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Understanding and resolving trematode life cycles is increasingly recognized as an important objective in helminth research, as many species are known only from certain developmental stages or hosts. For the diverse but incompletely documented trematode fauna of southern Africa, life cycle data are particularly scarce. This study aimed to reconstruct trematode life cycles by investigating conspecific developmental stages in first intermediate snail and second intermediate fish hosts across South Africa and Zambia. Snails ( Bulinus sp.) and fishes Enteromius oraniensis (Barnard, 1943) and Clarias gariepinus (Burchell, 1822) were collected between 2019 and 2025. Molecular sequencing (28S rDNA, ITS1–5.8S–ITS2, nad 1 and cox 1) confirmed conspecificity among developmental stages of two trematode taxa: previously published Petasiger sp. 5 (Echinostomatidae) and Tylodelphys sp. 2 (Diplostomidae). Cercariae and metacercariae of both lineages were morphologically examined and descriptions provided. For Petasiger sp. 5, life cycle reconstruction linked cercariae from Bulinus sp. with metacercariae infecting the gills of E. oraniensis , representing the first confirmed record of Petasiger sp. 5 metacercariae infecting cyprinids of the Smiliogastrinae in Africa. For Tylodelphys sp. 2, we provide the first record of its cercarial stage and identify Bulinus sp. as the first intermediate host, genetically linking it to metacercariae recorded from the cranial cavity of C. gariepinus . These findings underscore the value of molecular matching for life cycle reconstruction of digenean trematodes. By elucidating previously unknown developmental stages and host associations of Petasiger sp. 5 and Tylodelphys sp. 2, this study advances the understanding of trematode diversity and transmission in African freshwater ecosystems and provides essential data on first and second intermediate host stages for future species descriptions of these taxa. • Life cycle reconstruction of two aquatic southern African trematode species • Molecular data linked different larval and adult stages • First record of Petasiger sp. 5 metacercariae in African fishes
Hüsken et al. (Fri,) studied this question.