Parasitology International 66 (2017) 272–278
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Myxobolus chushi n. sp. (Myxozoa:Myxosporea) parasitizing Schizothorax niger (Heckel), a native cyprinid fish from Wullar Lake in Kashmir Himalayas Shoaib Ali Dar a,b, Harpreet Kaur a,c,⁎, M.Z. Chishti b a b c
Department of Zoology and Environmental Sciences, Punjabi University Patiala, India Centre of Research for Development, University of Kashmir, Srinagar, India Department of Zoology, Panjab University, Chandigarh, India
a r t i c l e
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Article history: Received 12 April 2016 Received in revised form 16 February 2017 Accepted 19 February 2017 Available online 21 February 2017 Keywords: Myxobolus 18S rDNA Kashmir Himalayas Homogeneity
a b s t r a c t In the study, a new species, Myxobolus chushi n. sp. infecting gills of wild specimens of Schizothorax niger (Heckel) inhabiting Wullar Lake in Kashmir Himalayas, (J&K) India has been described based on morphology of the myxospore and using partial 18S rDNA sequencing. Pathological changes in the gills have been studied with the help of histological sections stained with Luna's method. Twenty fish specimens were examined, out of which four had oval, white plasmodia in gills measuring 2.0 × 0.5 mm. The myxospores were spherical to ovoidal in shape with slightly attenuated posterior end, measuring 11.17 ± 0.23 (10.60–11.40) μm in length and 9.14 ± 0.06 (8.80–9.20) μm in width, having a prominent pore at the anterior end. The polar capsules were pyriform in shape, measuring 4.25 ± 0.15 (4.00–4.40) μm in length and 2.38 ± 0.27 (2.00–2.65) μm in width having polar filaments forming coils up to 5 in number. Parietal folds 9 in number present on the posterior part of the shell. The intensity of infection was recorded to be moderate as indicated by gill plasmodial index (GPI = 2). The plasmodium was located in the vascular network occupying whole of the gill lamella therefore typed as intralamellar vascular type, LV3. Analysis of 18S small subunit (SSU) rDNA sequence of the isolate demonstrated 90% homogeneity with M. sp. KLT-2014 infecting scales of Labeo rohita from Myanmar and 89% with M. dermiscalis infecting scales of Labeo rohita from India. © 2017 Elsevier B.V. All rights reserved.
1. Introduction The Wullar lake is the largest fresh water lake in Asia located in the northern part of Kashmir Himalayas harboring tremendous fish fauna in cold water sector. Schizothoracines are the local endemic cyprinid fishes are commonly found in the cold water of Kashmir Himalayas. These fishes are highly valuable in the region because of its highly nutrition value and low cost. It provides a source of livelihood for a large section of people in the valley. These fishes are infected by variety of parasitic pathogens [1]. Myxozoan parasites are commonly found in fish worldwide [2] and many species have been recorded to cause serious damage to economically important fresh water and marine fish. The whirling disease M. cerebralis has been considered as the most significant disease in trout [3] which infect salmonids causing heavy losses. There are 905 species under the genus Myxobolus [4] and is the most dominant group within the Subphylum Myxozoa (Phylum Cnidaria). The present study was ⁎ Corresponding author at: Department of Zoology and Environmental Sciences, Punjabi University Patiala, India. E-mail address:
[email protected] (H. Kaur).
http://dx.doi.org/10.1016/j.parint.2017.02.004 1383-5769/© 2017 Elsevier B.V. All rights reserved.
designed to study myxozoan infections in an economically important local fish, Schizothorax niger which is not only widely distributed but is also high in demand as food fish in Kashmir. So far, no study has been done on this parasitic group from fresh water fishes of Kashmir valley. Although many species of myxozoan parasites have been recorded from North India, mostly from freshwater fishes of wetlands of Punjab [5–27]. Therefore this study was aimed at the collection, identification and distribution of myxozoan parasites infecting cyprinid fishes in the temperate region of India. In the present study, a new species of myxozoan parasite belonging to the genus Myxobolus has been described infecting the gills of S. niger using morphological, histological and molecular data. 2. Materials and methods Fresh specimens of Schizothorax niger were collected from various catchment sites of Wullar Lake in Kashmir Himalayas. The fish specimens were brought to the Parasitology Laboratory at Centre of Research for Development (CORD), University of Kashmir in an ice-box. After complete parasitological examination the infected gills were removed and placed in a petridish containing 0.9% saline. The plasmodia were
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2.1. Molecular analysis
Fig. 1. Plasmodium of infected gills of Schizothorax niger.
