Dactylogyrus kolodynensis sp. n. (Platyhelminthes: Monogenea) infecting gills of Osteobrama cotio (Hamilton, 1822) (Cypriniformes: Cyprinidae) from India

Dactylogyrus kolodynensis, a new monogenean parasite species, is described from Osteobrama cotio (Cyprinidae) collected from Lawngtlai (Mizoram) and Lucknow (Uttar Pradesh), India, using morphological examination and sequencing of partial 28S rRNA gene. The new species is morphologically characterized and distinguished from closely related congeners by a combination of the following characters: copulatory tube a loose coil of one complete clockwise ring, jaw-shaped accessory piece comprising variable sheathes enclosing and guiding the copulatory tube, and sclerotized vaginal tube, with a terminal flower-bud-shaped vaginal pore. The molecular analyses of specimens of D. kolodynensis collected from two different localities using 28S rRNA gene showed identical genotype that did not match any of the known sequences in GenBank, confirming our initial morphological identification. Dactylogyrus cotius, a sympatric species on the gills of O. cotio, is regarded as a species inquirenda because of its poor description. This is the first report of a monogenean species from Mizoram, in northeast India, bringing the total number of Dactylogyrus species in India to 57.


Introduction
Osteobrama cotio (Hamilton 1822) is an Asian freshwater benthopelagic fish species inhabiting the rivers, lakes, ponds, and ditches throughout Pakistan, India, Nepal, and Bangladesh (Froese and Pauly 2021). In India, O. cotio is widely distributed in states of Assam, Manipur, Mizoram (Kar and Sen 2007), West Bengal, Madhya Pradesh, Punjab, and Uttar Pradesh (Vishwanath and Shantakumar 2007). This small indigenous fish provides a nutritional supplement to a large section of the economically backward population (Kumar and Goswami 2013). Two monogenean species have previously been described from the gills of O. cotio: Dactylogyrus cotius (Jain 1957) Gusev, 1973 andDactylogyroides osteobramii Agrawal, Pandey andTripathi, 2002. During a recent parasitological investigation of the cyprinid fishes, several specimens of monogeneans were found on the gills of O. cotio collected from the Kolodyne river in Mizoram and Gomti river in Uttar Pradesh. A morpho-molecular examination revealed that these specimens represent a new species of Dactylogyrus, which is described and illustrated in this paper.

Fish sampling
Thirty-five specimens of O. cotio [(total weight: 8.71-10.91 gm, length: 7-11 cm)] were collected using gillnets between August and December 2021 from the Kolodyne river in Lawngtlai, Mizoram, northeast India (11 specimens) and the Gomti river in Lucknow, Uttar Pradesh, north India (25 specimens). Fish were transported alive to the laboratory in oxygenated containers filled with riverine water and dissected under a stereo zoom microscope according to standard parasitological procedures. Their gills, along with the flatworms, were fixed and preserved separately in 5% formalin and 95% ethanol for morphological and molecular analysis. The scientific name, including taxonomic authority and date, of fish followed Fishbase (Froese and Pauly 2021).

Collection and morphological identification
Formalin-preserved monogeneans were slightly flattened under a coverslip, mounted unstained in Hoyer's medium and glycerine for examination of the sclerotised structures. Some of them were stained with Horen's trichrome, dehydrated in ascending series of ethanol, cleared in xylene, and mounted in DPX (Dibutylphthalate Polystyrene Xylene) for permanent preservation. The mounted specimens were studied, photographed, and measured using a light microscope (Leica DM4B) equipped with phase-contrast and Differential Interference Contrast (DIC) optics, a digital camera (Leica DFC7000 T), and image analysis software (LAS X; Leica Microsystems Ltd.). An illustration plate was prepared using a drawing tube attachment fixed to the microscope. The measurement (straight-line distances between two extreme points), terminologies, and identification of the flatworms were adapted from Gusev (1976), with the following modifications; the term ''anchor length'' was used instead of 'dorsoapical length', and the terms 'thumb''' and 'shank' were used in place of 'heel' and 'handle', respectively. The copulatory tube, on the other hand, was measured as the total distance along the median line of the coiled tube and the direction of its coil (clockwise vs counterclockwise) was determined using the procedure suggested by Kritsky et al. (1985). All measurements are expressed in micrometres and are presented as the mean with the range and number (n) of specimens measured in parentheses. Numbering and distribution of hooks followed Kulwiec (1927). Prevalence and mean intensity of infection were calculated according to Bush et al. (1997). Type specimens were deposited in the Helminthological collections of the Zoological survey of India, Kolkata, India.

DNA extraction, PCR amplification and sequencing
Genomic DNA was extracted from two 95% ethanol-fixed monogenean parasites (one individual randomly selected from each locality) with Extracta DNA Prep for PCR-Tissue (Quantabio, Beverly, US), according to the manufacturer's instructions. Partial fragment of 28S ribosomal RNA gene was amplified by employing the universal primers c1 forward and d2 reverse (Hassouna et al. 1984) and the thermo-cycle profile of Simkova et al. (2006). The PCR products were purified (on 1.5% agarose using the QIAquick PCR Purification Kit from Qiagen, USA) before being Sanger sequenced in both forward and reverse directions by a commercial facility (Eurofins Genomics India Pvt. Ltd.) using the identical primers that generated the PCR products. The resulting sequences were analysed with SnapGene version v.5.3 (http://www.snapgene.com) and a consensus sequence was obtained using BioEdit (Hall 1999). To achieve species-rank identification based on 28S rRNA gene, the consensus sequence was compared with all sequences from related species as retrieved from the NCBI GenBank database using BLAST (https://blast.ncbi. nlm.nih.gov/Blast.cgi) (see accession numbers in Table 1). Etymology: The species is named after the river ''Kolodyne'', the type locality of the species, with the Latin suffix -ensis denoting a location.

