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Description of myxosporeans (Cnidaria: Myxozoa) infecting the popular food fish Notopterus notopterus (Pisces: Notopteridae) in Malaysia and India

This study was a co-operative investigation of myxosporean infections of Notopterus notopterus, the bronze featherback, which is a popular food fish in the South Asian region. We examined fish from Lake Kenyir, Malaysia and the River Ganga, Hastinapur, Uttar Pradesh, India, and observed infections with two myxosporeans: Myxidium cf. notopterum (Myxidiidae) and Henneguya ganapatiae (Myxobolidae), respectively. These species were identified by myxospore morphology, morphometry and host tissue affinity, and the original descriptions supplemented with small-subunit ribosomal DNA sequences and phylogenetic analysis. Free myxospores of M. cf. notopterum were found in the gallbladder, and measured 14.7 ± 0.6 μm long and 6.3 ± 0.6 μm wide; host, tissue and myxospore dimensions overlapped with the type, but differed in morphological details (spore shape, valve cell ridges) and locality (Malaysia versus India). Plasmodia and spores of H. ganapatiae were observed in gills, and myxospores had a spore body 9.7 ± 0.4 μm long, 4.5 ± 0.5 μm wide; sample locality, host, tissue, spore morphology and morphometry matched the original description. Small-subunit ribosomal DNA sequences were deposited in GenBank (M. cf. notopterum MT365527, H. ganapatiae MT365528) and both differed by >7% from congeneric species. Although the pathogenicity and clinical manifestation of myxozoan in humans are poorly understood, consumption of raw fish meat with myxozoan infection was reported to be associated with diarrhea. Identification of current parasite fauna from N. notopterus is an essential first step in assessing pathogen risks to stocks of this important food fish.


Introduction
The bronze featherback, Notopterus notopterus Pallas, 1769 (Osteoglossiformes) is a member of a group commonly called "knife fishes", which are distributed widely in Africa, South and Southeast Asia (Talwar and Jhingran, 1991). Knife fish have commercial value for recreational anglers, and species of Notopterus, Chitala, Papyrocranus and Xenomystus have been categorized as a com- Food and Waterborne Parasitology 20 (2020) e00092 was examined for myxozoan parasites with special attention to infections of the gill. Due to degradation of the gill tissue, location and structure of plasmodia could not be characterized in detail. Spores were collected from damaged plasmodia by scraping gill filaments, then preserved in 4% formalin for morphological studies, and in 90% ethanol for molecular analysis. Twenty two fixed spores from a single fish were measured and described for Myxidium species using Nomarski differential interference contrast and photographed. All measurements are expressed in micrometers (Tables 2 and 3).

DNA extraction, amplification and sequencing
Total genomic DNA was extracted from spores preserved in ethanol. The spores were centrifuged at 9600 ×g for 10 min and the supernatant removed. For the Myxidium spores, total DNA was extracted from the spore pellet using a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), while for Henneguya, total DNA was extracted using a Genaid Tissue Genomic DNA Mini Kit (New Taipei City, Taiwan), following the manufacturers' instructions, with a 100 μl final elution step. ssrDNA was amplified using a nested PCR described in detail by . Universal eukaryotic primers ERIB1 and ERIB10 (Barta et al., 1997) were used in the first round PCR. Myxozoan specific primers Myx1F (Hallett and Diamant, 2001) and SphR (Eszterbauer and Székely, 2004) were used in the second round PCR. The primer sequences are listed in Table 1. Amplicons were analysed by electrophoresis in a 1% agarose gel. The PCR products were excised from the gel, purified with the Gel/PCR DNA Fragments Extraction Kit (Geneaid, New Taipei City, Taiwan) and sequenced directly using the BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) with an ABI PRISM 3100 Genetic Analyser (Life Technologies), using the amplification and inner primers.

Phylogenetic analysis
The sequence fragments were assembled using MEGA 6 (Tamura et al., 2013) and ambiguous bases were clarified by visual examination of the corresponding ABI chromatograms. Sequences of the discovered myxozoans were aligned with reference sequences from the NCBI GenBank database (based on a BLAST similarity >88% and coverage >75% for H. ganapatiae, and similarity >89% and coverage >77% for M. cf. notopterum) with CLUSTAL W (Thompson et al., 1994). Final alignments of 24 sequences for M. cf. notopterum and 34 sequences for H. ganapatiae were tested using MEGA 6 for the nucleotide substitution model of best fit as indicated by the Akaike Information Criterion (AIC). Phylogenetic relationships for M. cf. notopterum and H. ganapatiae were inferred using the maximum likelihood (ML) method with the G + I and GTR + G + I substitution models respectively, and bootstrapped with 1000 replicates. Initial trees for the heuristic search were obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using the Nearest Neighbor Interchange (NNI) approach for M. cf. notopterum and Maximum Composite Likelihood (MCL) approach for H. ganapatiae. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories; +G, parameter = 0.3388). The rate variation model allowed for some sites to be evolutionarily invariable ([+I]; 0.0000% sites). All alignments with <75% site coverage were eliminated. Chloromyxum cristatum and Ceratonova shasta were chosen as the outgroups for H. ganapatiae and M. cf. notopterum analyses, respectively.

