Characterization of Clinostomum metacercariae using microscopic and molecular approaches.

One of the fundamental aspects in understanding the biology, diversity and epidemiology of a parasite lies in its proper identification. In the present study, morphological and molecular characterization of Clinostomum metacercariae recovered from an ornamental fish, Trichogaster fasciata, was carried out in order to ascertain its identity. To serve the purpose, scanning electron micrographs and gene sequences for two commonly used molecular markers, i.e., nuclear ribosomal internal transcribed spacer 2 (rDNA-ITS2) and mitochondrial cytochrome c oxidase subunit 1 (mtCO1) were obtained. The sequences were further used for generating similarity index matrix as well as inferring phylogenies. Light and electron microscopic observations on metacercariae of the parasite revealed that it belongs to the genus Clinostomum. Identification of the same up to the level of species was made possible through sequence and phylogenetic analyses. The ITS2 sequence analyses of our species (KX758630) showed similarity to unidentified Clinostomum sp. reported from Nigeria (KY865625) and China (KP110579), and C. tilapiae recorded from South Africa (KX034048) and Nigeria (KY649353). However, the CO1 gene analyses suggested it to be highly identical to C. philippinense and the same was also corroborated in the phylogenetic analysis. Thus, morphological and molecular characterization revealed that the recovered metacercariae belong to the species C. philippinense. Additionally, a brief description of secondary structures of ITS2 of various species of Clinostomum has also been discussed.