Molecular Identification of Seven Myxobolus Species (Myxosporea: Myxobolidae) in Cyprinids from India

Parasite Collection and Morphology

Carp fish were sampled from the Parikshitgarh and Kheri Manihar of district Meerut and Bairaj of district Bijnour of Uttar Pradesh, India in January and February 2017 (n = 58; total length 11-16 cm) and June to August 2018 (n =74; total length 12-15 cm) respectively. Information regarding the collections of specimens of myxozoans is presented in Table 1. Fish were brought to the laboratory at the Department of Zoology, Chaudhary Charan Singh University, Meerut, UP, India and kept in an aerated aquarium. To check myxozoan infections they were euthanized with clove oil and dissected to check various organs like gill filaments, kidney, liver, intestine, gall bladder and muscles for myxozoan plasmodia/ cysts under a Motic stereomicroscope (SMZ-168 series). Plasmodia were carefully removed from the gills, liver, kidney and intestine, opened with a fine needle on a slide and observed under an Olympus microscope (CH30) for further morphological analysis. Measurements based on 25-30 fresh myxospores were taken according to the guidelines of Lom J [36]. All measurements are shown in micrometres (μm) unless stated otherwise also followed by the range (mean ± SD) shown in Table 2, with a comparison to the same species described previously. Some spores were fixed in 95% alcohol and stored at -20°C for subsequent molecular study.

DNA Isolation and Amplification

For genomic DNA extraction, ethanol-preserved plasmodia/cysts were processed using a QIAGEN DNeasyTM tissue kit (animal tissue protocol, Qiagen, Hilden, Germany) according to the protocol recommended by the manufacturer. The ssrDNA was amplified using the universal primers ERIB1 and ERIB10 [49] at the first round of PCR. The reaction was carried out according to the protocol followed as PCR was carried out in a 25-µl reaction mixture with the following thermocycling profile: denaturation at 95°C for 3 min; followed by 35 cycles of amplification at 95°C for 45 s, 56°C for 1 min and 72°C for 1 min, completed with terminal extension at 72 °C for 7 min and then stored at 4 °C. This was followed by a second round of PCR with primers Myx1F- SphR [50, 51]. The total volume of the reactions was 50 µl with the following PCR amplification profile: 95 °C for 3 min, then 35 cycles at 95 °C for 50 s, 56 °C for 50 s, 72 °C for 1 min, terminated with an extension at 72 °C for 7 min and then resting at 4 °C. The PCR amplification products were electrophoresed in 1% agarose gel (Tris-Acetate-EDTA buffer) stained with 1% ethidium bromide and examined under ultraviolet light. PCR products were purified with the PurelinkTM Quick Gel Extraction and PCR Purification Combo Kit (Invitrogen). Amplicons were sequenced from both strands with the PCR primers mentioned above using the Big Dye Terminator vr. 3.1 cycle sequencing kit in ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA).

Phylogenetic Analysis

Obtained contiguous sequences were manually assembled, edited using BioEdit, version 7.2.5 [52] checked and submitted to GenBank for accession numbers and put forward to NCBI for BLASTn and comparison with other myxozoan sequences. Newly generated sequences of the ssrDNA of myxozoan species were aligned together with published sequences of myxozoan species, available on GenBank. The sequences were aligned by using Clustal W [53] implemented in the MEGA X [54]. The evolutionary history was analyzed by using the Maximum Likelihood method and the General Time Reversible model. The nodal support was estimated by bootstrapping (n=1,000). The best evolutionary model of HYPERLINK "https://www. sciencedirect.com/topics/biochemistry-genetics-and- molecular-biology/nucleotide"nucleotide substitution using the Akaike Information Criterion (AICs) was determined with the MEGA X. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.4686)). Genetic distance estimation was carried out using the p-distance model of substitution in MEGA X. A total of 59 nucleotide sequences with over 91% similarity were used for phylogenetic analysis. There were a total of 2075 positions in the final dataset. Evolutionary analyses were conducted in MEGA X. The Chlorormyxum cristatum (AY604198) was selected as out-group in the final analysis.

