Turkish Journal of Zoology Turk J Zool (2017) 41: 791-799 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1606-47

Morphological and molecular characterization of puntiusii n. sp. (: ) infecting sophore Hamilton, 1822 from Ranjit Sagar Wetland, Punjab ()

1 2, Aditya GUPTA , Harpreet KAUR * 1 Department of Zoology and Environmental Sciences, Punjabi University, Patiala Punjab, India 2 Department of Zoology, Panjab University, Chandigarh, Chandigarh, India

Received: 27.06.2016 Accepted/Published Online: 13.03.2017 Final Version: 28.09.2017

Abstract: The pool barb, Puntius sophore Hamilton, 1822, is a tropical freshwater cyprinid native to inland waters in Asia. Herein, a novel myxozoan from plasmodia on the caudal fin of P. sophore from Ranjit Sagar Wetland in Punjab, India is described. Myxospores were consistent with the genus Myxobolus, ellipsoidal in frontal view, lemon-shaped in lateral view; length 7.76 ± 0.28 µm, width 5.36 ± 0.15 µm. There were two polar capsules of unequal size: a larger polar capsule, length 3.01 ± 0.08 µm, width 1.83 ± 0.11 µm, with 6–7 turns of the polar filament, and a smaller polar capsule, length 1.71 ± 0.15 µm, width 0.94 ± 0.07 µm, with 3–4 turns of the polar filament. The 1183 bp 18S rDNA sequence was up to 95% similar to Myxobolus spp. from other cyprinid in India. Given the novel host, unique myxospore morphology, and 18S rDNA sequence, we propose Myxobolus puntiusii n. sp.

Key words: Myxobolus, 18S rDNA, caudal fin, phylogenetic analysis, Ranjit Sagar Wetland, Myxobolus puntiusii

1. Introduction Punjab (Kaur and Singh, 2009, 2011d, 2012a; Singh and Myxozoans are microscopic, multicellular, spore-forming Kaur, 2015; Kaur et al., 2014a, 2016; Kaur and Gupta, metazoan parasites belonging to the phylum Cnidaria. 2015; Gupta and Kaur, 2016). The present study describes Primarily parasites of fish, myxozoan plasmodia/ the morphological and molecular aspects of a new species, pseudocysts can be either histozoic or coelozoic, causing M. puntiusii, found parasitizing the caudal fin of Puntius disease in wild and cultured populations of economically sophore (Ham.) from Ranjit Sagar Wetlands, Punjab, India. important fishes (Bartholomew et al., 1997; Yokoyama et al.; 1998, Pote et al., 2000). Myxozoan infections often lead 2. Materials and methods to substantial economic losses in aquaculture and capture Puntius sophore specimens (n = 30) with an average fisheries as a result of morbidity and mortality associated length of 5–7 cm were procured from various catchment with disease as well as reduced marketability of the fish. sites of Ranjit Sagar Wetland (32°26′30″ N, 75° 43′30″ The genus Myxobolus () is the largest E). They were transported live, with artificial aeration, myxozoan genus, with a global distribution in both fresh to the parasitology laboratory at Punjabi University, and marine waters (Lom and Dykova, 1992). Of the Patiala. The were anesthetized with chloroform approximately 800 species of Myxobolus globally, 131 at a concentration of 40 mg/L, necropsied, and examined species have been recorded in India, most of which are for the presence of parasites using a stereomicroscope. characterized by morphological and morphometrics of Plasmodia present on the caudal fin were removed, teased the spores (Kaur and Singh, 2012a). Taxonomic placement on a slide, mechanically ruptured, and examined by phase of Myxobolus spp. is challenging, as many species are contrast microscopy (Magnus MLX; Magnus Analytics, morphologically similar (Chen and Ma, 1998). Efforts New Delhi, India). Myxospores were measured and have been made to address this issue by supplementing photographed. The mean abundance, or fin plasmodial morphological descriptions with 18S rDNA sequence data index (FPI), was calculated based on the number of (Kent et al., 2001) plasmodia present per fin, visible either grossly or In North India, many species of myxozoan parasites microscopically (Attri, 2015). Categories of infection were were reported in freshwater fishes from wetlands in as follows: 0 plasmodia (no infection, 0); 1–5 plasmodia * Correspondence: [email protected] 791 GUPTA and KAUR / Turk J Zool

