Morphological and Histopathological Description of Myxobolus Adlardi N

Bulletin of Pure and Applied Sciences. Vol.35 A (Zoology),Issue (No.2) 2016:P.39-52 Print version ISSN 0970 0765 Online version ISSN 2320 3188 DOI 10.5958/2320-3188.2016.00009.7

MORPHOLOGICAL AND HISTOPATHOLOGICAL DESCRIPTION OF MYXOBOLUS ADLARDI N. SP. (CNIDARIA: MYXOSPOREA: MYXOZOA) INFECTING AN INDIAN MAJOR CARP, LABEO ROHITA HAMILTON, 1822 FROM A COLD WATER WETLAND IN PUNJAB (INDIA)

*׳Aditya Gupta¹ and Harpreet Kaur² ¹Department of Zoology and Environmental Sciences, Punjabi University, Patiala- 147002, Punjab, India.

²Department of Zoology, Panjab University, Chandigarh-160014, Chandigarh, India

*Corresponding author: [email protected]

Received on 10 August 2016 : Accepted on 23 November 2016

ABSTRACT

The present study was aimed to isolate myxozoan parasites infecting the freshwater fish Labeo rohita from Ranjit Sagar Wetland, Punjab from September, 2014 to August, 2015. Out of 50 fish specimens examined, 10 were found to be infected. The identification upto species level was done on the basis of morphological and morphometric attributes of the myxospores. Myxobolus adlardi n. sp. was characterized by the presence of ovoidal plasmodia of 0.2-0.9 mm in diameter in the gill filaments of Labeo rohita. Mature myxospores were pyriform and ellipsoidal in frontal view and lemon shaped in sutural view measuring 7.98×4.83 μm. Polar capsules were two, equal, bottle shaped measuring 3.50×1.76 μm in size. The prevalence of infection was found to be 20%. The intensity of infection or the mean abundance was determined by the gill plasmodial index (GPI) based on the number of countable plasmodia by stereozoom trinocular microscopy. Histopathological observations indicated the presence of intrafilamental-epithelial type, FE plasmodia in the gill filaments causing atrophy and destruption of the filaments.

Key words: Labeo rohita, gills, Myxobolus, GPI, histopathology

INTRODUCTION

Myxozoa are a group of aquatic parasites having a two host life cycle, involving a fish and an annelid worm or bryozoan. They were earlier considered as protozoans, but on the basis of the studies on their 18S ribosomal DNA, they were relocated under the phylum Cnidaria and unranked subphylum Myxozoa (Atkinson et al., 2015). Myxozoan infections in fish can cause economic loss to both aquaculture and freshwater fisheries and challenge the conservation of economically important fishes. The most significant diseases worldwide

Aditya Gupta and Harpreet Kaur

caused by myxosporeans are proliferative kidney disease caused by Tetracapsuloides bryosalmonae (Abd-Elfattah et al., 2014) whirling disease caused by Myxobolus cerebralis (Lodh et al., 2012), enteromyxosis by Enteromyxum leei (Bermudez et al., 2010), proliferative gill disease by Henneguya ictaluri (Wise et al., 2008), gill haemorrhagic disease by Thelohanellus bifurcata (Kaur and Katoch, 2014) and gill thelohanellosis by Thelohanellus theinensis (Gupta and Kaur, 2016). The lack of information on the life cycles of myxozoans is a barrier in understanding disease characteristics, such as transmission.

The identification of these parasites is mainly based on few morphological attributes of one life cycle stage, the myxospore, besides other features like host preference and tissue specificity. In recent studies on the group have reported many species with marginal differences in the morphology and morphometrics. The epidemiological studies on Myxozoa should include a combination of data from fish hosts, invertebrate hosts and the environment.

In North India many species of myxozoan parasites were recorded from freshwater fishes of wetlands of Punjab on morphological basis (Kaur and Singh, 2008, 2009, 2010a, 2010b, 2011a, 2011b, 2011c, 2011d, 2011e, 2011f, 2012a, 2012b; Singh and Kaur, 2012a, 2012b, 2012c, 2012d, 2014, 2015; Kaur et al., 2013a, 2013b, 2014a, 2014b, 2016; Kaur, 2014; Kaur and Katoch, 2016). Recently, molecular characterization of 18S rDNA gene has been done for the identification of myxozoa from northern part of India (Kaur and Attri, 2015; Kaur and Gupta, 2015, 2017a, 2017b; Gupta and Kaur, 2016). The present study describes the morphological and histopathological aspects of a new species, M. adlardi, found parasitizing the gill filaments of Labeo rohita (Ham.) from Ranjit Sagar Wetland, Punjab, India.