visible under the naked eye and appeared as creamish white pustules on the gills. The plasmodia were counted and teased on a clean slide to liberate myxospores, examined and photographed under Magnus MLX phase contrast microscope. The description of myxosporean species was done according to the guidelines of Lom and Arthur [28]. Fifty to hundred myxospores were studied in fresh as well as stained preparations. 10 to 15 fresh myxospores were measured with the help of calibrated occulo-micrometer. These myxospores were stained with Ziehl Neelsen to elucidate the morphological features. Tissue samples from infected gill were fixed in Bouin's fixative, embedded in paraffin wax, sectioned (5–7 um thick) and were stained with Luna's method [29]. The plasmodia were typed as per their tissue location within the gills according to Molnar [30]. Gill plasmodial index (GPI) was determined using the method given by Kaur and Attri [27].
The plasmodia preserved in ethanol were teased with the help of a sharp needle in a watch glass containing double distilled water, and transferred into 1.5 ml microcentrifuge tubes. DNA was extracted using Qiagen DNeasy Tissue Kit. The product was then quantified in a Nanodrop (Thermo Scientific, Wilmington. USA) spectrophotometer at 180 ng/μl. The universal primer Myx1 (present study) Forward (5′AACCGTGGGAAATCTAGAGC-3′) Reverse (5′-GAGTCAAATTAAGCCGCAGG-3′) were used for the amplification of 18S rDNA using an Eppendorf Master Cycler Pro S. The PCR was carried out, according to Andree et al. [31] at the final volume of 25 μl using the primers which amplified the fragments of approx. 1100 bp of the 18S rDNA gene. The amplification reactions were performed as per standard protocol [32]. The PCR products were analyzed on 2% agarose gel containing 0.5 μg/ml ethidium bromide in 1 × Tris-acetate-EDTA (TAE) buffer and size was estimated by comparison with the 100 bp plus DNA ladder. Following purification of amplified PCR product by EXOSAP treatment, the DNA was quantified and subjected to automated DNA sequencing on ABI 3730 × 1 Genetic Analyzer. Sequencing was carried out using BigDye® Terminator v3.1 Cycle sequencing kit. 2.2. DNA sequencing The PCR amplified products were sequenced at the Molecular Diagnostics and Research Laboratories (MDRL) Pvt. Ltd. Chandigarh, India. The primer Myx1 successfully amplified the 18S rRNA gene of approx. size 837 bp. The nucleotide sequence obtained from myxospores of Myxobolus chushi n. sp. was deposited in GenBank under the accession number KU885917. 2.3. Phylogenetic analysis The phylogenetic analysis was done on a selection of 18S rDNA sequences that comprised the new sequence KU885917 and 28 additional
Fig. 2. Line drawing of myxospores of Myxobolus chushi n. sp.
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sequences from closely related sequences showing 86% homogeneity or above in NCBI GenBank database using the basic local alignment tool (BLAST). Ceratonova shasta (AF001579) isolated from Onchorhynchus mykiss was taken as an out group. Genetic distance analyses were conducted using the Kimura 2-parameter model [33] in MEGA 6.0 software [34]. The Bayesian phylogenetic analysis was conducted using MrBayes v3.2.2 [35]. Sequence alignment was performed by Multiple Sequence Comparison by Log-Expectation (MUSCLE). The tree was generated using Neighbour Joining having 1000 bootstrap values and was proportional to the number of substitutions per site. 3. Results 3.1. Myxospore description 3.1.1. Plasmodia Elongately oval, large, white, 1–2 plasmodia per gill, visible within a naked eye, measure 2.0 × 0.5 mm, histozoic, present in gill lamella (intralamellar vascular type, LV3) (Fig. 1). 3.1.2. Myxospores Myxospores measure 11.17 ± 0.23 (10.60–11.40) μm in length and 9.14 ± 0.06 (8.80–9.20) μm in width spherical to ovoidal with slightly attenuated posterior end bearing a side view with a thickness of 6 μm. Shell valves 0.70 μm in thickness and symmetrical. Polar capsules drop-like, equal converging anteriorly, measuring 4.25 ± 0.15 (4.00– 4.40) μm in length and 2.38 ± 0.27 (2.00–2.65) μm in width. Polar filaments form 6 coils placed parallel to the polar capsule axis.
Fig. 3. Myxospores of Myxobolus chushi n. sp. a. Fresh myxospores. b. Myxospores stained in Ziehl-Neelsen.
Intercapsular process (ICP) absent. Sutural edge markings up to 9 in number, distinct at the posterior end. Sporoplasm agranular, homogeneous and hemispherical. Iodinophilous vacuole large, measuring 3.3 μm in diameter (Figs. 2, 3).
