Marine Leech Anticoagulant Diversity and Evolution

Leeches (Annelida: Hirudinea) possess powerful salivary anticoagulants and, accordingly, are frequently employed in modern, authoritative medicine. Members of the almost exclusively marine family Piscicolidae account for 20% of leech species diversity, and they feed on host groups (e.g., sharks) not encountered by their freshwater and terrestrial counterparts. Moreover, some species of Ozobranchidae feed on endangered marine turtles and have been implicated as potential vectors for the tumor-associated turtle herpesvirus. In spite of their ecological importance and unique host associations, there is a distinct paucity of data regarding the salivary transcriptomes of either of these families. Using next-generation sequencing, we profiled transcribed, putative anticoagulants and other salivary bioactive compounds that have previously been linked to blood feeding from 7 piscicolid species (3 elasmobranch feeders; 4 non-cartilaginous fish feeders) and 1 ozobranchid species (2 samples). In total, 149 putative anticoagulants and bioactive loci were discovered in varying constellations throughout the different samples. The putative anticoagulants showed a broad spectrum of described antagonistic pathways, such as inhibition of factor Xa and platelet aggregation, which likely have similar bioactive roles in marine fish and turtles. A transcript with homology to ohanin, originally isolated from king cobras, was found in Cystobranchus vividus but is otherwise unknown from leeches. Estimation of selection pressures for the putative anticoagulants recovered evidence for both positive and purifying selection along several isolated branches in the gene trees, and positive selection was also estimated for a few select codons in a variety of marine species. Similarly, phylogenetic analyses of the amino acid sequences for several anticoagulants indicated divergent evolution.