(light infection, 1); 5–10 plasmodia (moderate infection, 3. Results 2); 10–20 plasmodia (heavy infection, 3); 20 or more 3.1 Description of Myxobolus puntiusii n. sp. plasmodia (severe infection, 4). (Measurements based on 12–15 spores in frontal view.) Myxospores were collected from the infected fins and Vegetative stages fixed in absolute alcohol for molecular and phylogenetic Round to irregular, 0.8–1.0 mm in diameter, white, 5–6 analysis. Parasite genomic DNA (gDNA) was extracted plasmodia per fin, 100– 250 spores per plasmodium. using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Observed pathological signs on the fin consisted of white Germany) following the manufacturer’s instructions. multifocal cysts on the fin surface (Figure 1). Purified gDNA was quantified spectrophotometrically Mature spores (Nanodrop; Thermo Scientific, Wilmington, DE, USA) Spores (n = 12–15) ellipsoidal in frontal view, and spectrophotometer at 180 ng/μL. An approximately 1200 lemon-shaped in lateral view (Figures 2 and 3). Sutural bp fragment of the 18S rDNA gene was amplified using line distinct and slightly curved. Shell valves are thick, the My1F (CTAATCCCGGTAACGAACGA) and My10R symmetrical, and sutural edge markings on the posterior (CGTCCTCGCAACAAACTGTA) primers (Abidi et al., end. The spores had a mean length of 7.76 ± 0.28 µm, 2015) on an Eppendorf Master Cycler Pro S (Hamburg, width 5.36 ± 0.15 µm. Two polar capsules different in size. Germany). The 25 µL of PCR consisted of 3 μL DNA Large one pyriform, 3.0 ± 0.08 µm long and 1.83 ± 0.11 template, 2.5 μL of 10X Taq buffer (10 mM; Sigma-Aldrich, µm wide, tapering towards discharging canals of polar St. Louis, MO, US), 1 unit of Taq polymerase (1 U; Sigma- filament. Polar filaments coiled with 6–7 turns in polar Aldrich), 3.4 μL of deoxyribonucleotide triphosphates (20 capsule, situated perpendicularly to longitudinal axis of µM), 0.9 μL of each primer (5 pmol/μL), and 13.3 μL of capsule. Smaller polar capsule pyriform or elongated, 1.71 ± 0.15 µm long and 0.94 ± 0.07 µm wide, coiled with 3–4 water. Thermal cycling conditions were as follows: 95 °C turns. Polar filament 20–30 µm long when extruded. Two for 3 min, followed by 33 cycles of denaturation at 95 °C sporoplasmic nuclei and an iodinophilous vacuole also for 30 s, primer annealing at 58 °C for 30 s, extension at present (Figure 4). 68 °C for 1 min 20 s. The final extension was at 68 °C for 10 min. The PCR products were analyzed on a 2% agarose 3.2 Taxonomic summary gel containing 0.5 µg/mL ethidium bromide in 1X Tris- Type host: Puntius sophore (Hamilton, 1822) vernacular acetate-EDTA buffer and size was estimated by comparison name: pothi; common name: Pool barb (, ). with the 100 bp Plus DNA Ladder. The amplified products Type locality: Ranjit Sagar Wetland, Punjab, India. were commercially sequenced at Molecular Diagnostic Site of infection: Caudal fin. & Research Laboratories (Chandigarh, India) and the Type materials: Paratypes are spores stained in obtained nucleotide sequences were edited and aligned Ziehl–Neelsen and iron–hematoxylin, deposited with using BioEdit software (Hall, 2011). the supervisor. Slide no. M/ZN/16.10.2014 and M/ The 1183 bp 18S rRNA gene sequence, in addition IH/16.10.2014. to 25 sequences with >90% similarity identified by a Prevalence of infection: 20% (6/30). Blastn search of the National Center for Biotechnology Mean abundance or FPI: 2 (moderate infection). Information’s GenBank database, were downloaded Etymology: The specific epithet “puntiusii” has been and used for phylogenetic assessment (Altschul et al., given after the name of the host fish. 1990). (AF001579) isolated from 3.3 Phylogenetic analysis Oncorhynchus mykiss (Walbaum) was taken as an The 1183 bp 18S rRNA gene sequence ofM. puntiusii n. outgroup. Sequence alignment was performed by multiple sp. was deposited in GenBank under accession number sequence comparison by log-expectation. Genetic distance KU516662. BLAST analysis revealed M. puntiusii n. sp. analyses were conducted using the Kimura 2-parameter is most closely related to M. basuhaldari Szekely et al., model (Kimura, 1980) in MEGA6 software and identified 2014 recorded from the gill lamellae of Labeo rohita from as having the lowest Bayesian information criterion score India (KM029975; 95% similarity), M. kalavatiae Szekely (4430.972) (Tamura et al., 2013). All positions containing et al., 2014 from the gill lamellae of Cirrhinus cirrhosus gaps and missing data were eliminated. The Bayesian from India (KM029973; 93% similarity), M. mrigalhitae phylogenetic analysis was conducted using MrBayes v3.2.2 Basu & Haldar, 2003 from the gills of Catla catla from (Ronquist and Huesenbeck, 2003). The tree was generated India (KJ476880; 92% similarity) and M. rocatlae Basu & using maximum likelihood having 1000 bootstrap values Haldar, 2002 from the operculum of C. catla from India and was proportional to the number of substitutions per (KJ476878; 92% similarity). Phylogenetic analysis placed site. Tajima’s neutrality test for the nucleotide mutation the isolate within a clade containing Myxobolus spp. from was also performed. cyprinid fish in India, with high bootstrap (Figure 5).