3.1.3. Taxonomic summary Host: Schizothorax niger (Heckel) vern: chush; common name: snowtrout (Cyprinidae) Locality: Wullar Lake, Kashmir Himalayas Altitudinal zone: 1530 m Type Material: Paratypes are myxospore stained by Ziehl-Neelsen, deposited in the Parasitology Laboratory, Department of Zoology and Environmental Sciences, Punjabi University, Patiala, India- Slide no. S/ ZN/29.03.2016. Site of Tissue Development: Gill lamellae (Intralamellar vascular type, LV3) Prevalence of infection: 20% (4/20) Clinical symptomatology: Mucous laden Pathogenicity: Distortion of gill lamellae, cellular degeneration and hypertrophy Gill Plasmodium Index (GPI): 2 (moderate infection) Type of plasmodium: C (visible with naked eye)
Fig. 4. Histopathology of gills of S. niger infected with Myxobolus chushi n. sp. a. Section of gill lamellae of Schizothorax niger showing LV3 type plasmodium (100×). b. Section of LV3 type plasmodia showing myxospores (M) enclosed in plasmodium along with developing stages (400 ×). c. Magnified view of plasmodia showing thick cellular plasmodial wall (PW) and mature myxospores (M) (1000×).
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Etymology: The specific epithet ‘chushi’ derived from the vernacular name of the host 3.2. Molecular data The nucleotide sequence obtained from the myxospores of Myxobolus chushi n. sp. was deposited in the GenBank under the accession number KU885917. Analysis of 18S small subunit (SSU) rDNA sequence of the isolate demonstrated maximum homogeneity of 90% with M. sp. KLT- 2014 (KM401439) infecting scales of Labeo rohita from Myanmar followed by 89% with M. tsangwuensis infecting gills of Cyprinus carpio from China, M. dermiscalis infecting scales of Labeo rohita from India, M. musseliusae infecting the gills of Cyprinus carpio from China, M. carnaticus infecting gill lamellae of Cirrhinus mrigala from India and M. pavloskii infecting gill of Aristichthys nobilis from Hungary (Table 2). The phylogenetic tree based on the final alignment (837 bp) with Neighbour-Joining showed the highest bootstrap value of 90 and formed a separate clade thereby originating monophylogenetically (Fig. 5). 4. Pathogenesis The plasmodium of M. chushi n. sp. was located in the vascular network of the gill lamella of Schizothorax niger and typed as intralamellar
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vascular type, LV3 as per their location within the secondary gill lamella. The plasmodia were rounded in cross section, size ranging from 0.5 to 2.0 mm in diameter, bounded by thick cellular wall. Each plasmodium occupied whole of the gill lamella and due to its enlarged size were pushed into the interlamellar space thereby displacing the adjacent lamellae sideways. Histological sections revealed epithelial hyperplasia and inflammation. Gill epithelial lifting, necrosis, hypertrophy and hyperplasia of the goblet cells were common. This type of plasmodia seemed to cause total functional loss of infected gill lamellae (Fig. 4). 5. Discussion 5.1. Morphological comparison The present species is compared with the myxospore of morphologically similar species i.e. M. balatonicus [36] infecting gill filaments of Cyprinus carpio; M. muelleri [37] infecting gills of Mugil cephalus; M. macrocapsularis [38] infecting gills of Blicca bjoerkna; M.bliccae [39] infecting gill filaments of Blicca bjoerkna; M. rotundus [40] infecting gill lamellae of Abramis brama; M. ellipsoides [41] infecting fins of Rutilus rutilus; M. sommervillae [42] infecting gill filaments of Rutilus rutilus; M. diversicapsularis [43] infecting gill lamellae of Rutilus rutilus; M. caudatus [44] infecting tail fin of Barbus bynni; M. arrabonensis [45] infecting gill filaments of Chondrostoma nasus; M. dogieli [46] infecting
Fig. 5. Phylogenetic tree generated by Neighbour Joining analysis showing the phylogenetic position of Myxobolus chushi n. sp. (KU885917) with other myxosporeans. GenBank accession numbers are given and number above nodes indicated bootstrap confidence values. Scale bar: 0.01 mm.
