Integrative morphological and molecular characterization of Proterodiplostominae metacercariae (Digenea: Diplostomidae) infecting siluriform fishes in the Peruvian Amazon Basin

Proterodiplostominae Dubois, 1936 is a small but evolutionarily significant lineage of diplostomid trematodes that primarily parasitize crocodilians, with fishes acting as second intermediate or paratenic hosts. In Peru, knowledge of this group has been limited to the record of adult specimens of Proterodiplostomum longum (Brandes, 1888) recovered from Caiman crocodilus Linnaeus, 1758 (Sauropsida: Alligatoridae) in the Loreto region, and molecular evidence linking larval and adult stages has been absent. The present study provides the first integrative morphological and molecular characterization of Proterodiplostominae metacercariae infecting siluriform fishes in the Peruvian Amazon Basin. Metacercariae were collected from Hoplosternum littorale (Hancock, 1828) (Callichthyidae) and Pterygoplichthys pardalis (Castelnau, 1855) (Loricariidae) and examined using light and scanning electron microscopy, together with partial 28S rDNA and mitochondrial cox1 sequences. Phylogenetic analyses of 28S rDNA demonstrated that three isolates clustered within a strongly supported clade containing reference sequences of P. longum from crocodilian hosts, with minimal genetic divergence (≤0.536%), confirming their conspecificity. This study therefore provides the first molecularly supported record of P. longum metacercariae in Peruvian fishes and clarifies a previously undocumented component of its life cycle. In addition, phylogenetic analyses revealed two distinct evolutionary lineages (designated herein as Proterodiplostominae gen. sp. 4 and gen. sp. 5) exhibiting 9.882% mitochondrial divergence, suggesting previously unrecognized diversity within the subfamily. These findings expand the known host range of P. longum, provide new genetic data for larval stages of Proterodiplostominae in the Neotropics, and highlight the importance of integrative approaches for resolving life-cycle stages and species boundaries in Diplostomidae.