792 GUPTA and KAUR / Turk J Zool

Figure 1. Photomicrograph of infected caudal fin of Puntius sophore showing plasmodia of Myxobolus puntiusii n. sp.

4. Discussion and M. bengalensis, sister to M. basuhaldari. Moreover, Myxobolus puntiusii n. sp. has spores that are superficially estimates of evolutionary pairwise divergence among the similar to several Myxobolus spp., with overlap in assorted sequences of M. basuhaldari, M. kalavatiae, M mrigalhitae, spore dimensions. However, there are several characters M. rocatlae, and M. bengalensis was 0.01, 0.02, 0.02, 0.0 that aid in distinguishing this new species. Specifically, M. (nil), and 0.0 (nil) respectively. Tajima’s neutrality test for basuhaldari and M. kalavatiae were somewhat similar in the nucleotide mutation was also done. The D-value was size to M. puntiusii n. sp., but have equal polar capsules –0.584536, which indicates that some of the alleles were and are located in the gills, not the caudal fin, of different not consistent and were present at low frequencies. Due to host types. Similarly, M. dossoui is also similar in size to this low frequency, the population of myxozoan parasites M. puntiusii n. sp. and possesses dissimilar-sized polar might have increased with time. capsules as the present species, although the polar capsules The present study suggests tissue location, geographical themselves are nearly twice as long as in M. puntiusii n. region, and tissue specificity are strong phylogenetic signals. sp. Furthermore, the present species was comparable to The tree formed monophyletic clades involving M. puntiusii M. rachmani, M. diversus, M. alburni, M. gobiorum, and sp. nov. and M. kalavatiae, M. basuhaldari, M. mrigalhitae, M. drjagini in having unequal polar capsules, but differed M. rocatlae, and M. bengalensis infecting cyprinids carps due to variation in size of the spores. M. diversicapsularis, from India with a high bootstrap value. The other clades M. musseliusae, M. dossoui, and M. mrigalhitae have were limited to the myxosporeans from , Hungary, intercapsular process and hence are different from M. Canada, and Malaysia. Similar observations were found puntiusii n. sp. (Table). by Zhao et al. (2008) in M. ampullicapsulatus infecting BLAST analysis of M. puntiusii n. sp. 18S rRNA Carassius auratus auratus. Phylogenetic studies involving sequence showed a maximum similarity of 95% with M. LSU gene markers have been widely used for myxosporean basuhaldari (KM029975) infecting the gills of L. rohita , species identification, and discrimination of from India, 93% with M. kalavatiae (KM029973) infecting cryptic species (Atkinson et al., 2015). In a detailed study the gill lamellae of C. cirrhosus from India, and 92% with M. by Liu et al. (2016), a cryptic species complex was found mrigalhitae (KJ476880) infecting the gills of C. catla from between M. prolini and M. ellipsoids, having different India and M. rocatlae (KJ476878) infecting the operculum sized myxospores. This was earlier explained by Kaur and of C. catla from India. The phylogenetic tree placed M. Gupta (2017), where they found the sister taxa between T. puntiusii n. sp. within a monophyletic clade consisting of boggoti and T. pathankotensis infecting cyprinid carp from M. basuhaldari, M. kalavatiae, M mrigalhitae, M. rocatlae, India. It is predicted that DNA sequencing will continue

