Molecular Characterization of Muscle Infecting Myxobolus sp. Causing Outbreak in Labeo rohita, Rohu: First Report from Andaman Islands

Freshwater aquaculture receives more attention in Andaman and Nicobar Islands owing to its huge domestic demand for freshwater fishes. Quality seed, feed and improper pond management practices are the intimidating issues for the development of freshwater aquaculture in these Islands. Due to lack of intensified and organized research efforts on aquatic animal diseases, Andaman and Nicobar Islands were believed to be free from many of the aquatic animal diseases. Aquatic animal disease surveillance programme has been initiated in these Islands to address the knowledge gap which could unravel the presence of viral and parasitic diseases (Praveenraj et al., 2017; Saravanan et al., 2017a,b,c; Kiruba-Sankar et al., 2018; Praveenraj et al., 2019). Disease associated mortality paves way for instant production loss in aquaculture and major cause of concerns in rural farming. On the other hand, poor growth rate due to heavy parasitic infestations also claims a major toll in aquaculture in the form of substantially decreased production. Hence better management practices need to be adopted by the farmers to maintain healthy stocks for achieving enhanced pond productivity.     
     
Myxozoans are the spore-forming parasites of both marine and freshwater fishes, classified mainly based on spore morphology and also by its specificity to the host, organ and tissue-specific nature (Molnar, 1994; Rajesh et al., 2014). Globally, around 2,180 species belonging to 60 genera have been reported under the Class Myxosporea (Lom and Dykova, 2006) of which, Myxobolus is the dominant genus comprising of 744 species (Eiras et al., 2005). Myxozoan parasites are very common in Indian aquaculture and also considered as the emerging parasites of economically important aquaculture species (Singh and Kaur, 2012). It was reported that around 97 species of Myxobolus have been reported from the fishes in India of which, 29 species infest major carps and its hybrids (Kalavati and Nandi, 2007). In case of advanced stage of Myxobolus infestation, it will be a challenging task to deal with the control measures as the multi-cellular spores are resistant to most of the chemicals and environmental factors (Singh and Kaur, 2012).     

Molecular studies on the identification of Myxobolus sp. are highly encouraged due to the difficulties in conventional taxonomy which relies mainly on spore morphology (Abraham et al., 2015; Szekely et al., 2015). With this background, the present study was undertaken to deal with a disease outbreak occurred in a freshwater carp farm located at North and Middle Andaman and the causative agent was identified as Myxobolus sp. by using molecular  tools.

Disease outbreak with mass mortalities were observed in a carp farm (12°43' 17.02'' N; 92°53' 4.71'' E) located at Nimbudera, North and Middle Andaman district of Andaman and Nicobar Islands (Fig 1). The outbreak was observed in two cemented nursery tanks each measuring 10 x 6 x 1 m and stocked with ten days old fry of Labeo rohita (size range of 0.31 - 0.42 g) at the rate of 150 numbers/ m². Initially, the fish fry were fed twice a day ad-libitum with mixture of wheat flour, ground nut oil cake, milk powder, cooked chicken eggs and stored at 0°C up to 5 days. Stored feed was thawed in open air before placing it onto equidistantly kept feeding trays (4 numbers) hanged in water column of each tank. Visual health status of fish fry was observed everyday while feeding the fishes. Random fish sampling was carried out periodically in which, growth parameters of fry and water quality parameters were analyzed using standard protocol (APHA, 1998) and by following the manufacturer’s protocol for alkalinity and hardness test kits (Himedia, Mumbai). 
 

        
First infection on rohu fish fry was observed during 32 days of stocking of fish fry in cemented nursery tanks. The affected fish samples were collected and preserved in 70% ethanol for further laboratory analysis. The photographs of the affected fishes were taken by using 1200D Canon camera. Modified CTAB method (Bruce et al., 1993) was used to isolate the genomic DNA from the muscle tissue of affected fishes. The isolated DNA was used for PCR to amplify a region of 18S rRNA using forward primer (MC5-F) 5'-CTGAGAAACGGCTACCACATCCA-3' and reverse primer (MC5-R) 5'-ATTAGCCTGACAGATCACTCCACGA-3' by following the method of Molnar et al., (2002). PCR thermal cyclic condition consisted of 95°C for 5 min, 35 cycles of 95°C for 30 sec, 56°C for 30 sec, 72°C for 1 min and finally 72°C for 5 min in a thermal cycler (Bio-Rad, USA). PCR products were resolved in 1.2% agarose gel containing ethidium bromide and analysed using gel documentation system (Bio-Rad, USA). Amplified PCR products were purified and sequenced (Shrimpex Biotech Private Limited, Chennai) using gene-specific primers in ABI 3500 DNA analyser.
        
