Hirudins and ornatins in five species of the genus Placobdella (Annelida: Hirudinea: Glossiphoniidae).

Leeches of the genus Placobdella Blanchard, 1893 (Annelida: Rhynchobdellida: Glossiphoniidae) are mainly distributed across North and Central America, but at least two species are also present in the palearctic region: the European turtle leech Placobdella costata Müller, 1846, and the North African turtle leech Placobdella nabeulensis Ben Ahmed et al., 2023. All species of the genus are hematophagous and feed on vertebrates, but are known to preferentially target aquatic and semi-aquatic reptiles like turtles and snakes. Placobdella ornata Verrill, 1872, is the original source of ornatins, a class of potent platelet aggregation inhibitors. However, the whole repertoire of bioactive peptides, including anticoagulation factors remains largely underexplored for Placobdella, and functional characterizations of putative antithrombotics are scarce. Here we describe the genes and cDNAs that encode putative hirudins and ornatins in both of the palearctic and three American species of the genus Placobdella. A selection of putative hirudins and ornatins was recombinantly expressed and functionally characterized using appropriate coagulation and platelet aggregation assays, and our data confirm the expression of functional hirudins and ornatins in the studied leeches. In addition, our genetic analyses strongly support the hypothesis that blood feeding evolved only once in the evolutionary history of leeches.