punjab ( india ) has 3 main wetlands , i.e. harike , kanjli and ropar wetlands which are included in ramsar list of international importance . harike wetland ( 31 17 n latitude and 75 12 e longitude ) is the largest freshwater wetland ( in northern india ) of 4100  ha area with beas and sutlej as primary inflows . it is a habitat for as many as 26 species of fishes which include catla , cirrhinus , channa , mystus , chitala chitala , cyprinus and ambassis ranga . these wetlands are the major natural fisheries resource for food in whole of the punjab state . the study indicates that large variety of fishes in these wetlands are infested with myxozoan parasites . myxozoans include histozoic and coelozoic parasites infecting not only freshwater and marine fishes but have also been detected in molluscs , amphibians , reptiles , waterfowl and mammals ( moncada et  al . , 2001 , eiras , 2005 ) . as demonstrated firstly by wolf and markiw ( 1984 ) , it has been proven that myxozoan species require an alternate invertebrate host ( usually an annelid ) to complete the life cycle . among myxosporeans , ( 2005 ) reported 744 valid species , while lom and dykova ( 2006 ) counted 792 including 7 amphibian species . several other reports on myxobolus species are available from punjab and west bengal , india ( basu & haldar , 2004- basu et  al . , 2009 , about 131 species of myxobolus have been recorded in india ( kaur and singh , 2012 ) and are mainly differentiated by morphological , morphometric characteristics of spores , besides host and organ or tissue specificity . presently , the molecular analyses have been an important tool in the study of these parasites and this has expanded their taxonomy to the phylogenetic analyses . this has led to correct identification and differentiation of morphologically indistinguishable myxobolid species ( kent et  al . , 2001 , eszterbauer , 2002 , molnar et  al . , 2010 , the most revealing aspects of analyses based on ssu rdna sequences is the incongruence of phylogenetic trees with classification based 63on spore morphology alone ( bartosova et  al . , 2009 ) . so far , there are 14 sequences i.e caudatus 
 kc865607 ; myxobolus cuttacki 465682 ; m. orrissae 
 kf448527 ; myxobolus basuhaldari 
 km029974 , km029975 , km029976 ; m. kalavatiae 
 km029973 ; m. meerutensis 
 km029977;m . bhadrensis 
 km029968 , km029969 , km029970 , km029971 , km029972 and m. catlae 
 km029967 ( mondal et  al . , 2014 , szekely et  al . , 2014 , , 2014 , abraham et  al . , 2015 ) from india are available in the genbank . in this study , we present morphological and molecular characterization of m. dermiscalis n. sp . infecting the scales of l. rohita collected from harike wetland , punjab , india along with 18s rdna based phylogenetic analysis with myxobolus group and other related texa . in future , the molecular methods can be implied on the diagnostics of the economically important myxozoan parasites in this part of the world . fresh specimens of indian major carp , rohu , l. rohita were collected from the fisher - man at harike wetland during the period june 2013 to july 2014 . the infected scales were removed with the help of forceps in a petridish containing 0.9% saline . the pseudocysts were visible with the naked eye and appeared as creamish white patches on the scale . the plasmodium were teased on a clean slide to liberate spores and were examined under the microscope . fresh spores were treated with 8% koh solution for the extrusion of polar filaments . for permanent preparation , the prevalence ( in percentage ) of m. dermiscalis infecting scales of l. rohita was calculated according to bush et  al . the intensity of infection was determined by following the index proposed by kaur and attri ( 2015 ) for infection on the scales . 0  =  no infection ; 1  =  one pseudocyst per scale in 10% of total scales ( indicating light infection ) ; 2  =  two pseudocysts per scale in 1520% of total scales ( moderate infection ) ; 3  =  three to four pseudocysts per scale in 50% of total scales ( heavy infection ) ; 4  =  four to five pseudocysts per scale in 100% of scales ( severe infection ) . the pseudocysts ( 50 in numbers ) present on ethanol fixed scales were ruptured with the help of a sharp needle in a watch glass having double distilled water . the contents in the watch glass containing spores were collected in 1.5  ml micro centrifuge tubes . polymerase chain reaction ( pcr ) was carried out according to the