Morphological and molecular characterisation of three Indian Neascus-type metacercariae (Digenea: Diplostomidae)

Diplostomid digeneans are important parasites whose larval stages infect freshwater snails and fish as first and second intermediate hosts respectively. Diplostomid digeneans as adults are parasitic in many fish-eating birds and mammals. Our understanding of the species diversity of diplostomid digeneans remains incomplete, especially in the Indian sub-continent. Here, we describe three Neascus-type metacercariae (N. hanumanthai, N. gussevi, and N. xenentodoni) from freshwater fish specimens of Channa punctata (Bloch 1793), Trichogaster fasciata (Bloch and Schneider 1801) and Xenentodon cancila (Hamilton, 1822) respectively, collected in India. Next, we characterised these metacecariae using nuclear (28S and ITS1) and mitochondrial DNA (cox1) to determine their systematic and phylogenetic position. Molecular identification using interspecific variation for all three molecular markers revealed a closer relationship between N. hanumanthai and N. gussevi (1.9%-2.4%) than either of N. hanumanthai and N. gussevi to N. xenentodoni (3.1%-3.7% and 4.4%-4.0% respectively). In phylogenetic analyses, estimated by neighbour-joining (NJ) and maximum likelihood (ML) methods, N. gussevi and N. hanumanthai nested as sister groups of Posthodiplostomum Dubois, 1936 for all three markers used; N. xenentodoni, however, nested as a sister taxa of all other diplostomes when using 28S and ITS1 and nested as a sister taxa of Bolbophorus, Alaria and Neodiplostomum using cox1. These findings suggest that N. hanumanthai and N. gussevi are members of Posthodiplostomum, but that N. xenentodoni belongs to a separate and unknown genus. Similarly, by proteomics, we found that the cox1 protein sequences and structures were similar between N. hanumanthai and N. gussevi but distinct for N. xenentodoni.