Myxobolus bhadrensis Seenappa and Manohar [37], Szekely, et al. [6]

Spores description. Spores oval to ellipsoidal in frontal view with anterior and posterior rounded ends. In sutural view, spores pyriform at both the ends and convex at both sides. Length of spore 11.02-12.48 (11.87±0.44) (N=30); width, 7.28- 8.58 (7.95±0.38) (N=30) and thickness, 6.45- 7.02(6.74±0.21) (N=10). Polar capsules pyriform shape and unequal in size. Larger capsule 4.16-5.72 (4.86±0.46) long (N=30) and 1.82-2.86 (2.39±0.29) wide (N=30) while smaller capsule 2.86-4.16 (3.54±0.37) long (N=30) and 1.56-2.35 (1.94±0.25) wide (N=30). 3 polar tubule coils in smaller capsule and 4 coils in larger capsule. Sporoplasm large, binucleated having small iodinophilous vacuole. Intercapsular process not seen (Table 2; Figures 1a,1b & 2a,2b).

Click to enlarge

Figure 3: Myxobolus species phylogenetic relationships inferred by maximum likelihood analysis based on the ssrDNA sequences. Species in bold were newly sequenced in this study. GenBank accession numbers and their host name are listed along the species names. Numbers at nodes indicate the bootstrap confidence values (ML). Chloromyxum cristatum was used as an outgroup.

Present host and locality: Labeo rohita, Local name: Rohu; locality: Parikshitgarh Fish Farm (28°59′ N, 77° 56′ E), Meerut (U.P.), India.

Site of infection: Kidney.

Prevalence of infection: 22/42 in the 9-14 cm size group with a prevalence of 52%.

Material deposited: Digitized photographs were deposited in the Museum, Department of Zoology, Chaudhary Charan

Singh University, Meerut, under voucher number HSS/ZOO/ MYX/03/19.

Representative DNA sequence: The ssrDNA sequences of M. bhadrensis were submitted to NCBI with accession numbers MN994379 and MN994417.

• Remarks Spores of M. bhadrensis showed close resemblance in shape and size with spores of other Myxobolus species having unequal sized polar capsules. Spores of M. Mrigalae Chakravarty [39] showed morphological similarities with spores of M. bhadrensis but presence of several triangular marking in the valve of M. Mrigalae differentiates these two species. Spores M. indicum Tripathi [55] also resemble in size and shape but marked difference is seen in the size of polar capsules of both. Morphologically spores of M. bhadrensis shows close resemblance with spores of M. hosadurgensis Seenappa and Manohar [37] but spores of M. bhadrensis were oval to ellipsoidal and wider than spores of M. hosadurgensis which were elongated ovoidal in shape with anterior narrow end. Spores of M. vedavatiensis Seenappa and Manohar [37], M. lalithae Gupta, et al. [56] and M. haldari Gupta, et al. [45] also showed resemblance with spores of M. bhadrensis but spores of M. lalithae were oval or elliptical in shape while spores of M. vedavatiensis and M. haldari had an intercapsular process or ridge.

Myxobolus calbasui Chakravarty [21]

Spores description. Spores large size, rounded-oval in shape, anterior end pointed and posterior end much rounded in frontal view. Spores pyriform in sutural view with both sides convex. Length of spores 13.0-14.6 (13.8±0.4), width 8.8-10.7 (9.8±0.61), thickness 6.76-7.8 (7.3±0.28). Polar capsules oval-pyriform in shape, unequal sized. Larger capsule much larger than smaller capsule, showed marked differentiation in size, and reaches up to the anterior end of spore, but smaller capsule lies at some distance below the larger capsule. Larger capsule 4.68-6.0 (5.2±0.39) long and 3.12-3.9 (3.49±0.22) wide. Smaller capsule 2.34-3.0 (2.7±0.21) long and 1.3-2.3 (1.86±0.35) wide. Polar tubule coils seen 5-6 in larger capsule and 3-4 in smaller capsule in host fish C. mrigala while in spores obtained from C. reba no. of coils seen 6-7 in larger capsule and 4-5 in smaller capsule (Table 2; Figures 1c & 2c,2d).

Present host and locality: Cirrhinus mrigala (Ham.1822), Local name: Mrigal; locality: Parikshitgarh Fish Farm (28º59′N, 77°56′E), Meerut and C. reba (Ham. 1822), Local name: Raia; locality: Village Kheri Manihar Fish Pond (29º10′N, 77°92′E), Mawana, Meerut (U.P.), India.