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Table 1 Comparative description of Myxobolus chushi n. sp. with morphologically similar species (measurements are in micrometer). Species
Host
Site of infection
Locality
Myxospore size
Polar capsule size
Parietal folds
ICP
Myxobolus chushi n. sp. (Present study) M. balatonicus Szekely et al. (2015) M. muelleri Yurakhno (1997) M. macrocapsularis Reuss (1906) M. bliccae Donec and Tozyyakova (1984) M. rotundus Nemeczek (1911) M. ellipsoides Molnar et al. (2006) M. sommervillae Molnar et al. (2010) M. diversicapsularis Slukhai (1966) M. caudatus Ali et al. (2002) M. arrabonensis Cech et al. (2015) M. dogieli Molnar and Szekely (2008) M. carnaticus Banerjee et al. (2015) M. tsangwuensis Chen (1954) M. rutili Donec and Tozyyakova (1984)
Schizothorax niger
Gill lamellae
11.17 × 9.14
4.25 × 2.38
9
Absent
Cyprinus carpio
Gill filaments
J&K (India) Hungary
11.2 × 9.5
5.5 × 3.3
7
Present
Mugil cephalus Blicca bjoerkna Blicca bjoerkna Abramis brama Rutilus rutilus Rutilus rutilus Rutilus rutilus Barbus bynni Chondrostoma nasus Abramis brama Cirrhinus mrigala Cyprinus carpio Rutilus rutilus
Gills Gills Gill filaments Gill lamellae Fins Gill filaments Gill lamellae Tail fin Gill filaments Heart ventricle Gill lamellae Gills Gill filament
Turkey Hungary Hungary Hungary Hungary Hungary Hungary Egypt Hungary Hungary India China Hungary
8.3 × 7 13.6 × 9.7 12.4 × 10 10.6 × 9.5 11.72 × 9.7 11.8 × 9.7 10.3 × 8.7 13.3 × 8.4 8.7 × 7.8 14 × 12.7 9.49 × 8.27 11.2 × 9.3 13.1 × 9.9
3 × 1.8 7.3 × 3.4 6.2 × 3.4 4.9 × 3.2 4.77 × 2.6 6.0 × 3.3 5.4 × 3.5 7.0 × 2.8 4.8 × 2.9 4.13 × 2.2 3.09 × 2.07 5.0 × 3.0 5.9 × 3.4
6–8 5–7 5–7 NA NA 4–6 NA 5–6 4–6 NA NA NA 4–6
Present Present Present Present Absent Present Present Present Present Present NA Present Present
ICP - intercapsular process; NA - not available.
ventricle on the heart of Abramis brama; M. carnaticus [47] infecting gill lamellae of C. mrigala; M. rutili [39] infecting gill filament of Rutili rutilus and M. tsangwuensis [48] infecting gills of Cyprinus carpio but differ from all of the above in morphometric characteristics (Table 1). The myxospores of Myxobolus chushi n. sp. are characterized in having ellipsoidal shape in frontal view with rounded anterior and posterior end. In this respect, it is similar to M. balatonicus, M. muelleri, M. bliccae, M. rotundus, M. ellipsoides, M. sommervillae, M. diversicapsularis, M. arrabonensis, M. dogieli and M. tsangwuensis. The presence of prominent intercapsular process in M. dogieli, M. tsangwuensis, M. balatonicus, M. sommervillae, M. arrabonensis, M. muelleri, M. bliccae, M. macrocapsularis, M. caudatus and M. rotundus differentiate all of them from the myxospores of the present species in which intercapsular process is absent. The presence of unequal polar capsules in M. diversicapsularis, M. carnaticus and M. tauricus differentiates all of them from the myxospores of the species under study bearing equal polar capsules.