The homology of the generated sequences was analysed using the Basic Local Alignment Search Tool (BLAST) program (Altschul et al., 1990). The sequences were trimmed by CLC sequence viewer ver. 8 (QIAGEN) and submitted in the National Centre for Biotechnology Information (NCBI) GenBank database. Gene sequences were aligned using CLUSTAL W (Multiple Sequence Alignment with High Accuracy and High Throughput) (Edgar, 2004). The genetic distance between the Myxoblous sp. sequences was determined by the Kimura 2-parameter (K2-P) model (Kimura, 1980) in the software program MEGAX (Molecular Evolutionary Genetics Analysis) (Kumar et al., 2018). The best fit nucleotide substitution model was selected from 24 models, based on the one with the lowest BIC (Bayesian Information Criterion) scores, which was considered to describe the best substitution pattern (Nei and Kumar, 2000). The phylogenetic tree was constructed based on the maximum likelihood fits in MEGAX. Reliability of the phylogenetic tree was estimated using bootstrap values run for 1000 iterations. 

Among the myxozoan parasites, the genus Myxobolus is dominated with more numbers of species of which, most of them are fish pathogens (Kent et al., 2001; Feist and Longshaw, 2006). In this present study, first incidence of parasite infestation was recorded on 32 days of stocking of fish fry in the cemented nursery tanks. The details of growth and water quality parameters were given in Table 1. The affected rohu fry exhibited the clinical signs and symptoms such as lethargic movement, dark body colouration, loss of appetite, excess mucus secretion, 1-2 mm multifocal white nodules on the body surface and skeletal deformities including spinal curvatures (Fig 2). 
 

 

        
Molecular characterization was carried out using specific primers for 18S rRNA to determine the identity of pathogen. Around 1000 bp product was obtained in the PCR amplification (Fig 3). The sequence of the PCR product obtained in the present study revealed 95.45% identity with 18S rRNA sequence of Myxobolus musculi (GenBank accession number JQ388892) and 95.23% identity with Myxobolus pseudodispar (GenBank accession number KU340983). Molecular analysis along with the clinical signs identified the causative agent as muscle infecting Myxobolus sp. (Hahn, 1917; Longshaw et al., 2003). It was reported that high amount of sequence similarities were observed among the small subunit rRNA sequences of morphologically similar M. musculi, M. pseudodispar and M. cyprini (Molnar et al., 2002). Model test suggested the best fit nucleotide substitution model to be the General Time Reversible (GTR) with gamma distribution and assumption that a certain fraction of sites are evolutionarily invariable (+I) [(G+I), AICc = 27062.679, lnL = -13456.238, (+I) = 0.37, (+G) = 0.43]. It was found that the closest genetic congener of the Andaman isolate is Myxobolus musculi (GenBank accession number JQ388892) from which it differs by a K2-P distance of 4.6%. Genetic distance between the new species and members of other congeners are provided in the Table 2. The maximum likelihood tree generated was provided in Fig 4.
 

 

 

        
Molecular tool was employed for the identification of Myxobolus, since it is the preferred method to refine the taxonomic classification of myxosporea and to eliminate the ambiguities that arise from identifications based on morphology of myxospores alone (Andree et al., 1997; Molnar et al., 2002; Camus and Griffin, 2010; Liu et al., 2013). Further, spore morphology of Myxobolus may vary based on tissue location and therefore, it can be environmentally determined (Bahri et al., 2003). Hence the present study utilised the sequence data in solving complicated taxonomic decisions as reported earlier (Easy et al., 2005). The 18S rRNA sequence of Myxobolus sp. obtained in this present study has been submitted to NCBI GenBank under the accession number MK128509.
        
The first incidence of parasite infestation was recorded on 32 days of stocking of fish fry in the cemented nursery tanks and within a short period of time, the infestation further spread to 100% of population which indicates that the infectivity of Myxobolus sp. in Labeo rohita is quite high. These results revealed that rohu is the most susceptible species to Myxobolus infestation which is further corroborated with the earlier reports (Kalavati and Nandi, 2007; Kaur, 2014). The present study along with earlier report on Argulus outbreak (Saravanan et al., 2017a) provides detailed information about mass mortality of freshwater fishes due to parasitic diseases in these Islands. It is the need of the hour to implement strict biosecurity measures to prevent further spread of diseases and subsequent loss of biomass. Besides, better management practices are to be implemented in a stricter way so as to safeguard the freshwater aquatic biodiversity of the fragile Island ecosystem.

This work was carried out under the National Surveillance Programme for Aquatic Animal Diseases (NSPAAD), coordinated by ICAR-National Bureau of Fish Genetic Resources (NBFGR), Lucknow. The authors are thankful to Indian Council of Agricultural Research (ICAR) and National Fisheries Development Board (NFDB), Govt. of India for the financial support to carry out this work. We sincerely thank Mr. K. Lohith Kumar for preparation of map used in the study.