Quantity - but not diversity - of secreted peptides and proteins increases with age in the tree frog Pithecopus nordestinus

Amphibians inhabit the terrestrial environment, a conquest achieved after several evolutionary steps, which were still insufficient to make them completely independent of the aquatic environment. These processes gave rise to many morphological and physiological changes, making their skin (and cutaneous secretion) rich in bioactive molecules. Among the tree frogs, the secretion is composed mainly of peptides; but alkaloids, proteins and steroids can also be found depending on the species. The most known class of biologically active molecules is the antimicrobial peptides (AMPs) that act against bacteria, fungi and protozoans. Although these molecules are well-studied among the hylids, AMPs ontogeny remains unknown. Therefore, we performed peptidomic and proteomic analyses of Pithecopus nordestinus (formerly Phyllomedusa nordestina) in order to evaluate the peptide content in post-metamorphosed juveniles and adult individuals. Methods: Cutaneous secretion of both life stages of individuals was obtained and analyzed by LC-MS/MS after reduction and alkylation of disulfide bonds or reduction, alkylation and hydrolysis by trypsin. Results: Differences in the TIC profile of juveniles and adults in both treatments were observed. Moreover, the proteomic data revealed known proteins and peptides, with slight differences in the composition, according to the life stage and the treatment. AMPs were identified, and bradykinin-potentiating peptides were observed in trypsin-treated samples, which suggests a protein source of such peptide (cryptide). Conclusion: In general, skin secretion contents were similar between juveniles and adults, varying in quantity, indicating that the different stages of life are reflected in the number of molecules and not on their diversity.(AU)

Biochemical characterization and cytotoxic effect of the skin secretion from the red-spotted Argentina frog Argenteohyla siemersi (Anura: Hylidae)

Argenteohyla siemersi (red-spotted Argentina frog) is a casque-headed tree frog species belonging to the Hylidae family. This species has a complex combination of anti-predator defense mechanisms that include a highly lethal skin secretion. However, biochemical composition and biological effects of this secretion have not yet been studied. Methods: The A. siemersi skin secretion samples were analyzed by mass spectrometry and chromatographic analysis (MALDI-TOF/MS, RP-HPLC and GC-MS). Proteins were also studied by SDS-PAGE. Among the biological activities evaluated, several enzymatic activities (hemolytic, phospholipase A2, clotting, proteolytic and amidolytic) were assessed. Furthermore, the cytotoxic activity (cytolysis and fluorescence staining) was evaluated on myoblasts of the C2C12 cell line. Results: The MALDI-TOF/MS analysis identified polypeptides and proteins in the aqueous solution of A. siemersi skin secretion. SDS-PAGE revealed the presence of proteins with molecular masses from 15 to 55 kDa. Steroids, but no alkaloids or peptides (less than 5 KDa), were detected using mass spectrometry. Skin secretion revealed the presence of lipids in methanolic extract, as analyzed by CG-MS. This secretion showed hemolytic and phospholipase A2 activities, but was devoid of amidolytic, proteolytic or clotting activities. Moreover, dose-dependent cytotoxicity in cultured C2C12 myoblasts of the skin secretion was demonstrated. Morphological analysis, quantification of lactate dehydrogenase release and fluorescence staining indicated that the cell death triggered by this secretion involved necrosis. Conclusions: Results presented herein evidence the biochemical composition and biological effects of A. siemersi skin secretion and contribute to the knowledge on the defense mechanisms of casque-headed frogs.(AU)

Observation of predation of the giant fishing spider Ancylometes rufus (Walckenaer, 1837) (Araneae, Ctenidae) on Dendropsophus melanargyreus Cope, 1877 (Anura, Hylidae) / Observação de predação da aranha pescadora Ancylometes rufus (Walckenaer, 1837) (Araneae, Ctenidae) sobre Dendropsophus melanargyreus Cope, 1877 (Anura, Hylidae)

We report here an observation of predation of the giant spider Ancylometes rufus on the tree frog Dendropsophus melanargyreus in a southern region of Amazonia Forest. We also reviewed the available literature on predation of this spider species on vertebrates.

Nós reportamos aqui uma observação de predação da aranha pescadora Ancylometes rufus sobre a perereca Dendropsophus melanargyreus na região sul da floresta Amazônica. Nós também revisamos os dados de literatura sobre os eventos de predação envolvendo essa espécie de aranha e outros vertebrados.

Vocalization of the tree frog Polypedates maculatus (Rhacophoridae).

The period of calling activity of Polypedates maculatus lies between April and October. Males possess an indistinct subgular vocal sac which turns yellow during the breeding season. Mating calls type I, type II and distress calls have been identified. Mating calls type I and type II consist of a single pulse group. Type I call comprises of 7-22 pulses, whereas type II call consists of 4-6 pulses. Pulses are short. The frequency spectrum is broad and continuous. Distress calls, with 6 hormonics, are given by the females with their mouth open.