 30: McCraren, J.P., Landolt, M.L., Hoffman, G.L. and Meyer, F.P. (1975). Variation in response of channel catfish to Henneguya sp. Infctions (Proto zoa: Myxosporidia.) J. Wild. Dis, 11: Molnar, K. (2000). Myxobolus intrachondrealis n. sp. (Myxosporea : Myxobolidae), a parasite of the gill cartilage of the common Carp, Cyprinus carpio. Folio Parasitologica,47: Nemeczek,A. (1926). Beitrage zur Kenntnis der Myxosporidian fauna Brasiliens. Archiv fur Protistenkunde, 54: Pandey, K. and Shukla, J.P. (2005). Fish and Fisheries. 1st ed. Rastogi Publication, Meerut. pp Ravarti, N.M. (1994). Diseases of cultivable fresh water fishes and their control. International books and Periodicals supply service Pub. Delhi, 35: Reuss, H. (1906). Neue Myxosporidien von susswasserfischen. Bull. Acad. Imp. Sci. St. Petersburg. Ser., 5: Vol. 2 issue 05 May 2015

Department of Zoology, Punjabi University, Patiala, India

Department of Zoology, Punjabi University, Patiala, India Protistology 6 (2), 126 130 (2009) Protistology A new myxosporean species, Myxobolus eirasi sp. nov. and a known species M. venkateshi Seenappa and Manohar, 1981 from the Indian major carp fish Cirrhina