Molecular characterisation
Sequencing of partial 28S rRNA gene of D. kolodynensis resulted in amplicons of the same length size (508 bp) and showed no intraspecific nucleotide variations between different individuals collected from two different localities examined here. The sequence of D. kolodynensis sp. n. was deposited in GenBank database (http://www.ncbi. nlm.nih.gov), accession number OL964059. A BLAST search revealed that this sequence did not match 100% to any of the known any available sequences in GenBank, confirming our initial morphological identification (Table 1).

Differential diagnosis
Dactylogyrus kolodynensis sp. n. is differentiated from its closely related congeners by having a copulatory tube that is a loose coil of one complete clockwise ring, a jaw-shaped accessory piece with variable sheathes enclosing and guiding the copulatory tube, and sclerotized vaginal tube with a terminal flower-bud-shaped vaginal pore.

Discussion
Dactylogyrus Diesing, 1850 is a genus with the most species in the Monogenea (Platyhelminthes), with over 900 nominal species world-wide (Gibson et al. 1996), including 56 species from India (Wangchu et al. 2017). These species are well-known for causing chronic debility, poor development and growth, impaired respiration, and finally mass mortality of infested host fish, particularly those under intensive culture/captive conditions (Paperna 1963;Ramadan et al. 1995;Kritsky and Heckmann 2002;Lu et al. 2012). Dactylogyrus kolodynensis sp. n. can be easily confused with D. bucinus Gussev, 1976 from Barbus dorsalis (Jerdon, 1849) (now Puntius dorsalis) and D. parvianchoris Gusev, 1976 from Chaila bacaila (Hamilton, 1822) (now Salmostoa bacaila) in the general morphology of the haptoral and reproductive hard parts. However, the new species differs from D. bucinus mainly in having smaller anchors (24-32 in D. kolodynensis vs. 40-42 in D. bucinus), hooks (15-20 in D. kolodynensis vs. 16-27 um in D. bucinus), and vaginal tube (32-38 in D. kolodynensis vs 50 in D. bucinus), as well as the claw-shaped distal end (vs absent in D. bucinus). In addition, D. bucinus is a parasite of P. dorsalis in south India, while the new species was collected from O. cotio in northeast and north India.
It should be noted that no museum type specimens of D. bucinus and D. parvianchoris were available for examination. Inquiries to the Zoological Society of India, Kolkata and Zoological Institute, Russian Academy of Sciences, St Petersburg, where Gusev (1976) has deposited his specimens, were unsuccessful. Therefore, the comparison of D. kolodynensis sp. n. had to be based entirely on the published descriptions of D. bucinus and D. parvianchoris.
The most closely related species to D. kolodynensis sp. n., according to a BLAST results for our sequence OL964059, were two unpublished species from Pethia ticto (Hamilton, 1822) Gusev, 1976;D. longiacus Gusev, 1976;and D. subtilis Gusev, 1976. Due to this paucity of publicly available comparative molecular data as yet, we have not attempted to create a phylogeny including the new species, but have submitted the 28S gene sequence to GenBank for future comparison.
This study presented us with a unique problem involving D. cotius Jain (1957), one of the two monogenean species, previously described from the gills of O. cotio, along with D. osteobrami. Dactylogyrus cotius was originally described by Jain (1957) as Neodactylogyrus cotius from the gills of Rohtee cotio (now D. cotio) from Lucknow. Gusev (1973) transferred the species to the genus Dactylogyrus. While we consistently found D. kolodynensis sp. n. and D. osteobrami associated with O. cotio in both Mizoram and Uttar Pradesh, we never found D. cotius in either location. This absence of D. cotius, especially in material collected from Lucknow-the same host and locality as the previous one (Jain 1957) took us completely by surprise. This could be due to O. cotio being an atypical host for D. cotius, or it could be due to Jain (1957) misidentifying his host specimens as O. cotio and instead describing D. cotius from a different host species. A major problem with D. cotius is that its morphological description is mostly incomplete and its illustrations are highly diagrammatic, which means they do not correspond to each other. Additionally, Jain (1957) did not specify the location of his type specimens, indicating that they unlikely to have been deposited in a national or international museum, and thus be available for comparison. As a result, we believe it necessary to place D. cotius as a species inquirenda until the species is redescribed using specimens collected from the type host and locality.
The validity of D. kolodynensis sp. n. and its placement within the genus Dactylogyrus was thus supported both by morphological and molecular comparisons among related species. This is the first report of a monogenean parasite from the Mizoram, bringing the total number of nominal Dactylogurus species known from India to 57. Given that Mizoram is a biodiversity hotspot (Barman et al. 2018), with no fewer than 156 fish species, including approximately 78 cyprinids distributed in its diverse hilly terrain (Goswami et al. 2012), we anticipate a high species richness of monogenean parasites from this region.