Peptidomic analysis of the host-defense peptides in skin secretions of Rana graeca provides insight into phylogenetic relationships among Eurasian Rana species

Peptidomic analysis of norepinephrine-stimulated skin secretions from the Greek stream frog Rana graeca Boulenger, 1891 led to the identification and structural characterization of a range of host-defense peptides. These comprised brevinin-1GRa, brevinin-1GRb and an N-terminally extended form of brevinin-1GRb, ranatuerin-2GR together with its oxidized form and (11-28) fragment, temporin-GRa, temporin-GRb and its non-amidated form, and a melittin-related peptide, MRP-GR and its (1-18) fragment. The most abundant peptide, MRP-GR significantly (P < 0.001) stimulated insulin release from BRIN-BD11 clonal β-cells at concentrations ≥0.1 nM. Rana graeca (formerly Rana graeca graeca) and the morphologically similar Italian stream frog Rana italica Dubois, 1987 (formerly Rana graeca italica) were originally regarded as sub-species. However, the primary structures of the host defense peptides from both frogs support the claim based upon comparisons of the nucleotide sequences of S1 satellite DNA that R. graeca and R. italica are separate species. Cladistic analyses based upon the primary structures of the brevinin-1 and ranatuerin-2 peptides from Eurasian frogs indicate a close phylogenetic relationship between R. graeca and Rana latastei whereas R. italica is most closely related to Rana dalmatina.