Site of infection: Cirrhinus mrigala and C. reba: intestine and liver respectively.

Prevalence of infection: 26/43 and 11/17 in the 11-16 cm size group of Cirrhinus mrigala and C. reba, with a prevalence of 60% and 64.7% respectively.

Material deposited: Digitized photographs obtained from both host C. mrigala and C. reba were deposited in the Museum, Department of Zoology, Chaudhary Charan Singh University, Meerut, under voucher number HSS/ZOO/ MYX/11/19 and HSS/ZOO/MYX/10/19 respectively.

Representative DNA sequence: The ssrDNA sequences obtained from both the host C. mrigala and C. reba were deposited in GenBank with accession numbers MT012462, MT012463 and MT009485, MT012423.

• Remarks The characteristic large, rounded-oval spores of this species, with pointed anterior end and much rounded posterior end shows resemblance with those Myxobolus species, having different size polar capsules, infecting the cyprinid fishes. Spores of Myxobolus calbasui were identified on the basis of available morphological and morphometric data. Initially, this species was reported from the gall bladder of L. calbasu, L. rohita and C. mrigala at Calcutta district, West Bengal, India by Chakravarty [39]. Earlier, Ray [57] reported this species from liver of C. mrigala. Later, Gupta [38] (thesis) redescribed this species from different organs of four species of cyprinid fishes, L. rohita, L. bata, L. dyocheilus and C. mrigala. Spores of M. indicum Tripathi [55] described from the liver, intestinal wall and muscles of same host C. mrigala show close similarity in morphological characteristics but further studies are needed for their identification as a synonymous species due to the poor illustration of the spores. Spores of M. opthalmusculata Basu and Haldar [40], M. mrigalhitae Basu and Haldar [41], M. buccoroofus Basu and Haldar [42] obtained from different carp hosts are close in morphology with spores of M. calbasui. But the presence of a small depression at the anterior end of spores of M. opthalmusculata, the presence of parietal folds in the posterior region of the spores of M. mrigalhitae and narrow and bent spores of M. buccoroofus at anterior side, differentiate these species from M. calbasui. Spores of M. patialensis Kaur and Singh [43] are smaller in size with asymmetrical spore valve, having 4-5 parietal folds in the posterior part of spores as compared to spores of M. calbasui.

Myxobolus catlae [45] Szekely, et al. [6]

Spore description. Spores large-sized and elongated in shape. Anterior end of spores pyriform, sharply tapered and blunt in both frontal and sutural view. Posterior ends of spores ovo-rounded in both views. Spores measurement lies between: length 17.4-19.2 (18.5±0.58), width of 7.28-8.3 (7.9±0.32) and thickness 6.74-7.28 (7.00±0.18). Spore valves thin, uniform and symmetrical. Polar capsules (PC) very long, elongated-pyriform shape and filled most of the spore cavity, leaving a small space for sporoplasm at the posterior part of spore. Size of large PC: length 10.14-11.4 (10.92±0.34) and width 2.7-3.12 (2.92±0.15), size of small PC: length 10.14- 10.92 (10.51±0.26) and width 2.6-2.86 (2.70±0.10). Both capsules parallel to the longitudinal axis of body. In most of the spores, polar capsules equal in size but in some spores slightly unequal in size. The number of polar tubule coils ranged from 11 to 15 in each polar capsule. Sporoplasm small, binucleated and lies at the posterior end of the spore cavity. Intercapsular process and mucous envelop were not seen. A small, variable-sized iodinophilous vacuole was also present in the posterior part of the sporoplasm (Table 2; Figures 1d, 1e & 2e, 2f).

Present host and locality: Cirrhinus mrigala (Ham.1822). Local name: Mrigal; locality: Parikshitgarh Fish Farm (28º59′N, 77°56′E), Meerut and C. reba (Ham. 1822). Local name: Raia; locality: Village Kheri Manihar Fish Pond (29º10′N, 77°92′E), Mawana, Meerut (U.P.), India.

Site of infection: Gills lamellae of Cirrhinus mrigala and C. reba.