793 GUPTA and KAUR / Turk J Zool

Figure 2. Line drawing of myxospores of Myxobolus puntiusii n. sp. a. Ziehl–Neelsen stain (frontal view). b. Fresh myxospore (sutural view). c. Myxospore showing sporoplasmic nuclei with extruded polar filament (iron–hematoxylin stain).

794 GUPTA and KAUR / Turk J Zool

Figure 3. Photomicrograph of myxospores of Myxobolus Figure 4. Photomicrograph of stained myxospores. a. Ziehl– puntiusii n. sp. a. Fresh myxospores under dark field microscope. Neelsen stain. b. Iron–hematoxylin stain. b. Fresh myxospores under phase contrast microscope.

795 GUPTA and KAUR / Turk J Zool

Figure 5. Phylogenetic tree generated by maximum likelihood showing the phylogenetic position of Myxobolus puntiusii n. sp. (KU156662) with other myxosporeans. GenBank accession numbers are given and number above nodes indicates bootstrap confidence values. Scale bar: amount of inferred evolutionary change along the branch lengths.

796 GUPTA and KAUR / Turk J Zool = = # = # # # # # # # # # Polar capsule Polar #) (= or 2.9–3.4 × 1.6–2.0 2.9–3.7 × 2.0–2.2 4.3–5.2 × 2.7–3.2 11.8–13.7 × 2.5–3.0 5.5–6.0 × 3.0–3.5 4.5–6.5(5.5) × 2.5–5.0(3.1) 5.2–6.5 × 2.2–3.0 6.8–8.1 × 3.0–3.5 3.9–4.0 4.8–6.0 × 3.0–3.6 7.2–7.5 × 3.7–3.9 3.8–5.5 × 2.2–3.7 2.95–3.07 × 1.75–1.91 1.60–1.82 × 0.89–0.99 Polar capsule Polar 6.8–7.7 × 4.8–5.8 6.8–7.8 × 5.1–6.2 10.2–11.3 × 7.6–8.1 17.5–19.3 × 5.6–6.2 13.0–14.0 × 9.0–10.0 8.5–11(9.9) × 8.0– 10.5(9.2) 11.0–13.0 × 9.0–10.0 12.0–15.3 × 10.0–12.0 10.5–11.1 × 8.8–10.0 13.2–16.8 × 7.8–9.6 13.5–14.3 × 11.2–12.0 8.5–13.0 × 8.5–12.5 7.56–7.96 × 5.25–5.47 Spores India India India India Amur basin Amur Benin Ukraine Russia Russia Hungary Central Asia Russia India Locality Gill lamellae Gill Gill lamellae Gill Gills Gills, gut wall gut Gills, Skin Gill arch cartilage arch Gill Fin Fin Gills Fins kidney Gills Gills Caudal fin Caudal Site of infection of Site C. cirrhosus L. rohita , L. rohita C. mrigala , L. rohita C. catla Hypophthalmichthys molitrix Hypophthalmichthys Tilapia zillii Tilapia Gobio gobio Alburnus alburnus C. carpio Carassius auratus Alburnoides tenniatus Rutilus rutilus Puntius sophore Host Slukhai in Slukhai Szekely et al., 2014 et al., Szekely Yakovchuk, 1979 Yakovchuk, Szekely et al., 2014 et al., Szekely Allamuratov, 1966 Allamuratov, n. sp. n. sp. Sakiti 1991 et al., unequal. # = equal, in micrometers): M. species similar of (measurements morphologically with puntiusii description n. sp. Comparative M. kalavatiae M. basuhaldari Basu & Haldar, 2003 Basu & Haldar, M. mrigalhitae Basu & Haldar, 2002 Basu & Haldar, M. rocatlae Akhmerov, 1954 Akhmerov, M. drjagini 1991 Lom, Landsberg and M. dossoui Donec in Shulman, 1984 in Shulman, Donec M. gobiorum Donec in Shulman, 1984 in Shulman, Donec M. alburni M. musseliusae Ni et al., 1973 et al., Ni M. diversus M. rachmani M. diversicapsularis 1966 Shulman, M. puntiusii Species Table.