andree et  al . ( 1999 ) at the final volume of 25  l using the primers mx5-mx3 which amplified the fragments of 1597  bp respectively of the 18s rdna gene . the amplification reactions were conducted with 3075  ng of genomic dna , 12.5  l of 1    reaction buffer ( hi media ) , 1.0  l of each primer , 1.0  l of total dna and 10.5  l of double purified water . amplification was done by initial denaturation at 95  c for 3  min , followed by 33 cycles of denaturation at 95  c for 30  s , annealing of primers at 58  c for 30  s , extension at 72  c for 1  s. the final extension was at 72  c for 10  s. the pcrproducts were analysed agarose gel electrophoresis , and size was estimated by comparison with the1 kb plus dna ladder ( fig .  1 ) . extracted from spores using the dneasy blood & tissue kit ( qiagen ) following the manufacturer 's instructions . the product was then quantified in a nanodrop ( thermocycler ) . in the present study , the pcr amplified products were sequenced at the molecular diagnostics & research laboratories ( mdrl ) pvt . 1597  bp of 18s rdna sequences of m. dermiscalis n. sp . were deposited in genbank with accession number km092529 . phylogenetic analysis involving 23 nucleotide sequences was performed using the bayesian , maximum likelihood ( ml ) , maximum parsimony ( mp ) and neighbour joining ( nj ) methods . the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( 1000 replicates ) . the evolutionary distances were computed using the kimura 2-parameter method kimura ( 1980 ) and units of the number of base substitutions per site . the best fit model for analysis for the current data was gtr  +  g with lowest bis ( bayesian information criterion ) was estimated by using model test tool in mega 6.06 . round to irregular , white , 4 - 5pseudocysts per scale , histozoic , present within a cavity and measure 0.53.6  mm in diameter . the spores measure 5.847.84    3.985.98  m , oval to spherical in frontal view having rounded anterior and posterior ends . parietal folds two , present on the posterio - lateral margins of the shell valves . polar capsules are two , equal , measure 3.985.98    1.853.85  m and are pyriform with distinct neck at the anterior end . polar filaments form 56 coils and are arranged obliquely to the polar capsule axis , 7.2  m in length when extruded . two capsulogenic nuclei are present beneath each of the polar capsule . sporoplasm agranular , homogenous occupying whole of the extracapsular space behind the polar capsules and contain one nucleus and a large iodinophilous vacuole type specimen : paratype are spores stained with ziehl  neelsen and iron - haematoxylin , deposited in the parasitology laboratory , department of zoology & environmental sciences , punjabi university , patiala ( india ) . intensity of infection with parasite species ( index ) : 4 ( severe infection ) . etymology : the specific epithet dermiscalis has been given on the basis of location of pseudocysts within the layers of dermal scales . the edited nucleotide sequence was 1597  bp of m. dermiscalis n. sp . which was deposited in gen - bank under the accession number of km092529 . blastn search ( www.ncbi.nlm.gov/blast ) with 21 myxobolus exhibited 9093% sequence homology and maximum homology of 98% recorded was recorded with myxobolus sp . ( km401439 unpubl ) infecting scales of a cyprinid , l. rohita in myanmar available in the genbank ( table  1 ) . in the phylogenetic tree based on the final edited alignment with neighbour  joining analysis ( fig . 5 ) , m. dermiscalis was placed with the m. sp . with highest bootstrap value and confirmed the clustering pattern of maximum likelihood and maximum parsimony analysis ( 100% in ml , 100% mp ) . the present species has been compared with m. squamae ( keysseltz , 1908 ) from scales of abramis brama ; m. mrigalae ( chakravarty , 1939 ) from scales of cirrhinus mrigala ; m. yogendrai ( tripathi , 1952 ) , m. rewensis ( srivastava , 1979 ) and m. vanivillasae ( seenappa and manohar , 1980 ) from scales of cirrhinus mrigala ; m. rohitae ( haldar et  al . , 1981 ) from scales of l.  rohita , labeo calbasu , labeo bata and puntius sarana ; m. indirae ( gupta and khera , 1988 ) from scale , tail , fin , cartilage and head of cirrhinus mrigala m. episquamae ( egusa et  al . , 1990 ) from scales of mugil cephalus ; m. squamaphilus ( molnar , 1997 ) from scales of a. brama ; m. dermatis ( haldar et  al . , 1983 ) from scales of l. rohita ; and m. saugati ( kaur and