Abstract
Trematode metacercariae are the most abundant and frequently encountered helminths in freshwater fish. Yet, accurate species identification remains challenging, potentially leading to an underestimation of trematode diversity. Using data from parasitological examinations of 1,030 fish (47 species) collected from diverse freshwater habitats in Lithuania (2022-2024), we assessed the contemporary diversity of trematode metacercariae, host associations, microhabitat preferences, and changes in diversity patterns and transmission dynamics. Through integrated morphological and molecular techniques, we identified metacercariae belonging to 51 species from eight families, more than doubling previously reported diversity (25 species). While trematode family composition remained largely unchanged - the Diplostomidae and Strigeidae remained the most diverse families - notable differences were observed at the species level. Metacercariae of the Echinochasmidae and Echinostomatidae were detected for the first time, while previously reported Clinostomidae were absent. Fish of the Leuciscidae hosted the highest trematode diversity. Host specificity of metacercariae was generally low, with most species being euryxenous. At the microhabitat level, eyes harboured the highest number of species, while muscles showed the highest metacercarial density. Notably, we detected species first genetically characterised in North America (Echinoparyphium sp. 2 and Ichthyocotylurus sp. 2) and species potentially belonging to the genus Neogogatea, previously known only from Asia and North America, highlighting potential invasion risks and suggesting that European trematode diversity remains substantially underestimated. Future efforts should obtain molecular data from correctly identified adult specimens to resolve the identity of species currently identified only to the genus or family level, thereby enabling assessment of their geographical distributions and ecological roles.