797 GUPTA and KAUR / Turk J Zool to reveal that many myxozoans, especially those presently Identifier for this publication is: http://zoobank.org/ regarded as having restricted geographic and host ranges, urn:lsid:zoobank.org:pub:E61C53C8-C157-4FD5-9A87- are complexes of cryptic species. 29B1E6C6908F. The combination of morphological, morphometric, and molecular data obtained in the present study support Acknowledgements the description of a new species, M. puntiusii, which is The authors acknowledge financial support by University distinct from all of the other previously described species Grants Commission (UGC), Govt. of India under UGC- of the genus Myxobolus. CAS-II and a DST-Purse grant to the Department of Nomenclatural acts Zoology at Panjab University, Chandigarh, India. This work and the nomenclatural acts it contains have been registered in ZooBank. The ZooBank Life Science

References

Akhmerov AK (1954). On closely related species of a new Kaur H, Katoch A, Gupta M (2014a). Thelohanellus filli sp. n., a myxosporean genus. Dokl Akad Nauk SSS 97: 1101-1103. pathogenic myxosporean infecting gills of cultured carp, Labeo rohita (Hamilton 1822) in Punjab, India. Species 10: 31-38. Allamuratov B (1966). New species of parasitic protozoa found in the fish of South-Surkham Reservoir. Uzb Biol Zh 10: 58-60. Kaur H, Singh R (2009). A new myxosporean species, Myxobolus eirasi sp. nov., a known species M. venkateshi Seenappa and Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990). Basic Manohar (1981) from the India major carp fish Cirrhina local alignment search tool. J Mol Biol 215: 403-410. mrigala (Ham). Protistol 6: 126-130. Atkinson SD, Bartošová-Sojková P, Whipps CM, Bartholomew JL Kaur H, Singh R (2011d). Two new species of Myxobolus (Myxozoa: (2015). Approaches for characterizing myxozoan species. In: Myxosporea: ) infecting Indian freshwater fishes in Okamura B, Gruhl A, Bartholomew JL, editors. Myxozoan Punjab wetlands (India). Parasitol Res 108: 1075-1082. Evolution, Ecology and Development. Cham, Switzerland: Springer International Publishing, pp. 111-124. Kaur H, Singh R (2012a). A synopsis of the species of Myxobolus Bütschli, 1882 (Myxozoa: Bivalvulida) parasitizing Indian Attri R (2015). Taxonomic evaluation of myxozoan parasites fishes and a revised dichotomous key to myxosporean genera. infecting fresh water fishes in Punjab. PhD, Punabi University, Syst Parasitol 81: 17-37. Patiala, Punjab, India. Kaur H, Singh R, Katoch A, Attri R, Dar SA, Gupta A (2016). Species Bartholomew JL, Whipple MJ, Stevens DG, Fryer JL (1997). The diversity of the genus Thelohanellus Kudo, 1933 (Myxozoa: life cycle of shasta, a myxosporean parasite of Bivalvulida) parasitizing fishes in Indian subcontinent. J salmonids, requires a freshwater polychaete as an alternate Parasit Dis: 1-8. doi:10.1007/s12639-016-0836-8. host. J Parasitol 83: 859-868. Kent ML, Andree KB, Bartholomew JL, El-Matbouli M, Desser SS, Basu S, Haldar DP (2003). Observations on two new thelohanelloid Devlin RH, Feist SW, Hedrick RP, Hoffman RW, Khattra J et al. species (Myxozoa: Bivalvulida) from Indian major carps of (2001). Recent advances in our knowledge of the Myxozoa. J West Bengal, India. J Parasitol App Anim Biol 12: 15-24. Eukaryot Microbiol 48: 395-413. Chen CL, Ma CL (1998). Fauna Sinica. Myxozoa, Myxosporea. Kimura M (1980). A simple method for estimating evolutionary rate Beijing, China: Science Press Beijing. of base substitution through comparative studies of nucleotide sequences. J Mol Evol 16: 111-120. Gupta A, Kaur H (2016). Morphological, histopathological and molecular characterization of Thelohanellus theinensis n. sp. Landsberg JH, Lom J (1991). Taxonomy of the genera of the (Cnidaria: Myxosporea) infecting an Indian major carp, Labeo Myxobolus/Myxosoma group (Myxobolidae: Myxosporea), bata in a cold water wetland in Punjab (India). J Parasit Dis: current listing of species and revision of synonyms. Sys 1-10. doi:10.1007/s12639-016-0856-4. Parasitol 18: 155-186.