singh , 2011 ) from scales and gill lamellae of catla catla and l. rohita . the present species , m. dermiscalis n. sp . was compared with all the above mentioned species and was found different in spore morphology and morphometrics . the spores of the present species are characterized in having oval to spherical shape with rounded anterior end without intercapsular process and posterior end with two parietal folds . polar capsules are two , equal , pyriform with distinct neck . in this respect , the present species differed from m. squamae , m. yogendrai , m. rewensis , m. vanivillasae , m. rohitae , m. indirae , m.episquamae , m. squamaphilus , m. dermatis , and m. saugati in lacking the intercapsular process . furthermore , it differed from m. saugati , m. rohitae , m. dermatis and m. indirae in having two parietal folds placed posterio - laterally on the shell valves . the present species is also different from m. mrigalae in which several triangular markings are present , in addition to unequal polar capsules . in view of the above differences in morphology and morphometrics , the present species under study the universal primer sets mx5 and mx3 successfully amplified approximately 1600  bp fragments of the 18srdnagene from the present sample of myxobolus ( fig .  3 ) . the edited nucleotide sequence was 1597  bp of m. dermiscalis n. sp . which was deposited in genbank under the accession number of km092529 . infecting scales of a cyprinid , l. rohita in myanmar available in the genbank ( unpubl ) , which showed 98% homogeneity ( table  1 ) . in the phylogenetic tree based on the final edited alignment with neighbour  joining analysis ( fig .  5 ) , . with highest bootstrap value and confirmed the clustering pattern of maximum likelihood and maximum parsimony analysis ( 100% in ml , 100% mp ) . the present study indicated close similarity based on 18s rdna suggesting sister species , however , the morphological comparisons could not be made due to lack of published record . minimum evolutionary divergence between the two species was estimated using bayesian information criterion ( bis scores ) using best model test tool in mega 6.06 ( fig . 4 ) . in order to estimate the pattern of nucleotide substitution involving 23 nucleotide sequences using gtr  +  g model in ( table  2 ) . rates of different nucleotide substitution(r ) from one base ( transitional -in bold ) and to another base(transversional - in italics ) were assessed . maximum transition was c g having 19.99 probability value followed by tc 14.38 , ag 13.21 , ga 12.62 . as for trasversions at , gt 5.31 ; ta , ca 5.26 , tg , cg 247 5.51 , ac , gc 3.82 . the nucleotide frequencies were 26.44%(a ) , 26.69%(t / u ) , 248(g ) 19.19%(c ) and 27.68%(g ) . furthermore , the information generated confirmed the importance of features such as host , tissue tropism , geographical location of the host and correlation with the results of molecular data based on comparison of 18s rdna sequence ( kent et  al . , 2001 , seo et  al . , 2012 , zhu et  al . , 2012 , shin et  al . , 2013 ) . index of intensity of infection by m. dermiscalis n.sp was recorded as 4 indicated by the presence of four to five pseudocysts per scale in 100% of scale . the prevalence of m. dermiscalis n. sp . infesting scales of l. rohita was 59.7% . this study is the first report the molecular and phylogenetic characterization of scale infecting m. dermiscalis n. sp . from indian subcontinent . as the list of indian species of the genus myxobolus have increased over the years ( basu et  al . , 2009 , bandyopadhyay et  al . , 2006/2007 ; singh & kaur , 20122014 ) thus the correct identification in future studies must focus on molecular and phylogenetic characterization together with morphological characteristics , host , organ and tissue specificity . the present study describe a myxobolid species commonly found infecting scales of an indian major carp , l. rohita in freshwater habitat . the intensity of infection was severe as indicated by the scale pseudocyst index and highly symptomatic to the fish host . further investigations are being carried out on the presence of gill infecting myxozoan parasites among major indian carps in polycultured conditions . this is important to have assessment of the impact on these parasites on the carp fishes . beside morphological features , genetic data has been provided in the present manuscript which can form basis for development of diagnostics of this species .