An investigation of the host-specificity of metacercariae of species of Apophallus (Digenea: Heterophyidae) in freshwater fishes using morphological, experimental and molecular methods

Metacercariae of species of the genus Apophallus Lühe, 1909, infecting the fins and skin of freshwater fishes, frequently cause black spot disease. Two species, Apophallus muehlingi (Jägerskiöld, 1899) and A. donicus (Skrjabin & Lindtrop, 1919), are known to occur in Hungarian fishes. It has generally been thought that metacercariae of A. muehlingi infect cyprinid fishes, whereas those of A. donicus develop in percids. As part of a morphological, experimental and molecular study, metacercariae were collected from 99 infected specimens of five cyprinid hosts (Abramis brama, Blicca bjoerkna, Chondrostoma nasus, Squalius cephalus, Scardinius erythrophthalmus) and 18 infected specimens of two percid hosts (Gymnocephalus cernua, Perca fluviatilis) in Hungarian natural waters (Lake Balaton, River Danube). Moreover, 1024 common carp (Cyprinus carpio) specimens collected from Hungarian fish ponds were investigated for Apophallus infection, but without positive results. For reliable species identification, experimental infections of chicks were carried in order to produce adult specimens from metacercariae collected from the fins and skin of the cyprinid and percid hosts. Within 8 days, adult specimens of both A. muehlingi and A. donicus developed in chicks infected with metacercariae from the cyprinid common bream (Abramis brama) and the white bream (Blicca bjoerkna) and the ruffe (Gymnocephalus cernua), a percid, respectively. The morphology of the collected metacercariae and adult individuals developed in the feeding experiments was characterised. A molecular analysis was extended to cercarial samples from the snail Lithoglyphus naticoides and to a single adult specimen of Apophallus from a fox. Sequences of 28 specimens were analysed using molecular methods (sequencing the internal transcribed spacer region and the cytochrome oxidase I subunit). Phylogenetic analysis was executed, and the Apophallus samples clustered into three distinct branches using both genes, A. muehlingi from cyprinids, A. donicus from percids and, a third, previously unknown, Apophallus clade, also from cyprinids.