Identification of Larval Eustrongylides (Nematoda: Dioctophymatoidea) sp. from Channa punctata Bloch, 1793 by Morphological and Molecular Techniques

Objective: This study aimed to identify the larval form of Eustrongylides sp. isolated from the visceral organs of Channa punctata (Bloch, 1793) using morphological and molecular methods. Methods: Fishes were collected from fish farms in Nadia and North 24 Paraganas for the collection of nematodes. The visceral organs were dissected and kept in 0.67% normal saline. Nematodes collected from the abdominal regions and visceral organs for light microscopy study were fixed in 70% ethanol. Morphological features were studied by placing the nematodes in lactophenol. The specimens were later preserved in 70% ethanol containing 5% glycerine. Specimens processed for scanning electron microscopy were fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetraoxide. Proper identification was done by using standard methology. Molecular studies were performed for the 18S rRNA, ITS1, 5.8S rRNA, ITS2 and 28S rRNA gene fragments using polymerase chain reaction amplification, sequencing and phylogenetic analysis. Results: The morphological characteristics of nematodes were described with the help of light and scanning electron microscopy. Additional features not described earlier like dimensions and shape of the cephalic papillae, absence of somatic papillae, presence of caudal papillae, were identified for the first time. Moreover, molecular studies with ITS regions further confirmed the identification of the nematode. Conclusion: Thus the use of morphotaxonomy along with molecular techniques would help in proper identification of Eustrongylides sp infecting edible fish. Studies on the nematode would help toexplore the intermediate as well as paratenic hosts of the parasite. Data in this regard would contribute significantly to the fish database in regard to parasites infesting edible fishes.