Classical morphology and DNA barcoding based identification of freshwater ectoparasite, Argulus foliaceus in rohu Labeo rohita

DNA barcoding coupled with classical morphological description have become promising approach for the species level identification. In the present study, 278 numbers of rohu Labeo rohita were screened, for Argulus infestation, among which 167 fish (60.07%) were found infested with Argulus. Morphological attributes of the parasites were studied by light microscope (LM) and Scanning Electron Microscope (SEM) which showed dorsoventrally flattened body comprising of head, thorax, abdomen, a pair of large compound eyes, suctorial organs with sclerotised support structures and two openings of spermatheca at posterior end. Posterior incision of abdomen do not reach the mid-line and the cephalo-thoracic carapace have not extended beyond the beginning of abdomen. Further validation was done using molecular tools for accurate parasite identification. The pair-wise genetic distance value, using Kimura-2 parameter showed a species level variation of 0.001 (1%) with that of A. foliaceus, while 0.083 and 0.052 (i.e. more than 2%) with that of A. indicus and A. japonicus respectively. Phylogenetic tree generated using Neighbour-Joining (NJ) and Maximum-Likelihood (ML) methods, with Kimura-2 parameter were also in agreement with pairwise distance values. The mitochondrial cytochrome c oxidase subunit 1 (COI) sequences of A. foliaceus formed one cluster with the present studied samples, whereas sequences of A. japonicus formed a sister group. Integrating morphological and rapid DNA barcoding tools, the species was delineated as A. foliaceus.