Prevalence of infection: 30/43 and 09/17 in the 11-16 cm size group of Cirrihinus mrigala and C. reba with a prevalence of 69.7% and 53% respectively.

Material deposited: Digitized photographs obtained from both the host C. mrigala and C. reba were deposited in the Museum, Department of Zoology, Chaudhary Charan Singh University, Meerut, HSS/ZOO/MYX/07/19 and HSS/ZOO/ MYX/08/19 respectively.

Representative DNA sequence: ssrDNA sequences of M. catlae obtained from both the host C. mrigala and C. reba were submitted to NCBI with accession numbers MT002743, MT003664 and MT002747, MT002748 respectively.

• Remarks Spores of M. catlae can be differentiated from other known Myxobolus species easily because of their large, elongated shaped spores with the large number of polar tubule coils in the polar capsules. Identification was confirmed on the basis of morphological as well as the molecular data also. Spores of M. bengalensis Chakravarty, et al. [58] were much smaller in length as compared to spores of M. catlae. In M. cuttaki Haldar, et al. [59]9 number of coils is less as compared to M. catlae while the spores of M. catmrigalae Basu, et al. [42] much broader and have more coils in polar capsules. Spores of M. zoohuri Majumder, et al. [60] were comparatively smaller in size and have more coils in the polar capsule as compared to the M. catlae.

Myxobolus kalavatiae Szekely, et al. [6]

Spore description. Spores medium size, ovoidal, elongated both in frontal and sutural view, with anterior end, tapering and blunt while posterior end broad and rounded. Length of the spore 11.4-12.74 (12.12±0.39) (N=25); width 5.84-6.76 (6.34±0.28) (N=25); and thickness 4.94-5.46 (5.22± 0.19) (N=15). Spore wall thick, uniform and symmetrical. Polar capsules long, pyriform shaped and equal- sized, obliquely located on either side of the midline, filling around more than half of the spore cavity. Capsules 4.94-5.72 (5.45±0.23) long (N=20) and 1.56-1.82 (1.67±0.094) (N=20) wide.Polar tubule coils are 5 in each capsule. Sporoplasm small, binucleate having a small iodinophilous vacuole. Intercapsular process and mucous envelope not seen (Table 2; Figures 1F, 1g & 2g, 2h).

Present host and locality: Labeo rohita (Ham. 1822), local name: Rohu; locality: Parikshitgarh Fish Farm (28º59′N, 77°56′E) Meerut (U.P.), India.

Site of infection: Gills.

Prevalence of infection: 37/42 in the 9-14 cm size group with a prevalence of 88%.

Material deposited: Digitized photographs were deposited in the Museum, Department of Zoology, Chaudhary Charan Singh University Meerut, under voucher number HSS/ZOO/ MYX/04/19.

Representative DNA sequence: The ssrDNA sequences were submitted to NCBI with accession numbers MN994420 and MN994422.

• Remarks Originally Szekely, et al. [6] collected spores of M. kalavatiae from gills of the host fish C. cirrhosus but we have collected spores of M. kalavatiae from the gills of the host fish L. rohita. As compared to the original description, spores were larger in size, having polar tubule coils, which were not seen by Székely, et al. [6]. Comparison of spores of M. kalavatiae was done with other Myxobolus sp. having equal polar capsules. Comparison of morphological characters of M. kalavatiae showed that it is different from M. shetti Seenappa and Manohar [37], M. venkateshi Seenappa and Manohar [37], M. basuhaldari Szekely, et al. [6], M. meerutensis Szekely, et al. [6]. As spores of M. shetti were smaller and broader than spores of M. kalavatiae, having a small intercapsular process and lacking the polar tubule coils. Spores of M. venkateshi were egg-shaped with a well-developed intercapsular process. Spores of M. basuhaldari showed close resemblance with spores of M. kalavatiae, as both have two equal size polar capsules, equipped with 5 coils, except spores of M. basuhaldari were smaller in size and ovoidal in shape and had a small knob at the anterior end, as compared to the spores of M. kalavatiae. In the original description given by Székely, et al. [6] these two spp. showed close resemblance in their size but spores of M. kalavatiae, obtained in the present study were longer and elongated than the spores of M. basuhaldari. In sutural view spores of M. meerutensis are lemon-shaped while spores of M. kalavatiae are elongated. No. of polar tubule coils seen 6 in M. meerutensis and 5 in M. kalavatiae. A small knob-like intercapsular appendix was also present in the spores of M. meerutensis but not seen in the spores of M. kalavatiae.