Discussion
The parasite fauna of fishes belonging to Notopteridae is poorly studied, with data available only for the bronze featherback, N. notopterus. Our cooperative work on finding myxosporean infections in this host from Malaysia and India resulted in detecting two species. We used morphology, morphometry and ssrDNA sequencing, to identify these taxa as M. cf. notopterum and H. ganapatiae, respectively.
Myxidium notopterum Sarkar, 1996 was described from the liver of N. notopterus from India. We identified morphometrically very similar myxospores from the gall bladder of the same host, from Malaysia. We regard the tissue difference as minor, as spores in the gall bladder probably originated in the liver, and no developmental stages were observed in the gall bladder itself. Morphological features differed from the type species (overall sigmoid versus cylindrical shape, more elongate polar capsules, more tubule turns, and valve cell ridges), however no molecular data are available from the type species for comparison. Thus we regard the currently available data as insufficient for identifying M. notopterum, or describing a new species. We recommend that the parasite be collected and re-described from the original biotope in India, and a molecular comparison made. As for the other congeners, morphometry, host and geographic origin differentiate M. cf. notopterum from M. cuneiforme, M. amazonense, M. scripta, and M. truttae. Nevertheless, some measurements of spore features overlap among species, especially polar capsule dimensions and the count of polar tubule turns (Eiras et al., 2011;Li et al., 2016;Mathews et al., 2015;Roberts et al., 2008). This is Gill filaments 20.0-30.0 2.1-3.0 4.9-6.7 0.7-1.1 Uttar Pradesh, India Bhatt and Siddiqui, 1964 the first report of Myxidium species from a Malaysian freshwater fish, and only one other congener is known from this region, the marine Myxidium incomptaverni, which is a hyperparasite of Diplectanocotyla gracilis (Monogenea) (Freeman and Shinn, 2011). Generally, myxospore morphology is a lesser important correlate in phylogenetic relationships, with myxozoans from vertebrate hosts yielding the strongest evolutionary signals, followed by aquatic hosts and tissue tropism (Carriero et al., 2013;Rocha et al., 2018;Kent et al., 2001;Eszterbauer, 2004;Holzer et al., 2004;Fiala, 2006). Due to their paraphyletic and polyphyletic nature, similar morphological features, and closely-related genetic identity, suggestions have been made to merge the genera Myxidium and Zschokella (Fiala, 2006;Li et al., 2016). Phylogenetic analysis from this study produced results that are congruent with previous observations of polyphyly of these two genera, with M. cf. notopterum in a sister clade to several Zschokkella species. The phylogeny supported tissue tropism as an evolutionary signal for M. cf. notopterum, as this species clustered with other gall bladder/bile duct infecting myxosporeans. We found only weak correlation of vertebrate host with myxozoan species in this group, as multiple host taxa/groups (e.g. Siluriformes; Cypriniformes) were represented by myxozoans that clustered with M. cf. notopterum (host = Osteoglossiformes). Although we have added sequence data from the first Myxidium from an osteoglossiform, further clarity of phylogenetic relationships should be gleaned from future sequencing of related myxozoans, particularly from South Asian fishes.
Henneguya spp. are important disease agents in both wild and farmed food fish, with a considerable increase in the number of novel Henneguya species identified recently (Eiras and Adriano, 2012). Henneguya is within the family Myxobolidae, and is distinguished on the basis of myxospores having two caudal processes. Five myxobolid myxozoans are known from the host, Notopterus spp.: three Henneguya spp. (H. ganapatiae, H. notopterae, H. singhi) and two Myxobolus (M. meglitschus and M. notopterum). As we only encountered one of the Henneguya spp. recorded from gills of the bronze featherback, we were unable to test the validity of the other two species with either a redescription of morphology, or addition of molecular data. We confirmed that the species we found, H. ganapatiae, shows morphometric differences to H. notopterae and H. singhi in polar capsules and spore body. However, as with the Myxidium species we found, no sequence data were available from Henneguya spp. from N. notopterus in India, for comparison. From available data, our phylogenetic analysis showed that H. ganapatiae was most similar (90.7%) to three Henneguya species from gill filaments, gill lamellae and muscles, respectively, of Lates calcarifer from Malaysia, and Henneguya chaudharyi (89.4%) from gill filaments of Channa punctatus from India.
Due to difficulties of obtaining live infected N. notopterus, we were unable to prepare histological sections and thus could not study developmental stages or the specific site preference of H. ganapatiae on the gills; both of which are important non-molecular characters for species descriptions. We identified the parasite found as H. ganapatiae Quadri, 1970 on the basis of host, geographic locality, tissue tropism and similarity of myxospore morphology and morphometry. We supplement the type description with molecular data and analysis of the phylogenetic position of this species. Additional myxozoan surveys from South Asian fishes are needed to improve both taxonomy and broader context of known and novel myxozoan parasites from this region. Identification and surveillance for species that parasitize N. notopterus is important for assessing risks to both wild and cultured stocks of this important food fish species.

Declaration of competing interest
The authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.