MATERIAL AND METHODS

Live fish specimens were procured from various catchment sites of Ranjit Sagar Wetland (32 ̊ 26 ̍ 30 ̎ N Latitude and 75 ̊ 43 ̍ 30 ̎ E Longitude), Punjab and were brought to the laboratory for further investigation. Fishes were 8-12 months old and 50 fish specimens were studied. The fishes were examined and dissected under the trinocular stereozoom microscope. The organs examined were gills, liver, intestine, stomach, kidneys, gall bladder, scales and fins. Plasmodium was removed, teased on a clean microscopic slide and examined under the light microscope at 100X oil objective (Magnus inclined Trinocular microscope MLX-Tr) for the presence of myxospores. For permanent preparations, air dried smears were stained with Ziehl-Neelsen and Iron-haematoxylin. Identification up to generic level was done with the help of key given by Lom and Dykova (1991). Complete description of the species was prepared according to the guidelines of Lom and Arthur (1989). The spore characteristics such as shape and size of the spores and polar capsules, presence or absence of the intercapsular appendix and iodinophilous vacuole etc. were taken into consideration.

RESULTS

Vegetative stages (Plasmodia)

Minute, visible under stereozoom binocular microscope, ovoidal, creamish white, measure 0.2-0.9 mm in diameter, histozoic, 2-4 plasmodia in number per gill, 50-100 myxospores per plasmodium. Gills pale and mucous laden (Fig. 1).

Taxonomic summary of Myxobolus adlardi n. sp.

Type host: Labeo rohita (Hamilton) vern. rohu, Family- Cyprinidae

Type locality: The fish specimens were procured from Ranjit Sagar Wetland, Punjab, India. It lies at an altitude of about 540 msl at 32˚26΄30ʹʹ N Latitude and 75˚43΄30ʹʹ E Longitude and is spread over an area of 87.60 sq km

Site of Organ Development: Gills (Intrafilamental-epithelial type, FE)

Tissue location: Gill filaments

Type Material: Paratypes are myxospore stained in Ziehl-Neelsen and Iron-haematoxylin, deposited with the supervisor. Slide no. HM/ZN/10.08.2015 and HM/IH/10.08.2015

Prevalence of Infection: 20% (10/50)

Gill Plasmodial Index (GPI): 1 (Indicating light infection)

Category of Plasmodium: B (Plasmodia visible under stereozoom microscope, size range 0.2-0.9 mm)

Clinical symptomatology: Creamish white pustules on the gills and mucous laden body surface

Pathogenicity: Degeneration of gill filaments

Etymology: The specific epithet “adlardi” has been given after the name of Dr. Robert Adlard. He is a principal scientist in the field of Parasitology at Queensland Museum, Australia.

Myxospore description (Table I)

(Measurements based on 12-15 myxospores in frontal view)

Myxospores measure 7.98×4.83 μm, pyriform, ellipsoidal in frontal view and lemon shaped in sutural view. Both shell valves symmetrical, measure 0.80 μm in thickness. Parietal folds absent. Polar capsules two, equal, bottle-shaped, running parallel to each other, measure 3.50×1.76 μm. Polar filament coils arranged perpendicular to the polar capsule axis forming

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6-7 filaments in the polar capsule. Sporoplasm agranular and homogenous occupy extracapsular space behind the polar capsules. Iodinophilous vacuole absent (Fig. 2, Fig. 3).

Table I: Measurements (μm) and ratio of M. adlardi n. sp.

