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

Abstract

DNA barcoding coupled with classical morphological description have become promising approach for the species levelidentification. In the present study, 278 numbers of rohu Labeo rohita were screened, for Argulus infestation, amongwhich 167 fish (60.07%) were found infested with Argulus. Morphological attributes of the parasites were studied by lightmicroscope (LM) and Scanning Electron Microscope (SEM) which showed dorsoventrally flattened body comprising ofhead, thorax, abdomen, a pair of large compound eyes, suctorial organs with sclerotised support structures and two openingsof spermatheca at posterior end. Posterior incision of abdomen do not reach the mid-line and the cephalo-thoracic carapacehave not extended beyond the beginning of abdomen. Further validation was done using molecular tools for accurateparasite identification. The pair-wise genetic distance value, using Kimura-2 parameter showed a species level variationof 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. japonicusrespectively. Phylogenetic tree generated using Neighbour-Joining (NJ) and Maximum-Likelihood (ML) methods, withKimura-2 parameter were also in agreement with pairwise distance values. The mitochondrial cytochrome c oxidase subunit1 (COI) sequences of A. foliaceus formed one cluster with the present studied samples, whereas sequences of A. japonicusformed a sister group. Integrating morphological and rapid DNA barcoding tools, the species was delineated as A. foliaceus. Keywords: Argulus foliaceus, COI gene, DNA barcoding, Labeo rohita, Morphology