Myxobolus haldari Gupta, et al. [45]

Spores description. Spores long, ovoidal or elliptical in frontal view, biconvex in sutural view with round anterior and posterior ends. Length of spore 10.7-11.6 (11.16±0.27) (N=25), width 7.9-8.63 (8.3±0.19) (N=25) and thickness 5.52-5.8 (5.67±0.08) (N=15). Polar capsules (PC) unequal sized and pyriform shaped. Large PC length 4.6-4.8 (4.7±0.06) (N=25); width - 2.24-2.39 (2.32±0.04) (N=25) and small PC length 3.56-3.68 (3.63±0.05) (N=25); width 1.84-2.0 (1.94±0.05) (N=25). Number of polar tubule coils 4-5 in smaller capsule, while 6-7 coils in larger capsule. Intercapsular process was not seen (Table 2; Figures 1h, 1i & 2i, 2j).

Present host and locality: Labeo rohita (Ham.1822), local name: Rohu; locality: Parikshitgarh Fish Farm (28º59′N, 77°56′E), Meerut (U.P.), India.

Site of infection: Kidney.

Prevalence of infection: 33/42 in 9-14 cm size group with a prevalence of 78.6%.

Material deposited: Digitized photographs were deposited in the Museum, Department of Zoology, Chaudhary Charan Singh University, Meerut, under voucher number HSS/ZOO/ MYX/05/19.

Representative DNA sequence: The ssrDNA sequences of Myxobolus haldari were submitted to NCBI with accession numbers MT002391 and MT002392.

• Remarks Spores of Myxobolus haldari were found very close to the spores of M. haldari in morphometric traits and other Myxobolus species with unequal polar capsules. Spores of Myxobolus haldari can be differentiated from spores of M. mrigalae Chakravarty [39], M. seshadri Lalita Kumari [61], M. carnaticus Seenappa and Manohar [62] and M. vedavatiensis Seenappa and Manohar [37] due to their size differences and by the presences of several triangular markings in shell valve of M. mrigalae, presences of inter capsular ridge in spores of M. seshadri, M. carnaticus and M. vedavatiensis. Spores of Myxobolus haldari and M. bhadrensis Seenappa and Manohar [37] showed differences in the length of their polar capsules.

Myxobolus saranae Gupta, et al. [46]

Spore description. Spores elongated, oval-shaped with blunt- oval anterior and posterior ends in frontal view. In a sutural view, spores lentiform or convexo-convex in shape. Spores 10.6-11.96 (11.3±0.39) long (N=25); 7.0- 8.1 (7.52±0.34) wide (N=25) and 5.2-5.8 (5.5±0.22) thick (N=15). Polar capsules unequal sized, longer, pyriform or pear-shaped, converging towards the anterior end of spores. Larger capsule 4.94-6.2 (5.52±0.35) long (N=25) and 2.6- 3.38 (2.92±0.26) wide (N=25) whereas length of small polar capsule 3.12-3.9 (3.52±0.24) (N=25) and width 1.56-1.86 (1.7±0.09) (N=25). Polar tubule coils were seen in both polar capsules and no. of coils ranges 5-6 in larger capsule and 3-4 in smaller capsule. Sporoplasm small, cresentric shaped, uni or binucleated. Intercapsular process and mucous envelop not seen. A variable-sized iodinophilous vacuole was seen in the posterior part of sporoplasm (Table 2; Figures 1j & 2k, 2l).

Present host and locality: Labeo bata (Ham. 1822), Local name: Bata; locality: Bairaj, Bijnor, (29°23′ N, 79°11′ E), (U.P.), India.

Site of infection: Gills.

Prevalence of infection: 23/30 in the 15- 20 cm size group with a prevalence of 76.6%. Material deposited: Digitized photographs deposited in the Museum, Department of Zoology, Chaudhary Charan Singh University, Meerut, under voucher number HSS/ZOO/ MYX/06/19.