Characters Range Mean Values SD CV

LS 7.80-8.18 7.98 0.26 0.07

WS 4.75-4.91 4.83 0.11 0.01

LPC 3.40-3.60 3.50 0.14 0.02

WLPC 1.65-1.87 1.76 0.15 0.02

Ratio: LS/WS 1.65

ICP Absent

Number of filament 6-7 turns

Parietal folds Absent

DISCUSSION

The present species has been compared with the myxospores of morphologically similar species in having pyriform and ellipsoidal shape. The data for comparison was obtained from the original descriptions as following: M. pfeifferi Thelohan, 1895 infecting muscles and gills of Barbus barbus; M. miyairii Kudo, 1919 infecting intestine of Parasilurus asotus; M. orientalis Shulman, 1962 infecting gills of Carassius auratus gibelio; M. poljanskii Shulman, 1962 infecting gills of Pseudogobio rivularis; M. shulmani Donec, 1962 infecting brain and kidneys of Notropis cornutus; M. ampullaceus Lalitha Kumari, 1969 infecting dorsal and ventral fins of Barbus kolus; M. narzikulovi Dzhalilov & Ashurova, 1971 infecting kidneys of Nemacheilus stolitzkai; M. tripathi Kalavati, Sandeep & Narasimhamurti, 1981 infecting wall of the gut & visceral organs of Clarias sp.; M. chakravartyi Haldar, Das & Sharma, 1983 infecting internal eye musculature of Catla catla; M. bliccae Donec & Toziyakova, 1984 infecting gills of Blicca bjoerkna and Abramis sapa; M. trichogasteri Sarkar, 1985 infecting gall bladder and bile of Trichogaster fasciatus; M. magurii Sarkar, 1993 infecting arborescent organ of Clarias magur; M. saraswatii Gupta & Saraswat, 1993 infecting kidney of Clarias batrachus; M. coeli Haldar, Samal & Mukhopadhyay, 1996 infecting gall bladder of Chanos chanos; M. fossilii Gupta, Gupta & Saraswat, 2001 infecting cartilage and brain of Heteropneustes fossilis; M. catmrigalae Basu & Haldar, 2004 infecting gill lamellae of Catla mrigala; M. szekeli Kaur & Singh, 2011 infecting inner wall of the stomach of Wallago attu; M. catli Kaur & Singh, 2011 infecting gill lamellae of Catla catla; M. kalmani Kaur & Singh, 2011 infecting gill lamellae of Cirrhinus reba and M. richardsoni Kaur & Dar, 2016 (Unpb.) infecting gills of Schizothorax richardsoni, however differed from all of the above species in morphometric characteristics (Table II).

The myxospores of the present species have been characterized in having pyriform, ellipsoidal shape in frontal view and lemon shape in sutural view. Both the polar capsules were equal and bottle-shaped, running parallel to each other. The present species differed from M. chakravartyi, M. coeli and M. catmrigalae in having unequal polar capsules.

The present species lacked an intercapsular process, hence differed from M. pfeifferi, M. schulmani, M. poljanskii, M. orientalis, M. narzikulovi, M. bliccae and M. kalmani in having a prominent intercapsular process. The present species differed from M. ampullaceus in the presence of 5-6 parietal folds at the posterior region. Furthermore, the present species was close to M. magurii (LS/WS: 1.82), M. saraswatii (LS/WS: 1.51), M. trichogasteri (LS/WS: 1.66) and M. catli (LS/WS: 1.76) but differed from M. magurii in which the polar capsules were banana shaped and occupied more than half of the myxospore body, from M. saraswatii in which myxospores were spherical to oval in shape, from M. trichogasteri in which the anterior end of the myxospore was broadly pointed and from M. catli in which the myxospores were oval to egg shaped with blunt anterior end. The LS/WS ratio in the present species was 1.65, hence differed with M. tripathi (LS/WS: 0.77) and M. szekeli (LS/WS: 2.1). The present species differed from M. miyairii (13.75 µm) in presence of large sized myxospores.

The present species was most close to M. fossilii and M. richardsoni in the presence of bottle shaped polar capsules, but differed as the myxospore was round in shape with darkely stained outer shell valve in M. fossilii, and pyriform and ellipsoidal shaped myxospores with a prominent intercapsular process in M. richardsoni, which was absent in the present species.

The comparisons made above have clearly demonstrated the unique morphology and morphometry of the myxospores of M. adlardi n. sp., therefore considered as a new species.

Table II: Comparative description of M. adlardi n. sp. with morphologically similar species (measurements in micrometer).

Species Host Site of Locality Myxospores Polar PC (= ICP infection capsule or ≠)

M. adlardi n. sp. Labeo rohita Gill filaments Punjab (India) 7.98×4.83 3.50×1.76 = Absent

(Present study)

M. pfeifferi Barbus barbus Muscles, gills Hungary 11.5×10.6 5.35 = Present Thelohan, 1895

M. miyairii Kudo, Parasilurus Intestine Japan 13.75×6.5 4.5 = Absent 1919 asotus

M. orientalis Carassius Gills China 19.3×10.75 12.75×3.9 = Present Shulman, 1962 auratus gibelio

M. poljanskiii Pseudogobio Gills China 14.15×9.3 7.0 = Present Shulman, 1962 rivularis

Aditya Gupta and Harpreet Kaur

M. shulmani Donec, Notropis Brain, kidney Canada 17.55×13.3 4.5×2.5 = Present 1962 cornutus

M. ampullaceus Barbus colus Dorsal & Andhra 9.8×7.1 5.8×2.8 = Absent Lalitha Kumari, 1969 ventral fins Pradesh (India)

M. narzikulovi Nemacheilus Kidney Central Asia 13.0×8.0 5.5×4.0 = Present Dzhalilov & stolitzkai Ashurova, 1971

M. tripathi Kalavati, Clarias sp. Wall of the Andhra 10.1×13.0 5.5×2.5 = Absent Sandeep & gut & Pradesh Narasimhamurti, visceral (India) 1981 organs

