Raja Ben Ahmed, Amina Abilov, Christian Müller

• Medicinal leeches of the genus Hirudo inhabit large areas of the Palaearctic realm. The distribution range of Hirudo troctina includes the southern Iberian peninsula and the northwestern regions of Africa. H. troctina is used for medical purposes, but only very little is known about the components of its salivary gland secretion. Hirudins, bivalent inhibitors of thrombin, are probably the best known leech-derived bioactive factors. Hirudin-like factors (HLFs) represent another class of salivary gland components that share characteristic genetic and structural markers with hirudins. Hirudin is not a single entity but exists in at least four different variants. However, there are differences among the European members of the genus Hirudo with respect to the actual number of hirudin and HLF genes that are present within their genomes. Here, we describe the identification and molecular cloning of 11 genes that encode for putative hirudin and HLF variants in H. troctina . Three of the genes consist of exons and introns that originate from different “archetype” genes and are likely the result of recombination events. The diversity of hirudin and HLF genes in H. troctina surpasses that of all other European members of the genus Hirudo . The putative hirudin variants and representatives of the HLFs of H. troctina were expressed as recombinant proteins, purified and functionally characterized for their thrombin-inhibiting potencies. Phylogenetic analyses based on hirudin and HLF gene sequences of the leech genera Hirudo , Hirudinaria , and Whitmania supported the hypothesis that hirudins and HLFs diverged early in leech evolution.