Hall T (2011). BioEdit: An important software for molecular biology. Liu XH, Batueva MD, Zhao YL, Zhang JY, Zhang QQ, Li TT, Li AH (2016). Morphological and molecular characterisation GERF Bulletin of Biosciences 2: 60-61. of Myxobolus pronini n. sp. (Myxozoa: Myxobolidae) from the Kaur H, Gupta A (2015). Genetic relatedness provides support for a abdominal cavity and visceral serous membranes of the gibel species complex of myxosporeans infecting the Indian major carp Carassius auratus gibelio (Bloch) in Russia and China. carp, Labeo rohita. Anim Biol 65: 337-347. Parasit Vectors 9: 562. doi: 10.1186/s13071-016-1836-3. Kaur H, Gupta A (2017). Molecular and phylogenetic characterization Lom J, Dykovà I (1992). Myxosporidia (Phylum Myxozoa). In: of Thelohanellus boggoti Qadri, 1962 (Cnidaria, Myxosporea, Protozoan Parasites of Fishes. Developments in Aquaculture Bivalvulida) infecting the fin of Indian minor carp Labeo dero and Fisheries. Lom J and Dykovà I, editors. Amsterdam, the (Hamilton, 1822). Mol Biol Res Comm 6: 13-21. Netherlands: Elsevier, pp. 159-235.

798 GUPTA and KAUR / Turk J Zool

Pote LM, Hanson LA, Shivaji R (2000). Small subunit ribosomal Székely C, Cech G, Chaudhary A, Borzak R, Hridaya S S, Molnár K RNA sequences link the cause of proliferative gill disease in (2014). Myxozoan infections of the three Indian major carps in channel catfish to Henneguya n. sp. (Myxozoa: Myxosporea). J fish ponds around Meerut, UP, India, with descriptions of three Aquat Anim Health 12: 230-240. new species, Myxobolus basuhaldari sp. n., M. kalavatiae sp. n. and M. meerutensis sp. n., and the redescription of M. catlae Ronquist F, Huelsenbeck JP (2003). Bayesian phylogenetic inference and M. bhadrensis. Parasitol Res 114: 1301-1311. under mixed models. Biogeosciences 19: 1572-1574. Tamura K, Peterson D, Peterson N, Stecher G, Nei M Kumar S (2013). Sakiti N, Blanc E, Marques A, Bouix G (1991). Myxosporidies MEGA6: Molecular evolutionary genetics analysis using (Myxozoa: Myxosporea) du genre Myxobolus Bütschli, 1882 maximum likelihood, evolutionary distance and maximum parasites de poisons cichlidae du lae Nokoue au Benimm parsimony methods. Mol Biol Evol 28: 2731-2739. (Afrique de l’ Ouest). J Afr Zool 105: 173-186. Yakovchuk TA (1979). A new species of the genus Myxobolus Shulman SS (1966). Myxosporidia of the USSR. Moscow and (Myxosporidia: Myxobolidae) from the gill filaments of the Leningrad, Russia: Nauka. carp. Parasitol 13: 635-636. Shulman SS (1984). Parasitic protozoa. In: Key to Parasites of Yokoyama H, Liyanage YS, Sugai A, Wakabayashi H (1998). Freshwater Fishes of the USSR. Vol. I. Bauer ON, editor. Hemorrhagic thelohanellosis of color carp caused by Leningrad, Russia: Nauka. Thelohanellus hovorkai (Myxozoa: Myxosporea). Fish Pathol Singh R, Kaur H (2015). Two new and one already known species of 33: 85-89. the genus Thelohanellus (Myxozoa: Myxosporea: Bivalvulida) Zhao Y, Sun C, Kent ML, Deng J, Whipps CM (2008). Description parasitizing fresh water fishes in wetlands of Punjab, India. of a new species of Myxobolus (Myxozoa: Myxobolidae) based Biologia 70: 85-93. on morphological and molecular data. J Parasitol 94: 737-742.

799