M. chakravartyi Catla catla Internal eye West Bengal 12.3×9.1 6.05×4.15 ≠ Present Haldar, Das & musculature (India) (L) Sharma, 1983 4.70×3.30 (S)

M. bliccae Donec & Blicca Gills Ukraine 12.15×10.0 6.7×3.75 = Present Tozyykova, 1984 bjoerkna, Abramis sapa

M. trichogasteri Trichogaster Gall bladder, West Bengal 15.55×9.35 10.1×3.32 = Absent Sarkar, 1985 fasciatus bile (India)

M. magurii Sarkar, Clarias magur Arborescent West Bengal 14.13×7.75 7.53×2.34 = Absent 1993 organ (India)

M. saraswatii Gupta Clarias Kidney Uttar Pradesh 15.0×9.9 6.5×2.25 = Absent & Saraswat, 1993 batrachus (India)

M. coeli Haldar, Chanos chanos Gall bladder Orissa (India) 10.6×5.49 5.76×2.85 ≠ Absent Samal & (L) Mukhopadhyay, 1996 4.20×2.90 (S)

M. fossilii Gupta, Heteropneustes Cartilage, Uttar Pradesh 10.25×7.44 6.43×2.2 = Absent Gupta & Saraswat, fossilis brain (India) 2001

M. catmrigalae Basu Catla mrigala Gill lamellae West Bengal 20.4×16.3 11.9×2.3 ≠ Absent & Haldar, 2004 hybrid (India) (L)

11.0×2.3 (S)

M. szekeli Kaur & Wallago attu Inner wall of Punjab (India) 8.7×4.1 4.4×1.5 = Absent Singh, 2011 the stomach

M. catli Kaur & Catla catla Gill lamellae Punjab (India) 8.3×4.7 3.3×1.67 = Absent Singh, 2011

M. kalmani Kaur & Cirrhinus reba Gill lamellae Punjab (India) 10.0×4.7 3.4×1.67 = Absent Singh, 2011

M. richardsoni Kaur Schizothorax Gills Jammu & 11.1×7.6 6.1×1.9 = Present & Dar, 2016 (Unpbl.) richardsoni Kashmir (India)

HISTOPATHOGENESIS

The plasmodia of M. adlardii n. sp. were located within the gill filaments of Labeo rohita and were typed as intrafilamental-epithelial type, FE. The plasmodia were ovoidal, white, visible under stereozoom binocular microscope, 5-7 plasmodia per gill and measure 0.2 to 0.9 mm in diameter. Histological investigation of the gills of Labeo rohita revealed that the plasmodia were “intrafilamental epithelial type, “FE” as recounted by Molnar (2002). The plasmodia were located at the basal part of the gill filaments, hence were basilamellar in position. The plasmodia were completely crammed with mature myxospores. They were encapsulated in a thin wall enclosed by flattened endothelial cells. Externally, a hyperplastic epithelial layer was discerned around the plasmodia. Gill lamellae were atrophied and obliterated at the plasmodium site, and the plasmodial masses initiated some curling in the adjacent lamellae. Vacuolisations in the cartilaginous and central part of the filament, hyperplasia of gill, fusion of gill lamellae and hypertrophid nucleii were also observed (Fig. 4).

Aditya Gupta and Harpreet Kaur

Molnar (2002) stated that in addition to the morphometric characteristics of the spores, the exact site of infection and tissue specificity was essential for the differentiation of the large number of gill parasitic myxosporean species. According to Molnár (2002), there were two distinct types of infections in the gills, namely intrafilamental and intralamellar types. Ali (1999) described the infection with H. ghaffari as an “intralamellar type” to which the histological figures, however, show that the infection was in fact of the “intrafilamental type”, following the delineation of Molnar (2002). Furthermore, examination of the histological sections from the present study verified the assessment of infection with M. adlardi n. sp. as corresponding to the “intrafilamental epithelial type” and were similar to those of Henneguya rhinogobii (Kageyama et al., 2009) and H. suprabranchiae (Abdel-Baki et al., 2011). The present study showed no tissue reaction to Myxobolus infection, which might be due to the fact that the plasmodial wall is not antigenic (Haaparanta et al., 1993). The infection however could initiate epithelial hyperplasia, curling and atrophy of the respiratory lamellae at the cyst site, which confirms that M. adlardi n. sp. has a deleterious

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effect on its host by decreasing the functional respirat1ory surface of the gills as also demonstrated by Haaparanta et al. (1994).

ACKNOWLEDGEMENTS

The authors acknowledge financial support by University Grants Commission (UGC), Govt. of India under UGC-CAS-II and DST-Purse grant to the Department of Zoology, Panjab University, Chandigarh.

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