Representative DNA sequence: The ssrDNA sequences of Myxobolus saranae are submitted to NCBI with accession numbers MT002911 and MT002914.

• Remarks The morphometric details were similar to the M. saranae, which was described by Gupta, et al. [46] from the gills of Puntius sarana and Labeo calbasu. Later on, Kaur, et al. [47] re-described it from caudal fin of L. rohita. ssrDNA sequences generated from Myxobolus saranae did not resemble other available myxosporean sequences at the GenBank. Morphologically M. saranae shows some similarities with other Myxobolus species as Spores of M. mrigalae Chakravarty [39] have several triangular markings on the shell valves as compared to the spores of M. saranae. The presence of intercapsular ridge in spores of M. seshadri Lalitha Kumari [61] makes a differentiation in both the species. Spores of M. bhadrensis Seenappa and Manohar [37] were broader and its polar capsules were smaller than M. saranae. Spores of M. vedavatiensis Seenappa, et al. [37] larger in size and possesses a large inter capsular ridge. Spores of M. chakravartyi Halder, et al. [63] were larger in size with anterior flat end and a triangular thickening at the anterior end. In M. chakravartyi number of polar filament coils 8-9 in the larger capsule and 6-7 in the smaller capsule. Spores of harpreetae Ghosh, et al. [48] have long and less wide spores than M. saranae. Polar capsules of Myxobolus saranae are smaller in length as compared to M. harpreetae.

Myxobolus hosadurgensis Seenappa and Manohar [37]

Spores description. Spores long, ovoid or ellipsoidal in frontal view and biconvex in sutural view with round anterior and posterior ends. Length of spore 12.37-13.52 (12.86±0.41) (N=25), width 7.54-8.32 (7.94±0.25) (N=25) and thickness 5.46-5.98 (5.70±0.17) (N=10). Polar capsules (PC) unequal sized and pyriform in shape. Large PC length 4.65-5.3 (4.9±0.23) (N=25), width 2.66-3.1 (2.86±0.14) (N=25) and small PC length 2.96-3.43 (3.18±0.15) (N=25), width 1.56-1.82 (1.71±0.09) (N=25). Number of polar tubule coils, 3 in smaller polar capsule and 5 in larger polar capsule. Sporoplasm binucleated, having a small iodinophilous vacuole. Intercapsular process was not seen (Table 2; Figures 1k, 1l & 2m, 2n).

Present host and locality: Cirrhinus mrigala (Ham.1822) Local name: Mrigal; locality: Parikshitgarh Fish Farm (28°59′ N, 77° 56′ E), Meerut (U.P.), India.

Site of infection: Kidney.

Prevalence of infection: 21/43 in the 11-16 cm size group with a prevalence of 48.8%.

Material deposited: Digitized photographs were deposited in the Museum, Department of Zoology, Chaudhary Charan Singh University, Meerut, under voucher no. HSS/ZOO/ MYX/09/19.

Representative DNA sequence: ssrDNA sequences of Myxobolus hosadurgensis are submitted to NCBI with accession numbers MT002915 and MT002924.

• Remarks The spores of Myxobolus hosadurgensis showed close resemblance in morphometric traits with spores of Myxobolus hosadurgensis Seenappa and Manohar, 1981. Because of unequal sized polar capsules M. hosadurgensis showed its morphological similarities with M. mrigalae Chakravarty [39], M. pinnaurati Lalitha Kumari [61], M. carnaticus Seenappa and Manohar [37], M. bhadrensis Seenappa and Manohar [37] and M. vedavatiensis Seenappa and Manohar [37] with some difference in their size, shape and number of polar tubule coils in polar capsules. However, recently generated ssrDNA sequences of this species did not match with any other previous myxozoan sequences available at NCBI.

Phylogenetic Analysis

Molecular analysis of the ssrDNA gene confirmed the phylogenetic position of the studied species (Figure 3). Maximum likelihood (ML) generated a well-resolved tree topology and bootstrap values are presented (Figure 3).

