Food-borne trematode infections, which are mainly transmitted through consumption of inadequately cooked or raw fish and crabs, affect a large section of population, particularly in Southeast Asian countries, thus eliciting a remarkable morbidity and causing serious damage to health. In India, centering in several mountainous regions of the Northeast, the natives have the habit of consuming such fish or crabs that still sustain viable infective larval stage (metacercaria) of trematode flukes in their muscle tissue. The present study was undertaken to ascertain the spectrum of metacercarial diversity in commonly edible freshwater fishes and crab species in the northeastern state of Manipur and to adjudge their zoonotic potential, if any. Commonly edible fishes belonging to 15 species from 12 localities and crabs belonging to 2 species from 11 localities across Manipur state were surveyed for the purpose. The study revealed that 3 species of fishes (Channa punctatus, C. straitus and Wallago attu) harboured 4 different types of metacercariae belonging to 4 trematode families-Euclinostomum heterostomum (Clinostomidae); Lophosicyadiplostomum sp. and Posthodiplostomum sp. (Diplostomidae); and Polylekithum sp. (Allocreadiidae) in addition to adult flukes of Isoparorchis hypselobagri (Isoparorchiidae). Among these, metacercariae of Posthodiplostomum showed the highest prevalence (2.33 %) though a low abundance, while for other species the prevalence ranged between 0.25 and 1.19 %. The crab species (Barythelphusa lugubris masoniana and Potamiscus manipuriensis) were found infected with 4 different types of metacercariae representing the genera Paragonimus (Troglotrematidae) and Microphallus (Microphallidae). The paragonimids showed a higher rate of occurrence (*4-25 %) compared to microphallids (*15 %). The crustaceans surveyed emerged as prospective intermediate hosts for lungflukes. Identifying the potent vectors for zoonotic parasites helps in control measures towards their transmission to higher mammals.

Freshwater fishes in Manipur, Northeast India frequently harbour several types of metacercariae, which based on morphological criteria were identified as Clinostomoides brieni, Euclinostomum heterostomum (Clinostomidae) and Polylekithum sp. (Allocreadiidae). Molecular techniques utilizing PCR amplification of rDNA regions of larger subunit (LSU or 28S), smaller subunit (SSU or 18S) and inter transcribed spacers (ITS1, 2) were used for molecular characterization of these types. Sequences generated from the metacercariae were compared with their related sequences available in public databases; an analysis of the identity matrices and phylogenetic trees constructed was also carried out, which confirmed their identification. Similarly, the sequences generated from Polylekithum sp. were found to be highly similar to the species of the same genus. The rDNA ITS2 secondary structure provided additional confirmation of the robustness of the molecular marker as a tool for taxon-specific characterization.

141 SummaryThe spotted snakehead, Channa punctata Bloch, 1793, is a locally important fish species commonly consumed by the natives in the state of Manipur, Northeast India. The fish host C. punctata from Lamphel area revealed a diplostomid metacercarial infection. Morphologically, the recovered metacercaria was identified as a species of Posthodiplostomum Dubois, 1936. Molecular characterization using the ribosomal RNA genes (rDNA 18S, ITS2 and 28S regions) and the mitochondrial CO1 region supplements the identification. Molecular analysis revealed the metacercaria to be closely related to Posthodiplostomum sp. Japan isolate, with sequence similarity variation from 97.5 -99.7 % while considering for the three rDNA markers. The secondary structure of the ITS2 region further corroborated these results; the typical four-helix model, when compared to the taxon from Japan, showed differences only in twelve bases (with seven transitions and five transversions). In phylogenetic analysis also, the metacercaria claded with the genus Posthodiplostomum, coming closer to the Japanese isolate, thus supplementing the morphological identification of the metacercaria.

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