The pyriform shape of the myxospores of M. caudatus, M. bliccae and M. macrocapsularis differentiates them from the myxospores of the current species in having rounded and ellipsoidal shape of myxospores. Absence of iodinophilous vacuole in M. muelleri differentiates it from the myxospores of the present study in having large iodinophilous vacuole. Furthermore, the absence of parietal folds in M. ellipsoides differentiates it from the present myxospores in which up to 9 prominent parietal folds are present. In view of the above differences, the present species under study is proposed as new to the science and named as Myxobolus chushi n. sp. 5.2. Molecular comparison The primer Myx1 successfully amplified the 18S rDNA gene of approx. size 837 bp. Partial sequencing of the 18S rDNA gene of M. chushi n. sp. obtained from Schizothorax niger resulted in a total of
Table 2 Homogeneity of 18SrDNA gene sequences of M. chushi n. sp. and other myxospores available in GenBank. Myxozoan
Accession number
Organ infected
Host
Country
Query cover
Homogeneity (%) to M. chushi n. sp. (KU885917)
M. sp. KLT-2014 M. tsangwuensis M. dermiscalis M. musseliusae M. carnaticus M. pavloskii Thelo M diversicapsularis M. bjoerkna M. algonquiensis Thel M. arrabonensis T M. tauricus M. sommervillae M. muelleri M. dogieli M. shaharomae M. alburni M. erythrophthalmi M. feisti M. rutili M. balatonicus M. caudatus M. leuciscini M. macrocapsularis M. muellericus M. ellipsoides M. pfeifferi Ceratonova shasta
KM401439 KJ561441 KM092529 FJ710801 KF796620 HM991164 GU968199 KF314823 AF378335 KP025680 JQ388896 GU968202 DQ439806 EU003977 KF515729 FU567313 KF515727 JN252487 GU968201 KP205545 JQ388889 DQ439811 FJ716095 DQ439807 DQ439813 JQ388895 AF001579
Scales Gills Scales Gill filaments Gill lamellae Gills Gill lamellae Gill filaments Ovaries Gill filaments Gills, Fins Gill filaments Gill filaments Heart Kidney, liver Gill filaments Liver Gill rays Gill filaments Gill filaments Tail fin Gill filaments Gills Gill lamellae Fins Muscle Gall bladder
Labeo rohita Cyprinus carpio Labeo rohita Cyprinus carpio Cirrhinus mrigala Aristichthys nobilis Rutilus rutilus Blicca bjoerkna Notemigonus crysoleucas Chondrostoma nasus Barbus tauricus Rutilus rutilus Mugil cephalus Abramis brama Alburnus alburnus Alburnus alburnus Scardinius erythropthalmus Rutilus rutilus Rutilus rutilus Cyprinus carpio Barbus bynii Leuciscus cephalus Abramis bramma Leuciscus cephalus Rutilus rutilus Barbus barbus Oncorhynchus mykiss
Myanmar China India China India Hungary Hungary Hungary Canada Hungary USSR Hungary Turkey Hungary Hungary Hungary Hungary Hungary Hungary Hungary Egypt Hungary Hungary Hungary Hungary Hungry USA
97 97 97 97 96 97 97 97 97 81 81 78 78 81 79 79 80 79 78 81 77 78 76 82 79 78
1064/1064 (90) 1016/1016 (89) 1005/1005 (89) 1005/1005 (89) 990/990 (89) 990/990 (89) 948/948 (88) 941/941 (88) 922/922 (87) 854/854 (89) 819/819 (88) 809/809 (89) 806/806 (89) 793/793 (88) 787/787 (88) 785/785 (88) 774/774 (88) 774/774 (88) 769/769 (88) 752/752 (87) 750/750(88) 749/749(87) 745/745(88) 743/743(86) 736/736(87) 732/732(87) Outgroup
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Fig. 6. Estimates of evolutionary divergence between the sequences of Myxobolus chushi n. sp. and other myxosporeans available in NCBI GenBank.
837 bp and did not match any of the Myxobolus available in GenBank. The phylogenetic tree based on the final alignment showed it in a separate branch and stood alone in one clade confirming it to be a new species originating monophylogenetically. Moreover, estimates of evolutionary pair-wise divergence among the sequences of Myxobolus chushi n. sp. and M. sp. KLT-2014 were measured as 0.06, while it was 0.05 for M. musseliusae, 0.04 for M. arrabonensis, 0.03 for M. diversicapsularis and 0.00 for M. pavlovskii (Fig. 6). The difference in the divergence rate might be due to different geographic location in addition to various morphological attributes. In order to estimate the pattern of nucleotide substitution involving 28 sequences, K2 + G + 1 was used as the best substitution model having lowest Bayesian Information Criterion (BIC) of 4540.498. Nucleotide frequencies were estimated from the data (A = 27.67, C = 20.93, G = 27.84, T/U = 20.93), five rates of nucleotide substitution were [AC] = 3.63, [AG] = 14.29, [AT] = 4.09, [CG] = 4.83, [CT] = 19.51, [GT] = 4.09 proportion of invariable sites = 51.5895%; gamma distribution = 0.5170 estimated with five rate categories. All positions containing gaps and missing data were eliminated. There were 481 positions in the final dataset. 6. Conclusions This is the first report of a myxozoan parasite infecting a cold water cyprinid fish, Schizothorax niger from temperate region in northern India. The new species, Myxobolus chushi n. sp. has been characterized on the basis of morphology, morphometric, partial amplification of 18 s rDNA and also analyzed phylogenetically. In addition its pathogenicity has been described indicating necrosis of the gill lamellae thereby affecting the respiratory process. Acknowledgements The authors acknowledge the funding by Department of Science and Technology, Government of India, New Delhi in the form of major research project (MRP-SERB No-SR/SO/AS/67/2011). The help rendered
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