Myxobolus kalavatiae infecting the gills of rohu in the present study shows that ssrDNA sequences of both isolates did not show any intraspecific divergence and shared high identity (99.4%) with other isolates of the same species described by Székely, et al. [6] from gills of mrigal (Cirrihinus cirrohosus) (Figure 3). The phylogenetic analyses inferred from ssrDNA data set also show that M. kalavatiae is a sister to isolates of M. basuhaldari with strong homology (96.4%) reported from the gills of rohu and katla by Székely C, et al. [6]. The present phylogenetic analysis shows that M. kalavatiae was positioned within the well-supported clade that belongs to the gill infecting species in a clade A. Myxobolus catlae collected in the present study from gills of mrigal and reba was originally described by Chakravarty, 1943 from various hosts katla, rohu and mrigal. Later M. catlae was also reported by Székely, et al. [6] from the gills of Cirrihinus cirrhosus with a more comprehensive description. In the present study, we have collected M. catlae from mrigal and reba and ssrDNA sequences of these M. catlae isolates exhibited a similarity of 99.8 % with each other collected from two different hosts (Figure 3). Our isolates showed a 98.2 % similarity with M. catlae, which was isolated from the gills of Cirrihinus cirrhosus. In the clade B, Myxobolus bhadrensis ssrDNA sequence of two isolates collected from the kidney of rohu were found to be identical and showed a 99.2 to 99.4% similarity with other isolates of same species reported from the kidney and muscles of rohu, katla reported by Székely C, et al. [6] while showing similarity of 99.1% with isolate WBUAFSR3C2 infecting the kidney of rohu from West Bengal, India (Figure 3). The phylogenetic tree also showed that M. bhadrensis species formed a sister relationship with M. kingchowensis reported from the muscles of Carassius gibelio from China

with maximum homology (96.8 %) (Figure 3). According to the Székely, et al. [6] both the Myxobolus species (M. kingchowensis and M. bhadrensis) shared the same subclade and show affinity to tissue tropism towards the muscle and kidney respectively. Regarding Myxobolus saranae reported from gills of bata (Labeo bata) can be readily differentiated from other Myxobolus species studied here in the study based on molecular data of the ssrDNA gene and placed in the clade C (Figure 3). The tree topology strongly supported the independent branch formed by isolates of Myxobolus hosadurgensis shows no intraspecific variation among isolates. Myxobolus saranae forms a branch close to Myxobolus sp. (MK412937) infecting gills lamellae of rohu (Labeo rohita) shows only 81.3% similarity with each other and both are members of the same host family Cyprinidae (Figure 3).

Myxobolus haldari was originally described from the fins and gills of mrigal, rohu, bata and kali (Labeo dyocheilus) by Gupta, et al. [45]. Nonetheless, the molecular study of this species has not been performed previously. We have collected M. haldari from kidney of rohu and the molecular analysis of the ssrDNA gene confirmed its validation. The phylogenetic placement of M. haldari isolates clustered them together in an independent branch under subclade D and then clustered with sequences of Myxobolus hosadurgensis and M. calbasui isolates, parasites of kidney, liver and intestine of cyprinid host mrigal and reba (Figure 3). The retrieved similarity values of Myxobolus haldari isolates are 87.6% with isolates of Myxobolus hosadurgensis and 85.3% with M. calbasui isolates. Myxobolus hosadurgensis isolates share a common clade with M. calbasui collected in the present work from liver and intestine of reba and mrigal carp respectively in a clade D (Figure 3). Both the species share some morphological similarities as their myxospores have larger and shorter polar capsules. In turn, Myxobolus haldari which forms a branch close to Myxobolus hosadurgensis and M. calbasui have also significantly large and small polar capsules. The genetic distance estimation revealed the highest similarity of Myxobolus spp. to M. calbasui isolates (92.3%) (Figure 3). M. calbasui collected in the present study from the liver and intestine of reba and mrigal both have unequal polar capsules. Originally, this species was described by Chakravarty MM [39] from gall bladder of calbasu (Labeo calbasu), rohu and mrigal with small polar capsules have three to four filament turns while the large ones have five to six turns respectively. In all, M. calbasui isolates of reba and mrigal ssrDNA gene sequences revealed 100% similarity with each other (Figure 3). M. calbasui isolates share a clade with Myxobolus hosadurgensis from the kidney of mrigal and the genetic distance similarity values range 91.8% (Figure 3).